Analysis of trypsin digested IgG using capillary ......Analysis of trypsin digested IgG using capillary electrophoresis and mass spectrometry Ana Isabel Fernandes de Carvalho Thesis

Analysis of trypsin digested IgG using capillary

electrophoresis and mass spectrometry

Ana Isabel Fernandes de Carvalho

Thesis to obtain the Master of Science Degree in

Chemical Engineering

Supervisors: Prof. Åsa Emmer;

Prof. Miguel Nobre Parreira Cacho Teixeira

Examination Committee

Chairperson: Prof. Maria Matilde Soares Duarte Marques

Supervisor: Prof. Miguel Nobre Parreira Cacho Teixeira

Members of the Committee: Prof. Ana Margarida Nunes da Mata Pires de Azevedo

November 2018

ii

iii

Acknowledgments

I would first like to express my gratitude to Prof. Åsa Emmer for accepting me in her research group and

allowing me this great experience in KTH, for guiding me and for providing excellent conditions so that

my colleagues and I could have everything needed.

I wish to acknowledge Prof. Miguel Teixeira for his support, dedication, and interest during all this

process.

I am also grateful to all my colleagues of the Analytical Chemistry research group particularly to Sara

and Joakim for the friendship and guidance.

To António, thank you for your support and encouragement throughout all the process.

Finally, I would like to thank my family, specially my parents, for giving me this opportunity to study

abroad and for the great motivation along this journey.

iv

v

Abstract

Complex regional pain syndrome is a chronic condition, considered one of the most painful diseases.

Currently, there is no single diagnosis available which often leads to delaying the beginning of the

treatments. It could be hypothesized that mutations in the glycosylation of the protein Immunoglobin G,

the most abundant immunoglobulin in human plasma, could be a cause for this pathology.

This project includes the study of trypsin digested Immunoglobulin G. Capillary electrophoresis was

used for sample separation and mass spectrometry for identification. An evaluation of the systems and

optimization of the methods separately and in combination was carried out. Both electrospray ionization

mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry have been

investigated. Besides the implementation of the techniques mentioned, an effective method for coating

the capillaries with polyvinyl alcohol was developed.

Capillary electrophoresis revealed appropriate for the separation of Immunoglobulin G trypsin digested

peptides, with well-resolved electropherograms. With the electrospray ionization mass spectrometry, it

was not possible to identify IgG peptides neither performing a database search nor comparing with the

theoretical peptide mass list. Matrix-assisted laser desorption/ionization mass spectrometry proved to

be a powerful tool to identify the peptides. Numerous attempts were made to couple the separation

technique with mass spectrometry. However, it was not possible to develop a setup with reproducible

results.

Keywords: Capillary electrophoresis, mass spectrometry,

immunoglobulin G, complex regional pain syndrome

vi

vii

Resumo

A Síndrome de Dor Regional Complexa é uma condição crónica, considerada uma das doenças que

provoca mais dor. Atualmente, não existe um diagnóstico disponível, o que leva muitas vezes ao atraso

do início do tratamento. Uma hipótese para a causa desta doença são mutações na glicosilação da

proteína Imunoglobulina G, a imunoglobulina mais abundante no plasma humano.

Este projeto inclui o estudo da imunoglobulina G, digerida através da enzima tripsina. A técnica de

eletroforese capilar foi utilizada para separação de amostra e a espectrometria de massas para

identificação desta proteína. Foi realizada uma avaliação dos sistemas e uma otimização dos métodos

de separação e identificação, tanto separadamente como acoplados. Ambas as técnicas de

espectrometria de massas de ionização por electrospray e de ionização/dessorção a laser assistida por

matriz foram investigadas. Além da implementação das técnicas mencionadas, foi ainda desenvolvido

um método para o revestimento de capilares com álcool polivinílico.

A eletroforese capilar revelou-se adequada para a separação dos péptidos da imunoglobulina G, tendo

sido obtidos eletroferogramas bem resolvidos. Utilizando a espectrometria de massas de ionização por

electrospray, não foi possível identificar os péptidos, nem através de pesquisa em bases de dados nem

comparando com a massa teórica. A espectrometria de massas de ionização/dessorção a laser

assistida por matriz mostrou-se uma ferramenta eficaz para identificar péptidos. Diversas tentativas

foram feitas para acoplar as técnicas de separação e espectrometria de massas, no entanto, não foi

possível desenvolver uma montagem experimental com resultados reprodutíveis.

Palavras-chave: Eletroforese capilar, espectrometria de massas,

imunoglobulina G, síndrome de dor regional complexa

viii

ix

Table of Contents

Acknowledgments ................................................................................................................................ iii

Abstract .................................................................................................................................................. v

Resumo................................................................................................................................................. vii

Table of Contents ................................................................................................................................. ix

List of Figures ....................................................................................................................................... xi

List of Tables ....................................................................................................................................... xiii

List of Symbols ................................................................................................................................... xiii

List of Abbreviations .......................................................................................................................... xiv

1. Introduction .................................................................................................................................... 1

1.1. Context .................................................................................................................................. 1

1.2. Problem definition .................................................................................................................. 2

1.3. Objectives .............................................................................................................................. 2

1.4. Outline ................................................................................................................................... 3

2. Background .................................................................................................................................... 5

2.1. Fundamentals of Capillary Electrophoresis ........................................................................... 5

2.1.1. Capillary Wall Modifications ...................................................................................... 7

2.2. Fundamentals of Mass Spectrometry .................................................................................... 8

2.2.1. Electrospray Ionization ............................................................................................. 9

2.2.2. Matrix Assisted Laser Desorption/Ionization .......................................................... 10

2.3. Fundamentals of Immunoglobulin G .................................................................................... 11

2.4. Fundamentals of Proteomics ............................................................................................... 12

3. Materials and Methods ................................................................................................................ 15

3.1. Chemicals ............................................................................................................................ 15

3.2. Instrumentation .................................................................................................................... 15

3.2.1. Capillary Electrophoresis ........................................................................................ 16

3.2.2. Electrospray Ionization ........................................................................................... 17

3.2.3. Matrix Assisted Laser Desorption/Ionization .......................................................... 17

3.3. Sample Preparation ............................................................................................................. 18

3.4. Capillary Electrophoresis ..................................................................................................... 18

3.4.1. Capillary Wall Coating ............................................................................................ 19

3.5. Electrospray Ionization ........................................................................................................ 22

x

3.5.1. CE-ESI-MS Hyphenation ........................................................................................ 23

3.6. Matrix Assisted Laser Desorption/Ionization ....................................................................... 24

3.6.1. CE-MALDI-TOF-MS Hyphenation .......................................................................... 24

4. Results and Discussion .............................................................................................................. 29

4.1. Capillary Electrophoresis ..................................................................................................... 29

4.2. Electrospray Ionization ........................................................................................................ 34

4.2.1. CE-ESI-MS Hyphenation ........................................................................................ 39

4.3. Matrix Assisted Laser Desorption/Ionization ....................................................................... 40

CE-MALDI-TOF-MS Hyphenation ....................................................................................... 43

4.4. Comparison between ESI-MS and MALDI-TOF-MS ........................................................... 45

5. Conclusions and Future Works .................................................................................................. 47

6. Bibliography ................................................................................................................................. 49

A. Budapest Criteria for CRPS ........................................................................................................ 53

B. List of Digested Peptides from IgG ............................................................................................ 54

C. Biotools Analysis ......................................................................................................................... 69

xi

List of Figures

Figure 1 - Schematic representation of CE system ................................................................................. 5

Figure 2 – Schematic representation of tandem mass spectrometry ...................................................... 8

Figure 3 - Schematic representation of ESI-MS system (a) and of MALDI-MS (b)................................. 9

Figure 4 – Human IgG structure ............................................................................................................. 11

Figure 5 – Schematic representation of the mass-spectrometry/proteomic process ............................ 12

Figure 6 – Setup of the home-built CE instrument (voltage supply, main CE box, UV detector and

computer to collect the data, from left to right). ..................................................................................... 16

Figure 7 - Tool used for capillary wall coating ....................................................................................... 19

Figure 8 – CE electropherogram illustrating the impact of rinsing the capillary with NaOH ................. 20

Figure 9 – CE electropherogram of mesityl oxide 8,5 mg/mL; injection: 20 kV for 20 s; voltage: 25 kV;

buffer: sodium phosphate (pH=3.2; 100 mM) ........................................................................................ 22

Figure 10 – Apparatus used to verify the cut of the capillary ................................................................ 23

Figure 11 – Setup used for CE-ESI-MS analysis .................................................................................. 24

Figure 12 – CE-MALDI-MS setup ① .................................................................................................... 26

Figure 13 – CE-MALDI-MS setup ② .................................................................................................... 27

Figure 14 - CE electropherogram of digested bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s; voltage:

25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)............................................................................. 30

Figure 15 – CE electropherogram of intact bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s; voltage: 25

kV; buffer: sodium phosphate (pH=3.2; 100 mM) .................................................................................. 30


25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)............................................................................. 31


25 kV; buffer: sodium phosphate (pH=3.2; 50 mM) ............................................................................... 31

Figure 18 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s; voltage: 25


Figure 19 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s; voltage:

25 kV; buffer: ammonium acetate (pH=3.2; 100 mM) ........................................................................... 33


kV; buffer: ammonium acetate (pH=4.6; 100 mM) ................................................................................ 33


kV; buffer: ammonium acetate (pH=4.2; 25 mM) .................................................................................. 33

xii

Figure 22 - ESI-MS spectrum of digested human IgG 15 𝝁g/mL; scan mode: UltraScan; flow rate:

2 L/min; capillary voltage: 4 kV; dry gas flow: 6 L/min; dry gas temperature 300 °C; nebulizer pressure:

1 bar; mass tuning: 150 to 1500 m/z; target: 800 m/z ........................................................................... 34

Figure 23 – Zoomed in view of the peaks potentially related to IgG ..................................................... 36


2 L/min; capillary voltage: 4.5 kV; drying gas flow: 6 L/min; drying gas temperature 300 °C; nebulizer

pressure: 0.9 bar; mass tuning: 300 to 1000 m/z; target: 500 m/z ........................................................ 37


Figure 26 – ESI-MS spectrum of digested human IgG 100 𝝁g/mL; scan mode: UltraScan; flow rate: 2

L/min; capillary voltage: 4 kV; drying gas flow: 4 L/min; drying gas temperature 300 °C; nebulizer

pressure: 0.9 bar; mass tuning: 200 to 1500 m/z; target: 800 m/z ........................................................ 38


Figure 28 - MALDI-TOF-MS spectrum of digested human IgG; matrix: DHB; shot at 90% laser intensity

(1000 shots per burst, 6000 shots total), pulsed ion extraction by reflectron ........................................ 40

Figure 29 - Results obtained from Biotools database search for MS analysis (database: Swiss-Prot) 41

Figure 30 - Results obtained from Biotools database search for tandem MS analysis (database: Swiss-

Prot) ....................................................................................................................................................... 42

Figure 31 – Results obtained from Biotools database search for tandem MS analysis of peak 1186.65

Da (database: Swiss-Prot)..................................................................................................................... 42

Figure 32 - CE electropherogram of digested human IgG 1 mg/mL; injection: 20 kV for 20 s; voltage: 25


Figure 33 - CE electropherogram of digested human IgG 1 mg/mL from CE-MALDI experimental setup

①; injection: 20 kV for 20 s; voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM) ................. 44

Figure 34 - CE electropherogram of digested human IgG 1 mg/mL from CE-MALDI experimental setup

②; injection: 20 kV for 20 s; voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM) ................. 45

Figure 35 – Results obtained from Biotools database search for MS analysis of IgG 1 ....................... 69



Figure 38 - Results obtained from Biotools database search for MS analysis of IgG 4 ........................ 70

xiii

List of Tables

Table 1 – IgG sample list ....................................................................................................................... 18

Table 2 - Capillary parameters, wall coating procedure, symptoms and setup used ............................ 21

Table 3 – ESI peak list of digested human IgG 15 𝝁g/mL and comparison with theoretical data ......... 35

Table 4 – ESI peak list of digested human IgG 15 𝝁g/mL and comparison with theoretical data ......... 37

Table 5 – ESI peak list of digested human IgG 100 𝝁g/mL and comparison with theoretical data ....... 38

Table 6 - MALDI peak list of digested human IgG and comparison with theoretical data ..................... 41

Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 - 330 ................................. 54

Table 8 - Chain Immunoglobulin heavy constant gamma 2 at positions <1-326 ................................... 58

Table 9 - Immunoglobulin heavy constant gamma 3 at positions 1-377 ............................................... 62

Table 10 -– Immunoglobulin heavy constant gamma 4 at positions <1-327 ......................................... 66

List of Symbols

Symbol Meaning

𝐸 Applied Electric Field

𝑚/𝑧 Mass to Charge Ratio

𝑞 Charge

𝑟 Radius

𝑣 Velocity

𝜇𝐸𝑂𝐹 Electroosmotic Mobility

𝜇𝑒 Electrophoretic Mobility

𝜇 Viscosity

𝜁 Zeta Potential

xiv

List of Abbreviations

Abbreviation Meaning

AKD

BGE

Alkyl Ketene Dimer

Background Electrolyte Solution

CE Capillary Electrophoresis

CRPS Complex Regional Pain Syndrome

CZE Capillary Zone Electrophoresis

DHB 2,5-Dihydroxybenzoic Acid

EOF Electroosmotic Flow

ESI Electrospray Ionization

HCCA α-cyano-4-hydroxycinnamic acid

IgG Immunoglobulin G

MALDI Matrix Assisted Laser Desorption/Ionization

MS Mass Spectrometry

MC Miss-cleavage

OPG Osteoprotegerin

PMF Peptide Mass Fingerprinting

PVA Polyvinyl Alcohol

RSD Reflex Sympathetic Dystrophy

SA 3,5-Dimethoxy-4-Hydroxycinnamic Acid

S/N Signal-to-noise

SL Sheath Liquid

TOF Time of Flight

UV Ultra-Violet

1

1. Introduction

1.1. Context

This project is part of a larger one that aims to develop instrumental bioanalytical setups and procedures

for the study of glycoproteins of potential importance in the pathophysiology and diagnosis of Complex

Regional Pain Syndrome (CRPS).

CRPS is a severe long-term pain syndrome, ranked as the most painful disease according to the McGill

Pain Index1 [1]. This condition shows a great variety of symptoms such as allodynia or hyperalgesia,

temperature and skin color asymmetry or trophic and motor changes. Nevertheless, chronic pain is the

key symptom, either permanent or fluctuating, and most often in the deep tissue; affecting one limb

(arm, leg, hand, or foot) [3, 4]. As the name suggests, this is a regional syndrome, i.e., not in a specific

nerve territory or dermatome [4].

There are two types of CPRS. CRPS type I, formerly described as Reflex Sympathetic Dystrophy (RSD),

is developed after any type physical of trauma, especially fractures, and soft tissue lesion. CRPS type II,

once referred to as causalgia, occurs after major nerve damage, which is not found in type I [4]. One

distinguishable characteristic is that CRPS type II does not migrate from the original site of injury like

CRPS type I.

The fact of CRPS being a multi-faceted pathophysiology makes the treatment more complex. There is

not one treatment that is effective for all cases. However, according to Dimova and Birklein (2017), a

systematic and interdisciplinary approach based on the following basic therapeutic principles is used

[2]:

(1) “Medical and nonmedical pain therapy (acute and chronic phases);

(2) Physiotherapy, occupational therapy and training therapy (acute and chronic phases);

(3) Anti-inflammatory therapy (acute phase);

(4) Psycho- and socio therapy in a multimodal treatment setting (especially targeting pain-related

fears; all phases if necessary);

(5) A limited number of sympathetic nerve blocks (in selected cases after successful test blocks;

(6) Therapy of dystonia.”

1 The McGill Pain Index is a scale that shows the rating level of pain, based on McGill Pain Questionnaire

(Melzack, 1975). The last is a three-part pain assessment tool evaluating the sensory intensity, the

emotional impact and the cognitive evaluation of pain.

2

Data from a population-based study estimates that this condition has an overall incidence rate of 26.2

per 100,000 person-years. The probability of being affected is at least three times higher in women than

in men [5].Though, the female preponderance might also be due to women suffering 3 times more radial

fractures [2]. The most affected group were women with ages between 61 and 70 years [5].

1.2. Problem definition

Currently, there is no single test to confirm this pathology. The diagnosis is made based on the patient´s

medical history and through the signs and symptoms presented that are in line with the definition of

CPRS. In the last decade, a new diagnostic criteria, the Budapest Criteria, established by the

International Association for the Study of Pain (IASP) has become accepted. [2]. More information is

presented in the appendix A. Budapest Criteria for CRPS.

However, the large range of symptoms and its similarity with many other diseases makes the diagnosis

highly challenging. [3]. Consequently, the diagnosis is made in most of the cases too late; even years

after the symptoms begin. Evidence suggests that the earlier specific treatment is started, the more

successful it is likely to be; by opposition a delay in the diagnosis and thus in the treatment can result in

severe secondary complications jeopardizing the quality of life for CRPS patients. Thus, improvement

of the diagnosis method is urgently needed.

1.3. Objectives

Discovering effective CRPS biomarkers to improve and quicken the diagnostic process has been under

the focus of the scientific community. Results from past research suggest that the pathophysiological

mechanism is multifactorial, with neuroinflammation, autoimmunity, and nociceptive sensitization among

the mechanisms thought to be involved; and that a single marker for CRPS is not likely to be found.

Thus, multiple biomarkers are needed to enhance the diagnosis. [8,9]. Several glycosylated proteins

are considered to play central roles in autoimmunity, inflammation and disturbed bone turnover

processes, and are thus of interest in relation to CRPS. Osteoprotegerin (OPG) has already been

reported as a biomarker, with elevated levels in the early phase of CRPS being indicative of acute bone

modifications. [8].

Immunoglobulin G (IgG) has been used as a biomarker in multiple diseases. That said, it is important to

clarify the concept of biomarkers. According to Kyle Strimbu and Jorge Tavel (2011), “biomarkers are

merely the most objective, quantifiable medical signs modern laboratory science allows us to measure

reproducibly”. Another definition by the World Health Organization (2001) refers to a biomarker as “any

substance, structure, or process that can be measured in the body or its products and influence or

predict the incidence of outcome or disease”. [11, 12].

3

Regarding CRPS, in a small pilot trial some patients with CRPS were given intravenous immunoglobulin

treatment. The results have demonstrated a significant reduction in pain symptoms when compared with

those given a placebo [11]. Another study, by Valéria Tékus et al. (2014), where a patient’s serum or IgG

fraction was injected into mice also describes the relevance of IgG. The study reports that features

resembling the human disease were shown in mice, supporting both hypothesis “that autoantibodies

may contribute to the pathophysiology of CRPS, and that autoantibody-removing therapies may be

effective treatments for longstanding CRPS”. [12].

In the present work, the aim was to develop an effective method to separate, through Capillary

Electrophoresis (CE), and analyze digested IgG, through mass spectrometry (MS), using Electrospray

Ionization (ESI) and Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF). In

addition, the goal was to develop a protocol for the preparation of a coated capillary.

1.4. Outline

The main body of work is divided into five chapters.

It begins with the current chapter – 1. Introduction – in which the purpose of the research is presented,

as well as the goal of the study and the procedure used to achieve it.

The second chapter – 2. Background – describes the fundamentals of the three techniques used:

Capillary Electrophoresis, Electrospray Ionization Mass Spectrometry, and Matrix Assisted Laser

Desorption/Ionization Time of Flight Mass Spectrometry; along with a brief description of the protein

under study, Immunoglobulin G, and the methodology used when analyzing proteins.

The third chapter – 3. Materials and Methods – concerns the chemicals and instruments needed

throughout the experimental work. Moreover, all the preparation steps required to initialize the

experimental work and all the development of the different technical setups used are approached.

The fourth chapter – 4. Results and Discussion – presents the results obtained from the experiments

and their analysis.

The fifth and last chapter – 5. Conclusions and Future Works – is an evaluation of the obtained results.

Furthermore, it proposes several paths that should be followed as future work, aiming successful

capillary electrophoresis-mass spectrometry hyphenation.

4

5

2. Background

To accomplish a successful analysis of a protein as a potential biomarker, the protein needs to be

separated and fractioned into peptides, particularly if present in complex matrices as the present case,

blood plasma. Characterization is also required to determine the molecular weight and verify the identity

of each peptide. This chapter starts with a description of the instrumentation required to apply CE and

MS as well as the theoretical concepts and relevant variables to consider during the procedure.

Additionally, an overview of the protein IgG and of the proteomics, the global scale analysis of proteins,

is presented.

2.1. Fundamentals of Capillary Electrophoresis

CE is an analytical technique used to separate components in a mixture, based on the migrations of

ionic compounds in the presence of an electric field. One of the most significant advantages of this

technique, when compared with other analytical separation techniques, such as high-performance liquid

chromatography or gas chromatography, is its applicability range [13]. It evolved from being originally

only used for analyzing biological macromolecules to being used nowadays to separate compounds of

different natures, such as amino acids, vitamins, peptides and proteins, and so on. [14]

Its wide versatility derives also from the different modes of operations of CE [14]. Capillary Zone

Electrophoresis (CZE), the mode chosen for the present work, is the simplest one. In CZE, the capillary,

which typically consists of fused silica, is filled with a background electrolyte solution (BGE), used as a

buffer, and the sample is injected at one end of the capillary.

Little instrumentation is needed as shown in Figure 1. The main components are a capillary, whose

extremities are placed in BGE vials, a high voltage supply, electrodes to connect the high voltage supply,

a detector and a computer, with appropriate software. [15]

Figure 1 - Schematic representation of CE system

Source: Sergey N. Krylov Lab http://www.yorku.ca/skrylov/research.html (adapted)

6

The sample is loaded onto the capillary by placing the inlet end of the capillary in the sample vial and

applying either an electric field (electrokinetic injection) or external pressure/vacuum (hydrodynamic

injection). The detector used is commonly an Ultra-Violet (UV) detector, placed along the capillary, which

aims to monitor how long time each species takes to move from the inlet to the outlet of the capillary.

The computer controls the running parameters and gathers the information provided by the detector, the

signal is processed and an electropherogram is generated. The electropherogram presents in the x-axis

the retention time, i.e., the time it takes an analyte to move along the effective length of the capillary,

that is, from the beginning of the capillary until it reaches the detection window. In the y-axis, the

absorbance is displayed.

The phenomenon behind CE is electrophoresis. Its definition consists of the differential movement of

charged species by attraction or repulsion in an electric field. Separation is mainly influenced by the

differences in analyte velocity when responding to the applied electric field. The charge of the ions

defines the direction of its movement: cations move toward the negatively charged cathode, anions

move toward the positively charged anode, and neutral species remain stationary, having an

electrophoretic velocity of zero. The analyte velocity, 𝑣, can be computed by eq. (2.1). [14]

𝑣 = 𝜇𝑒 𝐸 (2.1)

Where the magnitude of the applied electric field, E, is obtained by dividing the applied voltage by the

capillary length (in volts/cm). The electrophoretic mobility, 𝜇𝑒, for a given ion and medium, is a constant

of that ion. It is determined by the electric force that the molecule experiences, balanced by its frictional

drag through the medium and it is defined by eq. (2.2).

𝜇𝑒 =𝑞

6 𝜋 𝜂 𝑟 (2.2)

Where q and 𝑟 stand for the analyte’s charge and radius, respectively and 𝜂 is the BGE viscosity.

Analyzing eq. (2.1) and (2.2), one can conclude that electrophoretic mobility and, therefore,

electrophoretic velocity, decreases with the growth of the particle’s size and increases with the charge

of the analyte. [14]

Besides electrophoretic mobility, there is another phenomenon contributing to the mobility of the analyte,

the electroosmotic flow (EOF). As mentioned earlier, the capillary is typically made of silica with silanol

groups (SiOH) exposed on the inner surface. Although the “pKa of the surface silanol groups is difficult

to determine and mostly not known, in general, EOF becomes significant above pH 4.” (Lauer, Rozing,

2014) [14]. Therefore, the silanol groups deprotonate above that pH, becoming negatively charged

(SiOH→SiO-).

Cations will migrate towards the negatively charged wall to forming a narrower first layer of positive

charges (Stern layer) that neutralizes most of the silanol ions. Then, a second diffuse layer neutralizes

the remaining negative charge creating a potential difference close to the wall, the zeta potential, 𝜁. EOF

describes the movement of the electrolyte’s ions, in the diffused layer, when an electrical field is applied.

7

The magnitude of the EOF can be expressed either in terms of velocity or mobility by eqs. (2.3) and

(2.4), respectively; where 𝜀 is the solution dielectric constant.

𝑣𝐸𝑂𝐹 =𝜀 𝜁 𝐸

𝜂

(2.3)

𝜇𝐸𝑂𝐹 =𝜀 𝜁

𝜂

(2.4)

Summing the contributions of the electrophoretic mobility with the electroosmotic flow, it is possible to

define the apparent mobility. [10,12]

2.1.1. Capillary Wall Modifications

Capillary wall modifications are a method used to minimize analyte adsorption to the inner surface of

the capillary. These may cause fluctuations in the EOF and, consequently, in the migration times in the

CE experiments. Adsorption may also lead to significant band broadening, compromising the separation

efficiency.

The ultimate goal of the coating is to enhance the performance of the separation. Therefore, some

requirements need to be fulfilled. The coating should demonstrate a high “shield” capacity by cutting the

analyte-capillary wall interactions, should not interfere with the analytes and ideally be stable over time,

allowing several runs. Likewise, it should be versatile, i.e., be stable with different buffers’ nature,

concentration and pH. If possible, it should be inexpensive and both preparation and regeneration

should be simple and fast. [17]

During the last two decades, many coating techniques have been proposed. The coating process can

be done either by permanent modification with covalently bonded or physically adhered phases or by

dynamic deactivation using running buffer additives. Both approaches have been somewhat successful,

although no single method is clearly superior. However, there is not a single solution suitable for all

experiments and the procedure should be optimized for each situation.

When CE-ESI-MS is used, a dynamic coating should be excluded since it is likely to compromise the

MS detection. If the chemicals used in the coating entered the mass spectrometer, severe background

noise could occur. Other drawbacks are the suppression of analyte signals and contamination of the ion

source and MS optics [17]. Therefore, a permanent coating was applied in this project, more specifically

with polyvinyl alcohol (PVA).

The protein IgG is the object of study and, when working with proteins as analytes, analyte-capillary wall

interactions are expected to occur. PVA coating is a neutral coating and highly hydrophilic. It reduces

significantly the adsorption by suppressing the EOF, making it appropriate for protein separation [18].

This coating has also previously been determined to be highly stable over a wide range of conditions,

however its performance is enhanced under an acidic setting [19]. Moreover, it tolerates the most

common organic solvents [17].

8

2.2. Fundamentals of Mass Spectrometry

MS is an analytical technique used for a qualitative and quantitative determination. The molecules under

study are desorbed from condensed phases, and ionized when introduced into the ion source, to acquire

positive or negative charges.

In the next step, the ions go through the mass analyzer, accelerated by a strong electric field. The mass

analyzer separates the ionized analytes in line with their mass-to-charge (𝑚/𝑧) ratios. There are several

types of mass analyzers, depending on the separation method. Separation can be achieved based on

the 𝑚/𝑧 resonance frequency, 𝑚/𝑧 trajectory stability and time ions of different masses take to fly from

the ion source to the detector. Examples of each mass analyzer are Orbitrap, quadrupoles and TOF,

respectively. [20]

When the objects of study are proteins, it is common to perform tandem mass spectrometry or MS/MS.

A given ion, selected in the first mass analyzer, the precursor ion, collides and fragments, generating

so-called product ions. New 𝑚/𝑧 are then considered in a second mass analyzer. This can be performed

several times. [21] Figure 2 illustrates the several steps on the MS process.

The signals generated are showed in a computer, which displays the signals graphically as a mass

spectrum presenting their relative abundance according to their 𝑚/𝑧. The y-axis represents the intensity,

which reflects the number of ionized analytes detected and, in the x-axis, the 𝑚/𝑧. If ions are singly

charged, the value of 𝑚/𝑧 represented corresponds to its real molecular mass plus one [M+H]+.

However, if doubly charged, the real mass will be double plus two [M+2H]2+ as the output value, et

cetera. [21]

This technique presents a high versatility and adaptability due to the possibility of choosing between

several types of each component of the instrument. There are a number of different ion sources,

analyzers and detectors.

There are two types of ionization methods, the “hard” ionization, in which the analytes get highly

fragmented and the “soft” ionization in which a low degree of fragmentation occurs. Macromolecules are

usually analyzed by the last, the soft methods. Note that, in this project, the two most common soft

ionization techniques were used: Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix

Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). With the

discovery of these two soft ionization methods, the molecular weight restriction of the analytes was

Figure 2 – Schematic representation of tandem mass spectrometry

9

mitigated. Only with MALDI and ESI, it became possible to analyze large molecules (beyond 1 kDa) with

a very high ionization efficiency, revolutionizing mass spectrometry. [22]

Figure 3 presents a schematic overview of the steps occurring inside each of the mentioned instruments.

2.2.1. Electrospray Ionization

Electrospray ionization is a robust and sensitive technique that provides reliable results. The sample is

injected directly into the system. Usually, diluted in a volatile solvent solution, referred to as sheath liquid,

as an extra liquid flow that increases the total volume under analysis when low quantity of sample is

available. In this way, it is possible to conduct a more stable experiment. The sheath liquid is typically a

mixed organic/ aqueous solvent. The injection is performed with a mechanical syringe pump through a

metal capillary needle, at a low flow rate (typically 1–20 μL/min). [23]

High voltage is then applied to an electrode surrounding the capillary tip, creating a strong electrical

field. Consequently, the solution is nebulized into a charged mist. A drying gas, nitrogen, in this case, is

used not only to improve the nebulization, assisting in the droplet formation but also to direct the spray

from the capillary to the mass spectrometer. Also, this gas evaporates the solvent, decreasing the droplet

size. As the solvent evaporates, the electric field density on the droplets increases, causing similar

charges to repel each other. The point where the droplet can no longer support the surface charge is

known as Rayleigh limit. Thus, the ions detach from the droplets, moving through a heat chamber (100–

300°C) at atmospheric pressure towards the mass analyzer, that is under high vacuum. Complete

desolvation of the ions is achieved. [23, 24]

Several parameters such as drying gas temperature, flow or applied voltage can be altered during the

experiment to optimize the detection.

Figure 3 - Schematic representation of ESI-MS system (a) and of MALDI-MS (b)

Source: Nolan Speicher http://www.idtdna.com (adapted)

10

The electrospray ionization, particularly in large analytes, results in protonated or deprotonated species,

depending on the mode (positive or negative), or complexed species. For example, in positive mode,

this results in forming a range of charged species for each molecule (+2, +3, +4, etc.). As this can make

the mass interpretation rather complex, deconvolution is performed. By deconvoluting the raw data,

multiply-charged species mass is recalculated into the singly-charged mass.

For the deconvolution, the 𝑚/𝑧 and the mass (M) of each analyte needs to be known. Eq. (2.5) is used

to compute the parameters from the 𝑚/𝑧 of the multiply-charged ion. Also, the charge state (Z) must be

calculated for each ion, which is typically accomplished by using the 𝑚/𝑧 values of two adjacent peaks

in eq. (2.6). [25]

(𝑚/𝑧)𝑧 =𝑀 + 𝑍

𝑍⇔ 𝑀 = (𝑚/𝑧)𝑧 ∙ 𝑍 − 𝑍

(2.5)

𝑍 =(𝑚/𝑧)𝑧−1 − 1

(𝑚/𝑧)𝑧−1 − (𝑚/𝑧)𝑧

(2.6)

ESI is easily coupled with other instruments. Having a previous step for separation, as, for example, CE,

simplifies the analysis of the mass spectra, which can ease the interpretation of the results.

2.2.2. Matrix Assisted Laser Desorption/Ionization

MALDI-TOF MS is one of the most used mass spectrometric techniques in biological molecules studies.

This technique is an effective tool for identification of relatively pure protein samples. However, the same

cannot be said when analyzing samples with higher complexity.

In contrast to ESI, when using MALDI, the sample is not inserted directly. It is co-crystalized with an UV-

absorbing crystalline matrix material and spotted onto a metal target plate. There are several types and

fabricants of MALDI plates depending on the final application.

In this project two were used: an AnchorChipTM from Bruker Daltonics (Bremen, Germany) and a

stainless-steel plate treated in-house with Alkyl Ketene Dimer (AKD). The plates are printed with small

spots, where both sample and matrix are deposited. [21, 22]

The plate is then inserted into the instrument, where a UV laser, most commonly a nitrogen UV laser,

irradiates each spot. The matrix absorbs the energy, heating and volatilizing the sample and ionizing it

at the same time [28]. In the scientific community, the exact mechanism of ion formation does not have

a single answer. Yet, the matrix is thought to be involved in the process by providing proton donating/

accepting or electron donating/accepting species upon bombardment with the laser. [28]

The choice of a matrix has a substantial influence on the analytical value of MALDI spectra. The most

used matrices for MALDI are 2,5-dihydroxybenzoic acid (DHB); α-cyano-4-hydroxycinnamic acid

(HCCA); 3,5-dimethoxy-4-hydroxycinnamic acid (SA). The choice is based on the final purpose.

According to the manufacturer Bruker Daltonics, DHB is appropriate to analyze peptides, glycans, and

glycopeptides; HCCA for peptides and smaller proteins and SA for proteins in general. [26]

11

Generally, MALDI imparts a single charge to proteins (with an occasional +2 or +3, as well), which both

simplifies and complicates downstream analysis. Also, the mass calculation is trivial, as 𝑚/𝑧 equals the

molecular mass plus one for z=1.

2.3. Fundamentals of Immunoglobulin G

Immunoglobulins are the major class of serum glycoproteins. Found in the blood and produced by

plasma cells, immunoglobulins consist of a group of proteins built from two heavy and two light chains.

Several papers have been published detailing its functions, and how their glycan pattern can affect it

[30]. The three main purposes of immunoglobulins are to neutralize the pathogen, activate other defense

cells and activate the complement system. [35]

IgG is the most prevalent isotype present in the human body. Human IgG is divided into four subclasses

(IgG 1, 2, 3 and 4) depending on the constant regions of their polypeptide chains. Subclasses 1, 2 and

4 have a mass of roughly 150 kDa; subclass 3 has a mass of around 170 kDa. [36] Bovine IgG only has

two subclasses IgG1 and IgG2 [37]. Figure 4 presents the generic structure of IgG.

As referred previously, each IgG subclass has two light (~25 kDa), and two heavy (~50-60 kDa) identical

peptide chains. In the heavy chains there is a specific asparagine (Asn) site, at position 297, where

glycans, during glycosylation, bind to IgG. There are also other amino acids which allow different types

of glycans to bind. In human IgG, only N-linked glycosylation occurs. That is, a given glycan binds to a

nitrogen atom in an amino acid residue. [29]

The concentration of IgG in healthy human serum ranges from 7–16 g/l [38]. It could be hypothesized

that the value increases in CRPS patients, as occurs with OPG levels. In a study carried out by Krämer

et al., it is reported a significative increase of OPG levels in CRPS patients when compared with not

only healthy individuals but also with patients after uncomplicated fractures [35, 36]. The behavior of

IgG could possibly follow the same tendency.

Figure 4 – Human IgG structure

VH – Variable Heavy Chain CH – Constant Heavy Chain

VL - Variable Light Chain VL - Constant Light Chain

12

2.4. Fundamentals of Proteomics

The term proteomics is a combination of the word protein with genome and it was coined by Marc Wilkins

(1994). He defined as “the study of proteins, how they're modified, when and where they're expressed,

how they're involved in metabolic pathways and how they interact with one another". Proteomics is then

the global-scale study of proteins. [41]. There are two main approaches used in proteomics: the “top-

down” and “bottom-up” approaches.

The “bottom-up” approach has for years been the standard. This approach consists of protein

characterization by analysis of the peptides that result from its digestion. Typically, proteins are digested

chemically or with an enzyme. [35, 36]. Figure 5 illustrates a generic “bottom-up” methodology approach.

By opposition, the “top-down” method is characterized by intact protein analysis, rather than peptides,

preserving all information of the intact protein.

When comparing both approaches, the “bottom-up” reveals a higher sensitivity and versatility. In

addition, as peptides are more easily fractionated, ionized and fragmented, this approach becomes

more likely to be used in protein analysis. Potential downsides include limited protein sequence

coverage by identified peptides. This occurs since some peptides are more easily ionized that others,

outcompeting those in the ionization step. Therefore, some peptides might not be detected. Likewise,

loss of small peptide and of post-translational modifications can occur as well as ambiguity of the origin

for redundant peptide sequences. [35, 36, 37]

As mentioned previously, in a “bottom-up” approach, in the sample preparation step, proteins are

extracted and then cleaved into peptides. Several enzymes are available; however, trypsin has been

the enzyme of choice for large-scale proteomics. Advantages of using this enzyme include high stability

in a vast range of conditions and a very high cleavage specificity. Trypsin cleaves exclusively on the

carboxy-terminal side of arginine and lysine residues, except if either of them is followed by a proline

residue. In this circumstance, the cleavage will not occur. [42]

After protein digestion, peptide separation and analysis, a database search is completed to identify the

peptides. Two common databases are NCBI or Swiss-Prot. Several parameters, including the enzyme

used in the digestion step, missed cleavages, taxonomy or mass weight limit, can be manually set by

the user to refine and narrow down the search results. Special attention should be taken as this may

affect the final output.

Figure 5 – Schematic representation of the mass-spectrometry/proteomic process

13

The database search is performed by peptide mass fingerprinting (PMF). PMF consists in comparing

the theoretical peptide masses present in the database with the peptide mass list obtained

experimentally. The degree of match is quantified according to a given scoring method, depending on

the database. To be considered a solid match a certain score needs to be achieved, meaning that false

positives are not likely to be obtained. [35, 36, 37]

.

14

15

3. Materials and Methods

The current chapter presents the chemicals and instrumentation used throughout this project,

particularly in the separation and mass spectrometry. Moreover, the experimental development of the

capillary coating procedure and CE-MS hyphenation setup is presented.

3.1. Chemicals

Milli-Q water, used as a solvent for all the solutions, was purified in a Millipore Synergy 185

(Massachusetts, USA) to a resistivity of 18.2 MΩ cm at 25 ℃.

Both IgG from human and bovine serum were digested with trypsin enzyme from bovine pancreas, using

ammonium bi-carbonate salt (NH5CO3) as a buffer, all purchased from Sigma Aldrich (Stockholm,

Sweden).

In the capillary preparation step, two types of polyvinyl alcohol ((C2H4O)n) (89-98 and 30-70 kDa) and

methanol (MeOH) were used, both purchased from Sigma Aldrich. Two buffers were used in CE: a

phosphoric buffer was prepared with sodium dihydrogen phosphate (NaH2PO4∙H2O) and the pH was

adjusted with o-phosphoric acid (H3PO4); an ammonium buffer was prepared with ammonium acetate

(C2H7NO2) and the pH was adjusted with acetic acid (CH3COOH), all purchased from Merck except the

last that was purchased from Honeywell Riedel-de Haën (New Jersey, USA). Sodium hydroxide (NaOH)

was used in the capillary equilibration and obtained from Merck.

The sheath liquid in ESI was composed by, among other chemicals already referred to in this chapter,

isopropanol (C3H8O) and diethylamine (C4H11N) obtained from Sigma Aldrich.

Specific for the sample preparation step for MALDI was used acetonitrile (C2H3N), trifluoroacetic acid

(C2HF3O2), purchased from Merck. The matrices DHB and HCCA along with the peptide reference were

purchased from Bruker Daltonics.

3.2. Instrumentation

Throughout the experiment, the following instruments were used: Thermomixer and Centrifugal Vacuum

Concentrator from Eppendorf (Hamburg, Germany), Sonorex Ultrasonic Bath purchased from Bandelin

(Berlin, Germany) and a pH/Ion meter from Metrohm (Herisau, Switzerland).

16

3.2.1. Capillary Electrophoresis

Fused silica capillaries, purchased from Polymicro Technologies (Arizona, USA) were used. The outer

diameter was 375 μm and the inner diameter 50 μm. Several lengths were used with the effective length,

i.e. the length from the inlet until the UV detection window, ranging between 21 and 81.5 cm, depending

on the apparatus. All capillaries were coated permanently with a PVA solution, according to the method

used by Martin Gilges et al. [45], with adaptations described in 3.4.1. Capillary Wall Coating. The oven

used was an HP Agilent G1530A 6890 Plus Gas Chromatograph System (California, USA). The capillary

was then inserted in the Agilent CE capillary cassette.

For the IgG separation, Agilent 7100-series CE system was used, operating in the CZE mode. The

instrument was equipped with a power supply able to reach 30 kV and a real-time UV-Visible diode-

array detector (190–600 nm). Each experiment was conducted with four different wavelengths: 200,

204, 210 and 214 nm, simultaneously. Using this instrument, an electrokinetic injection was applied.

Additionally, a home-built instrument was used. This instrument was essentially composed by four

blocks: a voltage supply, the main CE box, a UV detector and the computer to collect the data. Figure 6

presents the home-built CE instrument, with the four blocks mentioned, from left to right, respectively.

The UV detector model was a 100 UV Detector from Spectra-Physics (Darmstadt, Germany). The CE

box was also connected with an air pressure line, used for hydrodynamic injection of the analyte. Inside

the CE box, there was a “vial holder” and an electrode.

Keeping the humidity of a CE environment low is crucial to suppress current dispersion. As the CE box

was not properly sealed, a flask containing a satisfactory amount of calcium chloride was placed inside

the CE box.

Signals obtained from both CE instruments were recorded with Agilent Chemstation, Hewlett-Packard

(California, USA).

Figure 6 – Setup of the home-built CE instrument (voltage supply, main CE box, UV detector and computer to collect the data, from left to right).

17

3.2.2. Electrospray Ionization

The electrospray mass spectrometry was carried out in the Bruker amaZon Speed, in positive ionization

mode. In all analysis UltraScan mode (Gauss filter of 0.080 𝑚/𝑧) was used. The software TrapControl was

used to acquire the mass spectra and DataAnalysis to process it. In this software, the signal-to-noise

ratio (S/N) of 10 was defined as the cutoff to identify a peak, that is to distinguish a peak from the

background noise. Finally, BioTools with Mascot search was used for the peptide identification. As an

input to the search, it was set that trypsin was used for digestion with up to three possible miscleavage.

A miscleavage occurs when trypsin does not cleave on a site of IgG that was prone to, during protein

digestion. Also, the taxonomy was set to Homo sapiens.

To couple the CE instrument to the ESI, a CE-MS adapter kit from Hewlett-Packard was used. This

sprayer has three orifices, one for the nebulizing gas (nitrogen), one for the CE capillary and other for

the sheath liquid.

The capillary was changed to a CE-MS cassette from Hewlett-Packard.

3.2.3. Matrix Assisted Laser Desorption/Ionization

MALDI-TOF-MS analyzes were conducted on an ultrafleXtreme system from Bruker Daltonics.

Instrument settings and mass spectra acquisition are performed in FlexControl. The laser is a Nd YAG

laser. Mass spectra were acquired in reflector mode for peptide samples, and the laser was always shot

with an intensity of at least 75%. Mass spectra were processed on flexAnalysis, S/N value of 3 for the

peak identification. Peptide identification, as in 3.2.2. Electrospray Ionization, was completed in BioTools

with Mascot search. As an input to the search, it was set that trypsin was used for digestion with up to

one possible miscleavage. Also, the taxonomy was set to Homo sapiens.

Two different plates were used: an AnchorChipTM and a stainless-steel plate treated with AKD. The spot

size was 800 and 600 μm, respectively.

The first target plate contains “anchors”, hydrophilic patches surrounded by a hydrophobic surface. This

concentrates the sample by localizing the droplets in a very small area and preventing the sample from

spreading out. The concentration effect also improves the sensitivity when analyzing dilute samples.

[26].

The second is a new concentrating MALDI target plate. The AKD creates a highly hydrophobic coating

(contact angle slightly over 130°), in a stainless-steel plate. The manufacturing process includes the

application of AKD by an airbrush, and negative contact printing to create the concentration spots [27].

In the CE-MALDI-TOF-MS hyphenation, the MALDI plate was placed on a TIXY 200 XY positioning table

from Newport Spectra-Physics GmbH (Darmstadt, Germany), where the axes X and Y were regulated.

An extended MM53M5EX Motorized MicroMiniTM Stage purchase from National Aperture (Cambridge,

UK) was used for Z-axis. An Arduino DUE (Budapest, Hungary) microcontroller along with a home-built

circuit board was used to interface the axes with a computer, controlling the deposition parameters.

18

3.3. Sample Preparation

IgG digestion was performed using the enzyme trypsin. The enzyme/ protein molar ratio used was either

1:25 or 1:37,5. Ammonium bi-carbonate salt, with a concentration of 0,1 M, was added as a buffer, to

maintain a pH of 8. The solution was left to incubate overnight, for seventeen hours at 37°C. Digestion

quenching was achieved by increasing the sample temperature to 75º C and hold it for 5 minutes. Table

1 presents the different samples used throughout the project. The list includes the IgG nature (bovine

or human), sample’s concentration, if digested or intact and the enzyme/protein ratio.

3.4. Capillary Electrophoresis

Every morning, the capillary was rinsed with Milli-Q water for 20 minutes and then conditioned with the

BGE solution for 30 minutes. A daily fresh BGE was taken from a stock solution and stock solutions

were prepared as needed or monthly. In between experiments using the same buffer, buffer

concentration and pH, the capillary was flushed with the BGE for 20 minutes. In between experiments

with different buffer solutions the capillary was flushed for additionally 10 minutes of the new BGE, i.e.

after altering the buffer the capillary was rinsed for a total of 30 minutes with BGE.

Also, before shutting down the instrument, the capillary was rinsed with the BGE solution for 20 minutes

and with Milli-Q water for 30 minutes, to remove all particles that were possibly inside it. Then, the

capillary was stored with both inlet and outlet inside a Milli-Q vial.

Unless stated differently, all injections were performed electrokinetically, with an injection time of

20 seconds and an applied voltage of 20 kV. CE runs were performed with an applied voltage of 25 kV.

Table 1 – IgG sample list

Number Nature Concentration

(mg/mL) Digested Trypsin:IgG

① Bovine 1,5 1:37,5

② Bovine 1,5 -

③ Bovine 1 1:25

④ Human 1 1:25

19

3.4.1. Capillary Wall Coating

The capillary was prepared based on the procedure described in the reference [45] with the permanent

coating. Several attempts were made in order to achieve a consistent coating method.

One of the characteristics of PVA is its high viscosity, that along with the capillary having such a small

inner diameter, makes the coating process rather challenging. To simplify the injection of the PVA into

the capillary, instead of a regular syringe that was constantly getting clogged, the metallic tool presented

in Figure 7 was used.

This tool has a hole inside where it is possible to insert the 0.5 mL Eppendorf vial with PVA, and two

orifices on top. In one of them, the capillary was inserted ensuring that the inlet was inside the vial and

the other was connected to a nitrogen line. The pressure was then set to 4 bar, forcing the PVA to pass

through the capillary.

Caution must be taken to the outlet, verifying the PVA flow. Whenever there was no flow, one of three

things occurred:

(1) The capillary was clogged. One option to solve the clogging was to rinse with water, if this did

not solve the issue then the extremities of the capillary would be trimmed;

(2) The capillary was not correctly inserted in the vial. It was necessary to open the tool and verify

the position;

(3) Leaking in the system. As this tool was only adapted to serve this purpose, there were several

spots where leaking was occurring. Rubber pieces and plastic film were added to prevent

leakages. Likewise, all nuts should be checked and leaked tight.

Complete dissolution of PVA was rather difficult to achieve, the solution had to be in the ultrasonic bath

for several hours. Two batches were made: one dissolved in cold water and another in boiling water. No

difference was observed. Two capillaries (capillaries ① and ②) were then coated, and both got

clogged, leading to the conclusion that all the small particles of PVA were not dissolved. To solve this

Figure 7 - Tool used for capillary wall coating

(a) front view; (b) top view

(a)

(b)

20

matter, the concentration was reduced to 1% in capillary ③. Clogging did not occur, however, such low

concentration showed not to be sufficient to obtain consistent electropherograms.

The next step was to change the type of PVA to a water-soluble one. With this PVA, the average

molecular mass of the polymer was the same as the one reported in [45]. In a first attempt, capillary ④

with a concentration of 5% was made. However, from the second experiment, no peaks occurred in the

electropherogram. Again, the proteins were most certainly getting attached to the capillary inner wall.

Then, in capillary ⑤ the concentration was increased to 10%, similar to the one reported in [45]. When

this capillary was tested, a step was observed in the electropherogram due to a current drop. No

regeneration of the capillary was successful.

A second capillary was prepared following the same procedure. In the first experiment with capillary ⑥,

some peaks were visible. Later trials showed no peaks. The capillary was then regenerated by rinsing

with water, methanol, and BGE for 30 minutes each. Peaks were visible once more, although the

baseline was gradually increasing along the experiment. To overcome this issue, a solution of 0,1 M of

NaOH was flushed through the capillary for 5 minutes. This procedure was followed throughout the

project, every time needed. Note that, as the capillary is coated with PVA, only low concentrations should

be used not to damage the coating. Figure 8 illustrates the impact of rinsing the capillary with NaOH.

Afterwards, consistent results and stable current were obtained. For this reason, capillary ⑥ was used

in the initial CE experiments. After those, ESI-MS was performed. During that period, both capillary ends

were kept in water. Before coupling both instruments, the capillary was tested and identical

electropherograms were obtained. Therefore, the capillary was adapted to CE-ESI-MS, as described in

the chapter 3.5.1. CE-ESI-MS Hyphenation. After some experiments the capillary was damaged, so a

new capillary had to be prepared to be used in the CE-MALDI-MS apparatus.

Capillaries ⑦ to ⑭ were prepared using the same method as for ⑥. With capillary ⑦, no consistent

results were obtained. Capillaries ⑧ to ⑫ broke in half during the experiments with no clear cause

detected. Capillary ⑬ clogged overnight. Finally, capillary ⑭ showed characteristics suitable to be used

in the CE-MALDI-MS experiments. Relevant capillaries’ parameters can be found in Table 2.

Figure 8 – CE electropherogram illustrating the impact of rinsing the capillary with NaOH (a) before rinsing the capillary with NaOH; (b) after rinsing the capillary with NaOH

21

Table 2 - Capillary parameters, wall coating procedure, symptoms and setup used

Number PVA MW*

(kDa) PVA conc.

(% w/w) Solvent

Total/eff. length (cm)

Symptom Setup

① 89-98 5 Cold water 80/71,5 Capillary clogged. CE

② 89-98 5 Hot water 75/66,5 Capillary clogged. CE

③ 89-98 1 Cold water 75/66,5 Current dropped during the first run. After trimming

capillary ends, current back but results not consistent. CE

④ 30-70 5 Cold water 90/81,5 1st run working well, then no more peaks. CE

⑤ 30-70 10 Cold water 90/81,5 Current dropped during the first run. CE

⑥ 30-70 10 Cold water 88/79,5

88/40

1st run working well, then no more peaks. After rinsing with

the BGE, there were peaks again but with an inclined

baseline. Rinsed with NaOH (0,1 M). Afterwards,

consistent results and stable current. Very low EOF.

Damaged in ESI.

Used from 26.03.2018 to 25.04.201 8.

CE

CE-ESI

⑦ 30-70 10 Cold water 80/21 No consistent electropherograms. CE-MALDI

⑧ - ⑫ 30-70 10 Cold water 80/22 Broken during CE experiment. CE-MALDI

⑬ 30-70 10 Cold water 77,5/21 Capillary clogged overnight. CE

⑭ 30-70 10 Cold water 87,5/24 Consistent results and stable current. Very low EOF.

Used from 25.06.2018 CE-MALDI

*MW represents mass weight.

22

Whenever a new capillary was prepared, after 4 hours in the oven at 140º C, the capillary was flushed

using the following steps:

(1) MeOH for 20 min;

(2) Milli-Q water for 20 min;

(3) BGE for 30 min.

The effectiveness of the coating was tested using mesityl oxide. This component is neutral and therefore,

theoretically, no evident peaks should occur. As there was only one peak after almost one hour, one can

conclude that the PVA wall coating was effective, as the EOF was very low. The electropherogram

obtained is presented in Figure 9.

Throughout the development of a PVA coating capillary method, it was noted that wetting the capillary

with water before injecting the PVA, decreased the probability of clogging.

3.5. Electrospray Ionization

Three different types of sheath liquid were used: 50/50 (v/v) water/methanol with 5 mM ammonium

acetate; 50/50 (v/v) water/isopropanol with 5 mM ammonium acetate and 50/50 (v/v) water/isopropanol

with 5 mM ammonium acetate and 1,5% (v/v) of diethylamine. This last component was added to

decrease the ions charge state [46].

Before all analysis, the helium line, used in the ion trap, was flushed 2 times to guarantee the presence

of helium. Also, the syringe used in the mechanical syringe pump was cleaned by flushing several times

with acetonitrile, Milli-Q water, and isopropanol. Then, the same chemicals were flushed through the

system.

A blank experiment was performed to verify the background noise and potential contaminations. In case

of detection of contaminants, thoroughly cleaning would be executed by extensive flushing.

Blank and sample solutions were always ultra-sonicated for 10 minutes before the injection into the

instrument. IgG was analyzed with a concentration of 15 μg/mL in a solution with a volume of 20 μL.

Figure 9 – CE electropherogram of mesityl oxide 8,5 mg/mL; injection: 20 kV for 20 s; voltage: 25 kV;

buffer: sodium phosphate (pH=3.2; 100 mM)

23

3.5.1. CE-ESI-MS Hyphenation

In order to be used in the CE-ESI-MS hyphenation, the capillary had to be adapted. First, a new window

had to be made leaving the capillary with one window at 8.5 cm (CE) and a second at 45 cm (CE-ESI).

Extra caution had to be taken when handling the capillary and inserting it inside the cassette, as the

windows are especially fragile.

Second, the outlet tip, that was inserted in the CE-MS sprayer adaptor kit, needed to be flattened. The

precision of the spray depends on the quality of this cut. The cut itself, as all previous capillary cuts, was

made with a CE column cutter, and then flattened with a thin sandpaper. The apparatus shown in Figure

10 was used to verify the flatness of the capillary tip.

Finally, the capillary should protrude approximately 0.1 mm out of the sprayer tip. This length was

adjusted with the screw of the sprayer tip and then optimized by checking the actual length in the

computer screen. Then the cassette was carefully placed in the instrument to avoid any potential

deviations in the position.

Figure 11 presents the set-up used for this part of the study. On top it is possible to observe the entire

set, with the computer that controls the CE on the left and the ESI computer on the right. The height of

the capillary inlet was adjusted to be leveled with the MS spray tip outlet to avoid siphoning effects. The

instruments should also be kept as close as possible to minimize the total capillary length needed and

shorten the sample “travel” time along the capillary.

Figure 10 – Apparatus used to verify the cut of the capillary (a) detail on the capillary tip; (b) detail on lenses used; (c) overview of the setup

(a) (b)

(c)

24

3.6. Matrix Assisted Laser Desorption/Ionization

External calibration was performed before every analysis, using peptide calibration samples (Peptide

calibration standard) supplied by the manufacturer Bruker Daltonics. Additionally, internal calibration

was performed using the 𝑚/𝑧 of the autodigestion of the trypsin.

Samples were acidified with trifluoroacetic acid and applied to the MALDI target plate. DHB and HCCA

matrices were prepared according to the recommendations of the manufacturer [26].

The TA30 washing solution was prepared by mixing 30% acetonitrile together with 70% of

0.1% trifluoroacetic acid solution. A fresh solution was prepared for each experimental run. A solution of

DHB powder with a concentration of 20 mg/mL in TA30 was used. The DHB matrix solution (20 mg/mL

2,5-DHB in TA30) was vortexed until it became transparent, indicative of complete dissolution.

A volume of 0.5 µL of the sample solution (1 mg/mL IgG) was deposited onto each MALDI target plate

position. Then, after total solvent evaporation, 0.5 µL of the matrix solution was deposited on top of the

sample. The same procedure was applied to the peptide calibrants. Four replicate spots were used for

each sample and calibrant. The plate was then ready to be inserted in the instrument.

3.6.1. CE-MALDI-TOF-MS Hyphenation

The CE instrument was coupled off-line with the MALDI-TOF-MS instrument by using a positioning table

and a robotized arm (X-Y axes, and Z axis, respectively). The software allowed selecting several lineups

such as, deposition along a single row, deposition along a single column, or even customizable patterns.

The height of the robotic arm when changing from one spot to the next as well as the time of deposition

Figure 11 – Setup used for CE-ESI-MS analysis

25

on each anchor could also be chosen. The control program in Arduino, available from previous works

[27, 47], was adapted to the present work.

Several modifications were completed to improve the experimental setup. Initially, the capillary outlet

along with an external electrode were introduced into a vial, filled with the BGE solution, located on the

positioning table.

By analyzing the sample in CE, before hyphenation, the time that a given analyte takes to move from

the inlet until the detection window is known. Then, the minute in which the analyte should reach the

capillary outlet was computed. This calculation was done taking into account the effective and the total

length of the capillary, assuming that the electrophoretic mobility is constant. The following step was to

insert this information as an input to the Arduino platform that controls the deposition on all axes.

Changing spots would occur as follows: the robotic arm would raise the capillary, move to the following

spot and then lower the position. The capillary height was set to increase 2 mm, to ensure that the

droplet would not be dragged in the process.

The CE software was programmed, in a function named “Timetable” to change the applied voltage to

zero, 30 seconds before the first relevant analyte reached the capillary outlet and then back to the initial

voltage. This voltage interruption was meant to allow the user to manually move the separation capillary

from the vial to the starting spot on the MALDI plate, inserting it and fixing onto the robotic arm. Then,

the Arduino would be set to start the deposition according to the selected program.

As there was no significant EOF, a second capillary was used to create a “sheath liquid effect”,

generating a spray in the separation capillary tip and dragging the sample to the MALDI target plate.

In a first attempt, the second capillary had one of the extremities in a vial filled with BGE and the other

connected to the separation capillary, with a small plastic tube. A pressure of 1.5 bar was applied to

force the BGE through the second capillary. An electrode was also positioned in the vial. During the

separation, the pressure was always being applied. Note that this vial was grounded to the MALDI plate.

26

Figure 12 illustrates the experimental setup ①. The vial with the second capillary can be distinguished

by the red lid.

A second mode of operation was created to address two obstacles of the experimental setup ①. The

first was the fact that part of the liquid was rising along the outside of the capillaries and not being

deposited onto the plate. The second, setup ②, was related to the capillary being too short to reach the

MALDI plate, after breaking during one run. A solution was found in using a plastic piece with four orifices

and with a minor dead volume in the middle. A different capillary was inserted in each one of three

orifices and the remaining was covered.

Other modified was to remove the electrode inserted in the vial used to create the spray. For several

experiments the current was decreasing abruptly after a few minutes, the cause was not understood.

However, when the electrode was removed from the setup, a stable current was achieved.

With this mode of operation, the capillary was not initially in a vial but placed onto a spot in the MALDI

plate. Before starting the separation, a droplet of BGE was deposited in the spot to ensure electrical

contact during the process.

In contrast to the previous scenario in which the pressure was constantly applied, in mode ② the

pressure had to be disabled whenever the droplet size was too large to fit in one spot, to prevent

contamination in other spots.

Figure 12 – CE-MALDI-MS setup ①

(a) capillary used for the separation; (b) capillary used to create the spray effect

(c) vial filled with BGE where the capillary used to create the spray effect is inserted

(c)

27

In Figure 13, the experimental setup ② is presented.

Figure 13 – CE-MALDI-MS setup ②

(a) capillary used for the separation; (b) capillary used to create the spray effect

28

29

4. Results and Discussion

4.1. Capillary Electrophoresis

In this chapter the most relevant results from the experiments that were done are presented as well as

its analysis. It is divided in four sections, starting with the results from the bovine IgG separation using

CE. The second section is concerning human IgG analysis through ESI stand alone and coupled with

CE. Likewise, MALDI results are presented as well as the results from the off-line CE hyphenation

attempts. This chapter ends with the fourth section in which a brief comparison between both mass

spectrometry techniques is presented. Capillary electrophoresis was used to separate the protein IgG

into peptides. Initially, bovine IgG samples were used, given its similarity with human IgG and its lower

cost when compared with human IgG. Before performing the separation, the sample was digested with

trypsin left to incubate overnight.

The first CE investigation was done to understand the behavior of the sample when injected. The first

sample prepared, sample ①, had a concentration of 1.5 g/mL of trypsin digested bovine IgG, prepared

as described in chapter 3.3. Sample Preparation. The electropherograms presented in Figure 14 were

all obtained using sample ①, and conditions, leaving the vial inside the instrument during the four runs

as well as the rinsing step with the BGE between runs. Note that the initial conditions were chosen

based on non-published studies done with the same instrument and type of sample. Electropherograms

were labeled chronologically.

All electropherograms henceforth presented are collected registering a wavelength of 214 nm unless

stated.

Observing the electropherograms it is possible to see that the results are not reproducible, even though

no parameters were altered in between runs. In the first electropherogram, A, around 40 minutes, there

is a large hump suggesting incomplete digestion of the IgG. In more detail, the electropherogram of an

intact protein is characterized by one single broad peak. As for the case of a digested protein, multiple

peaks are expected, each corresponding to a given peptide. To test this hypothesis, intact bovine IgG

(sample ②) was analyzed, resulting in the electropherogram presented in Figure 15. The fact that the

peak of the intact protein occurs around the same time supports the hypothesis of incomplete digestion.

30

The last three electropherograms illustrated in Figure 14, B, C and D, show a different behavior, not

showing the large hump. In opposition, around 40 minutes, a cluster of peaks is observed. The high

number of peaks may indicate that the sample is somehow changing. As the sample is being analyzed

under the same conditions, it was expected to obtain replicates of the first electropherogram. However,

the large hump in A is evolving to several peaks in B, C and D. Possibly the quenching of the digestion

was not successful, though no conclusions on the cause of this cluster of peaks can be drawn.

Nevertheless, excluding the systematic peak at ~1 min and the group of peaks after 40 min, and

zooming in in the remaining, it is possible to observe almost all the same peaks in the four

electropherograms of Figure 14.

Figure 15 – CE electropherogram of intact bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s;

voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)

Figure 14 - CE electropherogram of digested bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s;


31

That is shown in Figure 16, where electropherograms B and D (from Figure 14) are presented as an

example of these similarities. The peaks were labeled to facilitate the comparison between

electropherograms, according to their shape and retention time. The labels are arbitrary and do not

correspond to any identified substances. Groups A and B correspond to peaks that could not be

numbered given their inconsistency in between electropherograms.

The next step was an optimization of the buffer concentration. Figure 17 presents the result obtained

when the concentration of the buffer decreases from 100 mM to 50 mM. On one hand the peak intensity

decreases (Figure 17, top panel), but on the other the resolution of the electropherogram improves as

the peaks are better separated (Figure 17, bottom panel).





32

By observing Figure 17 it is also possible to see that the retention time was shorter than in the previous

case, as the corresponding peaks occurred in a shorter time.

To improve digestion efficiency, the concentration of IgG was decreased to 1 mg/mL (sample ③), while

maintaining the same number of moles of trypsin. As expected most of the peaks that occurred in the

previous sample were also present in sample ③, as seen in Figure 18.

After 35 minutes, the behavior becomes quite different when compared with the previous

electropherograms. Peaks from number 22 to 28 seem to be missing and many more peaks appear

after minute 40, suggesting that the tryptic digestion was more effective.

At this point, based on the results presented in Figure 18, it can be concluded that. a proper method to

separate the digested IgG was established. The method was tested by analyzing three times the sample

under the same conditions and reproducible results were achieved. Then, the goal was to couple the

separation step to mass spectrometry, beginning with ESI. However, transferring a CE-UV method to

CE-ESI-MS, is not a straightforward task and special attention must be given to the method’s

compatibility with MS requirements.

In this project, the main constraint was to find an appropriate BGE that suited both the PVA coating of

the capillary, and that was constituted of volatile components, to avoid any contamination of the MS by

non-volatile salts and high background signals. Due to the last restriction, sodium phosphate could not

be an option.

Both ammonium acetate and formate are generally used in ESI, since they evaporate readily as a

volatile component. In this case, ammonium acetate was chosen as ammonium formate could interfere

with the coating. Several pH values and different buffer concentrations were tested to reach the optimal

conditions. The first attempt was to use the same conditions as in the previous buffer and this is

presented in Figure 19.

Figure 18 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s;


33

The baseline obtained in the previous electropherograms was highly unstable. To improve that, an

attempt with a higher pH (4.6) was carried out. The result can be observed in Figure 20. However,

increasing the pH did not improve the stability of the baseline.

A flatter baseline was only achieved when the concentration was decreased to a quarter of the initial,

i.e., 25 mM. The parameters with the best results were seen at a concentration of 25 mmol/L and pH 4.2,

used in Figure 21.

Despite the optimization attempts, the electropherograms using this buffer were much less resolved

than with sodium phosphate, and presented much fewer peaks. Further optimization could be carried

out, in the future, when performing CE-ESI-MS experiments.


25 kV; buffer: ammonium acetate (pH=4.2; 25 mM)





34

4.2. Electrospray Ionization

Before coupling the instruments, ESI was optimized separately. CE-ESI-MS can be used via a sheath–

liquid or a sheathless interface, but in proteomics studies a sheath–liquid interface is mostly used. As

previously mentioned, three different sheath liquid solutions were tested: 50/50 (v/v) water/methanol,

50/50 (v/v) water/isopropanol and 50/50 (v/v) water/isopropanol and 1.5% (v/v) of diethylamine, all

solutions with 5 mM ammonium acetate. Three mass spectra were obtained and compared in terms of

resolution, peak shape, S/N and charge state, but no relevant differences were found. Thus, 50/50 (v/v)

water/methanol with 5 mM ammonium acetate was chosen for being the simpler sheath liquid of the

three tested.

Figure 22 shows the mass spectrum of human IgG with a concentration of 15 μg/mL.

The data present in the mass spectrum was searched against a protein database, in this case, using

Mascot, but no significant matches were found. Therefore, the experimental peak list was compared

with a theoretical trypsin digested IgG mass list, present in the appendix B. List of Digested Peptides

from IgG. More specifically, the list includes the four types of IgG and a maximum charge state of +4

was assumed. Additionally, the peak list was compared with a contamination list available in the

reference [48], in which most common MS contaminants are specified.

A S/N of 10 was set as the cutoff to identify a peak. Table 3 presents the most relevant peaks

corresponding to the mass spectrum in Figure 22, that is, peaks which 𝑚/𝑧 matched either a

contamination or the theoretical IgG digestion, with a difference of 𝑚/𝑧 0.2 or lower. Additionally, the

table presents the intensity, S/N of each peak as well as mass and charge of the possible matches.


2 L/min; capillary voltage: 4 kV; dry gas flow: 6 L/min; dry gas temperature 300 °C; nebulizer

pressure: 1 bar; mass tuning: 150 to 1500 m/z; target: 800 m/z

35

Table 3 – ESI peak list of digested human IgG 15 𝝁g/mL and comparison with theoretical data

Experimental 𝒎/𝒛

Intensity S/N Contamination IgG type Charge

state

Theoretical mass

(Da)

353.17 376674 55.0 Triton - - 353.27

375.16 239256 34.9 - 1,3,4 +4 375.21/1497.85*

447.25 294566 43.0 - 1,2,3,4 +1 447.25

448.24 352608 51.5 - 1,2,3,4 +1 448.28

449.20 239726 35.0 - 2 +4 449.25/1793.99*

679.35 285981 41.7 PEG - - 679.41

787.64 239792 35.0 - 1

3,4

+2

+4

787.43/1573.86*

787.65/3147.58*

844.82 206832 30.2 - 3,4 +4 844.65/3375.60*

Note that peaks that were not allegedly matched with the theoretical nor with the contamination peak

list, were removed from the table. Also, all the values hereafter signalized with “*” represent the mass of

the singly charged peak, computed by the eq. (2.5).

From the mass spectrum (Figure 22), it is possible to observe six peaks with a molecular mass similar

to the theoretical trypsin digested IgG mass. One peak also presented a mass identical to the polymer

polyethylenglycol (PEG) and other to triton, a substance that is usually present in detergents.

The peaks possibly matching the contaminations (𝑚/𝑧 353.17 and 𝑚/𝑧 679.35) were not further

investigated, as it is not the purpose of the study. In opposition, and given that with only the information

present in Table 4 and in the mass spectrum in Figure 22 it is not possible to conclude if the peaks are

in fact from IgG, an analysis over each peak was carried out. Figure 23 presents a zoomed in view of

the six potentially related to IgG peaks. Unfortunately, it is possible to note a very poor and broad shape.

To verify the charge state of a given ion, the isotopic pattern is analyzed. In proteins, the most

predominant isotope is 13C, that is, carbon with one extra neutron, with a molecular mass of around

1 Da. When a given ion with mass M is singly charged, a second peak, the isotopic peak, should be

present at 𝑚/𝑧 ([M+H]++1)/1.

Following the same principle, if an ion is quadruply charged, the isotope peak should be detectable by

a 𝑚/𝑧 distance of 0.25. Analyzing the first peak (𝑚/𝑧 375.16) in Figure 23 (a), it is not possible to

observe the isotopic pattern, and therefore this peak is not be related to the theoretical one.

The next three peaks on the list are seen in Figure 23 (b). With such a broad peak, the analysis is rather

complex. All three peaks have a distance of roughly 1 𝑚/𝑧, possibly a singly charged isotopic pattern.

If 𝑚/𝑧 448.24 is indeed from the isotope of 𝑚/𝑧 447.25, then the last would be the only peak, of this

two, belonging to the peptide. The same follows to the next pair (𝑚/𝑧 448.24 and 𝑚/𝑧 449.20). If the

second peak is an isotope of the first, then only 448.24 is related to the listed IgG peptide mass. Also,

with such low intensities, the hypothetical isotopic pattern could be result from the noise.

36

However, to verify the isotopes existence, the presence of the peak at a certain 𝑚/𝑧 is not enough. It is

also necessary to verify the area of the allegedly isotopic peaks. The peak area is often interchanged

with the peak intensity, however as all three peaks are overlapping such analysis is not possible. That

said, it cannot be concluded that this ion is from IgG. Nevertheless, as the peak 𝑚/𝑧 447.25 is present

in several runs performed (including in the mass spectrum of Figure 26, even though with a very low

S/N), it is a possible match.

For the peak 𝑚/𝑧 787.64, two different possible matches were found. However, by analyzing the

zoomed in mass spectrum, this value is seen at a too high 𝑚/𝑧 to correspond to the “right” peak. This

peak is then excluded from the possible match list. The same follows with the last peak. In Figure 23 (d)

there are too many peaks overlapping and again, the peak listed is seen at a too high 𝑚/𝑧.

Consequently, this peak was also excluded.

Besides too few peaks matching the theoretically digested IgG, the contaminations detected represent

a concern to the quality of the results. To eliminate the contaminations, the solvent containers as well

as the syringe were cleaned thoroughly.

A second study was performed, using the same IgG stock solution. Figure 24 presents the mass

spectrum of human IgG with a concentration of 15 μg/mL. Table 4 presents the peak list corresponding

to the mass spectrum in Figure 24 along with the possible peak identification.

373.89

375.161+

376.021+

+MS, 7.2-8.7min #263-332

0.0

0.5

1.0

1.5

2.0

5x10

Intens.

371 372 373 374 375 376 377 378 379 380 m/z

447.25

448.391+

449.201+

+MS, 7.2-8.7min #263-332

0

1

2

3

5x10

Intens.

443 444 445 446 447 448 449 450 451 452 m/z

843.711+

844.401+

845.551+

846.251+

846.881+

+MS, 7.2-8.7min #263-332

0.75

1.00

1.25

1.50

1.75

2.00

5x10

Intens.

841 842 843 844 845 846 847 848 849 850 m/z

785.451+

786.751+

787.641+

789.011+

+MS, 7.2-8.7min #263-332

1.0

1.5

2.0

5x10

Intens.

783 784 785 786 787 788 789 790 791 792 m/z

Figure 23 – Zoomed in view of the peaks potentially related to IgG

(a) 𝒎/𝒛 375.16; (b) 𝒎/𝒛 447.25, 448.24, 449.20; (c) 𝒎/𝒛 787.64; (d) 𝒎/𝒛 844.82

(a) (b)

(c) (d)

37

Regarding the contaminations, triton was successfully eliminated, as opposed to PEG, for which the

peak 𝑚/𝑧 678.34 is still present. Figure 25 presents a zoomed in view of the first three peaks listed in

Table 4.


Experimental

𝒎/𝒛 Intensity S/N Contamination IgG type

Charge

State

Theoretical mass

(Da)

429.76 526886 20.3 - 1,2,3,4 3 429.56/1286.67*

625.73 383548 14.8 - 3,4 4 625.80/2500.17*

678.34 1205877 46.5 - 1,2,3,4 1 678.36

679.38 1203604 46.4 PEG - - 679.41


2 L/min; capillary voltage: 4.5 kV; drying gas flow: 6 L/min; drying gas temperature 300 °C; nebulizer

pressure: 0.9 bar; mass tuning: 300 to 1000 m/z; target: 500 m/z

429.762+

430.412+

+MS, 8.9-9.6min #435-479

1

2

3

4

5

5x10

Intens.

427 428 429 430 431 432 433 434 435 m/z

625.73 +MS, 8.9-9.6min #435-479

1.0

1.5

2.0

2.5

3.0

3.5

5x10

Intens.

622 623 624 625 626 627 628 629 630 631 m/z

678.342+

679.022+

679.382+

+MS, 8.9-9.6min #435-479

0.2

0.4

0.6

0.8

1.0

1.2

6x10

Intens.

675 676 677 678 679 680 681 682 683 684 m/z


(a) 𝒎/𝒛 429.76; (b) 𝒎/𝒛 625.73; (c) 𝒎/𝒛 787.64; (d) 𝒎/𝒛 678.34

(a) (b)

(c)

38

Regarding the peak 𝑚/𝑧 429.76, represented in (a), one possible match was found. The theoretical

peak is triply charged, and therefore an isotopic pattern of a triply charged ion should be observed to

confirm this match. In fact, it can be observed in Figure 25 (a), with a smaller peak roughly at 𝑚/𝑧

430.09 and other at 430.41, separated by about 𝑚/𝑧 0.33. Concerning the peak intensity as all three

peaks (𝑚/𝑧 429.76, 430.09 and 430.41) are overlapping such analysis is not possible. Therefore, it

cannot be concluded that this ion is from IgG.

Concerning the next peak, 𝑚/𝑧 625.73 is apparently a good match with IgG. However, zooming in, it is

evident that the S/N is too low to confirm (Figure 25 (b)). Additionally, no possible isotope peaks were

found, that is, at 𝑚/𝑧 [M+H]++0.25. Also, with a high charge state it is also likely to find the same peak

at lower charge stages and that did not occur. Given that, this peak is not likely to be a match.

In the third mass spectrum, Figure 25 (c), 3 main peaks can be observed: 𝑚/𝑧 values of 678.34, 679.02

and 679.38. The one possibility of match with the singly charged theoretically digested IgG would be if

𝑚/𝑧 679.38 was an isotope (roughly 678.34+1) and the peak in between those was related with different

ion. Moreover, the possible isotope has the same intensity as the main peak leading to the conclusion

that this is not likely to be a match.

A third experiment was carried out as an attempt to improve the number of IgG peaks identified, the

peak resolution and to mitigate all contaminations. Sample concentration was increased from 15 𝝁g/mL

to 100 𝝁g/mL. Figure 26 presents the mass spectrum of digested human IgG and Table 5 presents the

most relevant peaks along with the comparison with the possible matches.


Experimental

𝒎/𝒛 Intensity S/N IgG type Charge state

Theoretical mass

(Da)

340.20 283824 14.3 1,2,3,4 2 339.68/678.36*

447.10 91935 4.6 1,2,3,4 1 447.26

678.95 797889 40.3 1,2,3,4 1 678.36

Figure 26 – ESI-MS spectrum of digested human IgG 100 𝝁g/mL; scan mode: UltraScan; flow

rate: 2 L/min; capillary voltage: 4 kV; drying gas flow: 4 L/min; drying gas temperature 300 °C;

nebulizer pressure: 0.9 bar; mass tuning: 200 to 1500 m/z; target: 800 m/z

39

In similarity with the previous cases, the zoomed in peaks are presented in Figure 27.

In this mass spectrum, two peaks possibly from the same ion were detected: the singly charged

𝑚/𝑧 678.95 and the doubly charged 𝑚/𝑧 340.20. The mass difference of the singly charged ion is rather

high when compared to the theoretical peak (0.69 Da). Nevertheless, the existence of two peaks with

corresponding masses of different charge states is a strong indication for this match. Although it is not

possible to confirm, it is likely to be a positive match.

The peak 𝑚/𝑧 447.10 was previously analyzed, as it is also present in the mass spectrum of Figure 22.

In order to improve the mass spectra resolution, and once no contaminations were detected, the

following step was the hyphenation of this technique with the capillary electrophoresis.

4.2.1. CE-ESI-MS Hyphenation

After coupling the two instruments, several contaminations were visible in the mass spectra. The largest

concern was the PVA contamination. As a permanent coating, PVA was not expected to reach the mass

spectrometer, since it meant that the coating was being dragged from the capillary. Besides the possible

interferences in the MS, the proteins also seemed to be absorbed to the capillary inner walls.

In addition, the hyphenation itself was not successful given that when a given analyte was separated it

did not reach the mass spectrometer detector. In more detail, knowing the retention time in which a

given peak is visible in the UV-detector, the capillary length from the inlet to the UV-detector and from

447.10 +MS, 9.8-15.5min #699-1096

2

4

6

8

4x10

Intens.

443 444 445 446 447 448 449 450 451 452 m/z

678.95+MS, 9.8-15.5min #699-1096

0

2

4

6

85x10

Intens.

675 676 677 678 679 680 681 682 683 m/z

340.201+

340.63341.07

1+

+MS, 9.8-15.5min #699-1096

0.0

0.5

1.0

1.5

2.0

2.5

5x10

Intens.

336 337 338 339 340 341 342 343 344 345 m/z


(a) 𝒎/𝒛 340.20; (b) 𝒎/𝒛 447.10; (c) 𝒎/𝒛 787.64; (d) 𝒎/𝒛 678.95

(a) (b)

(c)

40

there to the outlet, and assuming that the electrophoretic mobility is constant, it is possible to determine

when it should be visible in the mass spectrometer detector.

The two reasons listed above: the capillary coating being damage, requiring to be replaced and the

hyphenation not being fully accomplished led to delays on this project. As this study was time-limited,

the decision was taken to try a different mass spectrometer, the MALDI-TOF. Therefore, as future work

further studies using the CE-ESI-MS hyphenation are suggested.

4.3. Matrix Assisted Laser Desorption/Ionization

Following the same optimization steps used in the ESI experiments, before testing the hyphenation, the

IgG samples were analyzed in MALDI to determine the 𝑚/𝑧 of each peptide, with no preceding

separation. The method was calibrated before each analysis with a peptide calibration standard.

Additionally, internal calibration was performed using the 𝑚/𝑧 ratio of peaks from the autodigestion of

the trypsin.

Figure 28 presents the MALDI-MS mass spectrum of trypsin digested IgG. A 50/50 sample/DHB matrix

mixture was deposited in each spot, on an AnchorChipTM MALDI plate.

The resultant data was then searched against a protein database to identify the peptide sequences and

further infer the protein content of the sample. As mentioned previously, there are four types of human

IgG. From these, only IgG 1 had a score over 56, the value required to positively identify a given protein.

Nevertheless, IgG 2, 3 and 4 also gave high scores, as observed in Figure 29.

Figure 28 - MALDI-TOF-MS spectrum of digested human IgG; matrix: DHB; shot at 90% laser

intensity (1000 shots per burst, 6000 shots total), pulsed ion extraction by reflectron

41

Sequence coverage was 39.4% for IgG 1, 27.3% for IgG 2, 25.7% for IgG3 and finally 26.6% for IgG 4.

More detailed information can be found at appendix C. Biotools Analysis. Table 6 presents the peak list

from the MS spectrum as well as the theoretical masses. Besides IgG, masses corresponding to bovine

trypsin and human keratin were also found.

Table 6 - MALDI peak list of digested human IgG and comparison with theoretical data

Experimental

m/z

Theoretical mass

(Da)

IgG

type Other

Experimental

m/z

Theoretical mass

(Da)

IgG

type Other

824.47 824.49 2 - 1677.84 1677.80 1 -

830.45 830.46 4 - 1690.86 1690.90 1 -

835.42 835.43 1,2,3,4 - 1794.02 1793.99 2 -

838.49 838.50 1,3 - 1808.03 1808.01 1,3,4 -

906.50 906.50 - Trypsin 1896.04 - - -

1020.51 1020.50 - Trypsin 1904.96 1904.94 2,3 -

1104.61 1104.61 1,2,4 - 1905.98 1905.89 2 -

1111.57 1 111.56 - Trypsin 1920.96 1920.94 2,3 -

1153.58 1153.57 - Trypsin 2082.03 2082.01 1 -

1172.61 1173.52 3,4 - 2163.07 2163.06 - Trypsin

1186.64 1186.65 1 - 2214.17 2214.19 2 -

1230.63 1230.63 2,3,4 - 2229.18 2228.21 1,3,4 -

1264.66 1264.66 1,3 - 2273.15 2273.16 - Trypsin

1364.66 - - 2545.05 2544.13 1,4 -

1365.68 1365.64 - Keratin 2550.14 2550.23 - Trypsin

1433.74 1433.72 - Trypsin 3773.79 - - -

Figure 29 - Results obtained from Biotools database search for MS analysis (database: Swiss-Prot)

42

After the determination of the 𝑚/𝑧 ratio of each peptide and analyzing the ones of interest, further

fragmentation was performed. The results from MS/MS mass spectrometry are presented in Figure 30.

Note that all MS/MS spectra were analyzed simultaneously in Mascot software. A score of 100 was

achieved for IgG 1. However, only 4 of the 11 peaks initially identified in the MS spectrum were positive

to IgG 1 in this database search. This could be due to the lower mass accuracy of tandem MS when

compared to MS or it could also mean that the remaining peaks were, in fact, false positives.

Figure 31 illustrates the information obtain from the Biotools database search for a given percursor ion,

in this case for 1186.65 Da.

Figure 30 - Results obtained from Biotools database search for tandem MS analysis (database: Swiss-Prot)

Figure 31 – Results obtained from Biotools database search for tandem MS analysis of peak

1186.65 Da (database: Swiss-Prot)

43

CE-MALDI-TOF-MS Hyphenation

Originally, the idea was to perform the hyphenation using the home-built CE instrument. As it is home-

made, it can be adapted so that it accomplishes the requirements. In this specific case, this instrument

had the advantage of allowing the user to apply voltage and pressure simultaneously, something that is

not possible using the commercial CE instrument. That said, the separation could occur without

interruptions and the pressure could guarantee a constant flow from the capillary inlet until the MALDI

plate.

After connecting all the parts of the home-built instrument (voltage supplier, main CE box and UV

detector) and inserting a test capillary, a very unstable current was detected. The connection with the

electrodes could be a potential issue, however after verifying it no improvements were obtained. Then,

all the parts were exchanged, one at a time, and the current was still not stable. It was concluded that,

as the CE box was not properly sealed together with the high humidity present is the air, it was not

possible to proceed used this instrument. Thus, hereafter, the commercial CE instrument was utilized.

As the AnchorChipTM plate could be damaged by the voltage applied during the off-line coupling, an AKD

plate was used. As described in chapter 3. Materials and Methods, multiple adjustments to the setup

were needed in order to maintain a stable current throughout the system. Another phenomenon that

occurred was that the CE separation worsen when moving the capillary outlet from inside the instrument,

as in stand alone CE, to the MALDI plate. Figure 32 presents the electropherogram of trypsin digested

human IgG, sample ④, obtained before coupling the instruments. This experiment was performed so

that the following electropherograms could be compared to this one and changes when coupling the

instruments could be detected. Also, as the CE software requires to be programmed prior to the run, it

was needed to know the retention time of the most relevant peaks.

The following step was to proceed with the off-line hyphenation of CE and MALDI-MS. Figure 33

illustrates the result of the first attempt to couple the instruments, the electropherogram obtained (top

panel) along with the respective current (bottom panel). The current was interrupted so that the user

could manually change the position of the capillary for the outlet vial attached to the outside of the CE

instrument to the robot arm. Two minutes were needed to change the position of the separation capillary.

Figure 32 - CE electropherogram of digested human IgG 1 mg/mL; injection: 20 kV

for 20 s; voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)

44

As explained previously, knowing the capillary length from the inlet to the UV-detector and from there to

the outlet, and assuming that the electrophoretic mobility is constant, it is possible to determine when

the analytes reach the MALDI plate. In the experiment of Figure 32, the capillary ⑭ was used (total

length: 87,5 cm; effective length: 24 cm).

At this moment, the goal was to detect the cluster of peaks with a retention time starting at 5 minutes.

Therefore, the deposition should be ready after around 14 minutes of separation. The voltage was

dropped to zero, the capillary was inserted in the robotic arm and a command was given in Arduino to

start the deposition. Note that experimental setup ① was used.

It is possible to observe that straight after turning the voltage back, the current suffered fluctuations,

then it got stable again and later dropped to zero in a step. No alteration was performed in the system

justificatory of this abrupt current drop. Resultant from this drop, the capillary broke close to the outlet.

The capillary ⑭ became too short to use mode ①, so the experiment setup was adapted to mode ②

of operation. Then a plastic piece with four orifices piece was used: one of them was covered and in the

others, capillaries were inserted. The separation capillary on the left, its remaining (the part that broke)

on the bottom and the capillary used to create a spray effect on the right. In this mode, in the beginning

of the experiment, the capillary is placed the MALDI plate.

Figure 34 illustrates the electropherogram obtained using experimental setup ② (top panel) along with

the respective current (bottom panel). Even with a stable current (first 7 minutes) and before starting the

deposition, that is, the capillaries were still on one MALDI plate spot; there were no peaks observed. It

was not expected that, only by changing the initial position of the separation capillary from the outlet vial

to the positioning table, the separation would be jeopardized. After 7 minutes of experiment, the current

suffers several fluctuations and latter ramped down to zero.

Figure 33 - CE electropherogram of digested human IgG 1 mg/mL from CE-MALDI

experimental setup ①; injection: 20 kV for 20 s; voltage: 25 kV; buffer: sodium

phosphate (pH=3.2; 100 mM)

45

Several more experiments were performed. One of three possible outcomes occurred: the capillary

broke during the run, no peaks were detected or too unstable current. That said, further setup

optimization is required in future works.

Even though a reproducible setup was not achieved, it could still be possible that the IgG sample was

reaching the MALDI plate. However, after analyzing the MALDI plate spots, it can be concluded that

either no IgG was present in the spots or the concentration was too low to be detected.

By analyzing digested IgG with MALDI-TOF-MS with no previous separation, as is the initial section of

the present chapter, but using the AKD target plate much lower intensities were obtained when

compared to the AnchorChipTM plate. Both DHB and HCCA matrices were used and similar poor results

were achieved. Later, it was verified that there were some difficulties encountered while the preparation

of AKD batch of that could also contribute to outcome of this investigation.

4.4. Comparison between ESI-MS and MALDI-TOF-MS

Overall, ESI and MALDI greatly improved biological mass spectrometry and particularly proteomics for

being soft ionization methods that can transform large biomolecules into ions. However, there are

numerous differences between the techniques.

MALDI results mainly in singly charged ions. In this project, this proved to be a large advantage as it

decreases the mass spectrum complexity when analyzing peptides. By opposition, ESI results in multiply

charged ions.

In MALDI much higher 𝑚/𝑧 were detected Though, this could be due to the higher charged that occurs

in ESI.

Figure 34 - CE electropherogram of digested human IgG 1 mg/mL from CE-MALDI

experimental setup ②; injection: 20 kV for 20 s; voltage: 25 kV; buffer: sodium

phosphate (pH=3.2; 100 mM)

[Atraia a atenção do seu leitor colocando uma boa citação no documento ou utilize

46

The steps before the analysis itself also vary. In ESI the molecules are ionized directly from the liquid

phase, the only preparation required is to mix the sample with the sheath liquid. As for MALDI, the

preparation step is much more time consuming. Waiting until the sample deposited in each spot is

completely dried to create the second layer of matrix, can take up to 30 minutes.

The hyphenation setup was clearly much simpler with ESI. The only thing to adapt was the CE cassette

and approximate the instrument as much as possible no decrease the required total capillary length.

However, introducing the capillary in the correct position of the ESI sprayer tip is not a straight forward

task. As for the off-line CE-MALDI-MS hyphenation, a setup had to be developed. In both cases, the

hyphenation was not successful. When coupling the separation with the first mass spectrometry

technique, it was noticed capillary coating was damaged, and therefore no further investigation was

possible. The ESI parameters need to be optimized for each analyte/setup. One possible justification

on why peaks were not being detected might be that the parameters were well not adjusted.

In opposition, in the hyphenation between CE and MALDI, the main issue was not in the second

instrument. In fact, no complete hyphenation was achieved. The setup was not fully developed and

therefore no conclusions can be taken from the hyphenation itself.

It is also important to refer that using ESI, it was not possible to identify the sample by performing a

database search. On the contrary, with MALDI IgG was not only identified, but the four types were

detected with very high scores. Hence, for this study, MALDI proved to be more effective.

47

5. Conclusions and Future Works

This final chapter introduces a summary of the main conclusions of the present project, as well as a

review of possible future investigations.

Before starting the analysis of IgG samples, an effective procedure to coat the capillary with PVA was

developed. The use of a specific tool that allowed a pressure injection was determinant in the procedure.

Several PVA concentrations were tested as well as methods to dissolve the PVA in water. The most

successful results were achieved using water soluble PVA (with a molecular mass of 30-70 kDa),

dissolved in cold water to achieve a concentration of 10% w/w. Throughout the development of a PVA

coating capillary method, it was noted that wetting the capillary with water before injecting the PVA,

decreased the probability of clogging.

In the first phase of the experimental work, a suitable method was developed for the separation of bovine

IgG through capillary electrophoresis. The best buffer proved to be sodium phosphate (pH=3.2;

concentration=100 mM).

A later step was to couple CE with MS. The first technique used was ESI. To do so, the buffer used in

the separation could not be a phosphoric buffer, as salts interfere with ESI. Therefore, the CE method

was optimized for the use of a buffer solution of ammonium acetate. The best results were obtained when

using a pH of 4.2 with a concentration of 25 mM, yet worse resolution and fewer peaks than with the

phosphoric buffer. To that extend, further optimization should be carried out to improve the outcome

when using this buffer.

Before coupling the instruments, tests with ESI stand alone were performed. Human IgG was analyzed,

yet no matches were detected when conducting a database search. Therefore, the experimental peak

list was compared with the masses of theoretical trypsin digested IgG and with the most common

contaminations in MS. Some peaks could possibly be from IgG, but no confirmation was possible given

the poor shape and resolution of the peaks. Additionally, triton and PEG contaminations were detected

and effectively suppressed.

The capillary was adapted to CE-ESI-MS and only then, hyphenation was put in place. In none of the

experiments done with this setup it was possible to detect peaks in ESI when expected based on the

retention times of CE and the capillary length (effective and total). Therefore, it is possible to conclude

that the hyphenation was not fully accomplished. Moreover, it was possible to observe peaks from

capillary coating in the mass spectrometer detector. As the capillary wall coating was damaged, the

investigation was not concluded. That said, future investigation could be focused on the optimization of

the ESI parameters, particularly when this instrument is coupled to the CE.

48

The decision was then made to try to another mass spectrometry technique: MALDI-TOF-MS. When

using this technique, excellent results were obtained. When performing a database search with the

results obtained, all four types of human IgG were a hit, with emphasis on IgG 1. Tandem MS was

carried out on the relevant peaks and again, when doing a database search the maximum score of

100% to IgG 1 was obtained.

Off-line coupling of CE and MALDI-TOF was not fully succeed in this project. Several capillaries broke

during the runs, but it was not possible to confirm the cause. Instabilities in the current could be related

with small fractures in the capillaries, however looking through a magnifying glass, fractures were not

detected. Due to these reasons it was not possible to conclude the runs. Therefore, a future work could

be to complement this study on the optimization of this setup with a detailed analysis on the feasibility

of using two different capillaries for the deposition onto the MALDI plate when there is no EOF. That is,

using one capillary for the separation itself and a second to obtain a spray effect and allow the analytes

deposition on the MALDI plate. In this project, the spray effect was accomplished using the BGE. Other

options should be investigated.

With the results from this project, it is possible to confirm that MALDI-TOF-MS is a powerful tool to

analyze peptides. However, one cannot conclude that ESI is not capable only because, in this specific

case, it could not identify the IgG peptides. It can simply be emphasized the complexity of implementing

a suitable and reliable experimental setup.

As CRPS is reportedly caused by mutations in the glycosylation of the IgG, when an effective

experimental setup is accomplished, glycosylation of IgG should be addressed. The final goal is to

investigate the glycosylation of IgG in blood samples from CRPS patients.

49

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53

A. Budapest Criteria for CRPS

Budapest clinical diagnostic criteria for CRPS [49]

(1) Continuing pain, which is disproportionate to any inciting event

(2) Must report at least one symptom in three of the four following categories:

• Sensory: reports of hyperesthesia and/or allodynia

• Vasomotor: reports of temperature asymmetry and/or skin color changes and/or skin color

asymmetry

• Sudomotor/edema: reports of edema and/or sweating changes and/or sweating asymmetry

• Motor/trophic: reports of decreased range of motion and/or motor dysfunction (weakness, tremor,

dystonia) and/or trophic changes (hair, nail, skin)

(3) Must display at least one sign at time of evaluation in two or more of the following categories:

• Sensory: evidence of hyperalgesia (to pinprick) and/or allodynia (to light touch and/or deep

somatic pressure and/or joint movement)

• Vasomotor: evidence of temperature asymmetry and/or skin color changes and/or asymmetry

• Sudomotor/edema: evidence of edema and/or sweating changes and/or sweating asymmetry

• Motor/trophic: evidence of decreased range of motion and/or motor dysfunction (weakness,

tremor, dystonia) and/or trophic changes (hair, nail, skin)

(4) There is no other diagnosis that better explains the signs and symptoms

54

B. List of Digested Peptides from IgG

The tables presented in this chapter include the list of trypsin digested peptides from human IgG, types 1 to 4, obtained through reference [50]. A maximum of

three miss-cleavages (MC) was allowed. In column MC the number of miss-cleavages is shown (from 0 to 3).

Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 - 330

Mass Position MC Peptide Sequence

9456.7722 1-93 3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTK

9411.7507 5-96 3 GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDK

9069.5604 5-93 2 GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTK

8372.2169 17-97 3 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKK

8244.1219 17-96 2 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDK

7901.9316 17-93 1 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTK

7579.8369 31-101 3 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK

7268.6031 106-171 3 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

7126.5782 31-97 2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

6998.4832 31-96 1 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK

6656.2929 31-93 0 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK

6070.9218 239-292 3 DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

6042.9821 102-157 3 SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

6040.9476 244-297 3 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

5609.8190 106-157 2 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

5484.6256 244-292 2 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

5483.6739 228-275 3 EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

5398.6048 276-322 3 TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

5393.5807 139-184 3 TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR

5198.4905 254-299 3 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

5119.5812 158-200 3 FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK

5039.5029 132-175 3 DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

https://www.uniprot.org/blast/?about=P01857%5b1-330%5d

55

Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 – 330 (continued)


4955.3573 254-297 2 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

4557.2064 132-171 2 DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

4433.2527 98-138 3 VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR

4399.0353 254-292 1 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

4313.1342 293-330 3 LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

4223.0865 139-175 2 TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

4216.0179 239-275 2 DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

3979.9940 102-138 2 SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR

3880.9980 172-203 3 TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

3756.8122 298-330 2 SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3744.9313 97-131 3 KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

3740.7900 139-171 1 TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

3629.8056 176-205 3 EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK

3629.7216 244-275 1 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

3616.8364 98-131 2 VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

3546.8309 106-138 1 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR

3543.7008 293-322 2 LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

3513.6790 300-330 1 WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3460.7971 172-200 2 TKPREEQYNSTYRVVSVLTVLHQDWLNGK

3398.7015 176-203 2 EEQYNSTYRVVSVLTVLHQDWLNGKEYK

3396.7943 224-253 3 GQPREPQVYTLPPSRDELTKNQVSLTCLVK

3330.5926 158-184 2 FNWYVDGVEVHNAKTKPREEQYNSTYR

3163.5777 102-131 1 SCDKTHTCPPCPAPELLGGPSVFLFPPKPK

2987.3788 298-322 1 SRWQQGNVFSCSVMHEALHNHYTQK

2978.5006 176-200 1 EEQYNSTYRVVSVLTVLHQDWLNGK

2958.5604 228-253 2 EPQVYTLPPSRDELTKNQVSLTCLVK

2898.4223 132-157 1 DTLMISRTPEVTCVVVDVSHEDPEVK

2887.5498 185-209 3 VVSVLTVLHQDWLNGKEYKCKVSNK

2819.4971 1-30 2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVK

2744.2456 300-322 0 WQQGNVFSCSVMHEALHNHYTQK

2730.4145 106-131 0 THTCPPCPAPELLGGPSVFLFPPKPK

2673.3770 276-299 2 TTPPVLDSDGSFFLYSKLTVDKSR

2544.1313 254-275 0 GFYPSDIAVEWESNGQPENNYK

2510.3361 222-243 3 AKGQPREPQVYTLPPSRDELTK

2459.3115 185-205 2 VVSVLTVLHQDWLNGKEYKCK

56

Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 – 330 (continued)

Peptide Sequence

Position MC Peptide Sequence

2432.2853 5-30 1 GPSVFPLAPSSKSTSGGTAALGCLVK

2430.2438 276-297 1 TTPPVLDSDGSFFLYSKLTVDK

2353.2986 218-238 3 TISKAKGQPREPQVYTLPPSR

2311.2040 224-243 2 GQPREPQVYTLPPSRDELTK

2228.2073 185-203 1 VVSVLTVLHQDWLNGKEYK

2160.0984 158-175 1 FNWYVDGVEVHNAKTKPR

2082.0059 139-157 0 TPEVTCVVVDVSHEDPEVK

1927.1044 204-221 3 CKVSNKALPAPIEKTISK

1924.0399 222-238 2 AKGQPREPQVYTLPPSR

1918.0466 201-217 3 EYKCKVSNKALPAPIEK

1905.1279 210-227 3 ALPAPIEKTISKAKGQPR

1895.1324 206-223 3 VSNKALPAPIEKTISKAK

1873.9218 276-292 0 TTPPVLDSDGSFFLYSK

1872.9701 228-243 1 EPQVYTLPPSRDELTK

1808.0064 185-200 0 VVSVLTVLHQDWLNGK

1724.9078 224-238 1 GQPREPQVYTLPPSR

1696.0003 206-221 2 VSNKALPAPIEKTISK

1690.9044 239-253 1 DELTKNQVSLTCLVK

1677.8019 158-171 0 FNWYVDGVEVHNAK

1671.8085 172-184 1 TKPREEQYNSTYR

1573.8584 1-16 1 ASTKGPSVFPLAPSSK

1497.8457 204-217 2 CKVSNKALPAPIEK

1466.8940 210-223 2 ALPAPIEKTISKAK

1375.7249 94-105 3 VDKKVEPKSCDK

1286.6739 228-238 0 EPQVYTLPPSR

1267.7620 210-221 1 ALPAPIEKTISK

1266.7416 206-217 1 VSNKALPAPIEK

1264.6565 17-30 0 STSGGTAALGCLVK

1189.5120 176-184 0 EEQYNSTYR

1186.6466 5-16 0 GPSVFPLAPSSK

1104.6081 244-253 0 NQVSLTCLVK

1098.5612 201-209 2 EYKCKVSNK

1085.6425 218-227 2 TISKAKGQPR

1033.5346 97-105 2 KVEPKSCDK

57

Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 – 330 (continued

Position MC Position Peptide Sequence

942.5618 94-101 2 VDKKVEPK

905.4397 98-105 1 VEPKSCDK

838.5032 210-217 0 ALPAPIEK

835.4342 132-138 0 DTLMISR

818.4730 293-299 1 LTVDKSR

788.4512 323-330 0 SLSLSPGK

678.3603 204-209 1 CKVSNK

670.3229 201-205 1 EYKCK

656.3838 222-227 1 AKGQPR

647.4086 218-223 1 TISKAK

605.3141 239-243 0 DELTK

600.3715 97-101 1 KVEPK

575.3399 293-297 0 LTVDK

501.3143 172-175 0 TKPR

489.3031 94-97 1 VDKK

472.2765 98-101 0 VEPK

457.2517 224-227 0 GQPR

452.1809 102-105 0 SCDK

448.2766 218-221 0 TISK

447.2562 206-209 0 VSNK

439.2187 201-203 0 EYK

406.2296 1-4 0 ASTK

361.2081 94-96 0 VDK

262.1510 298-299 0 SR

250.1220 204-205 0 CK

218.1499 222-223 0 AK

147.1128 97-97 0 K

58

Table 8 - Chain Immunoglobulin heavy constant gamma 2 at positions <1-326


9652.7301 1-93 3 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSNFGTQTYTCNVDHKPSNTK

9607.7086 5-96 3 GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSN FGTQTYTCNVDHKPSNTKVDK

9265.5183 5-93 2 GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSN FGTQTYTCNVDHKPSNTK

8881.3563 17-100 3 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDH KPSNTKVDKTVER

8396.0965 17-96 2 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDH KPSNTKVDK

8053.9062 17-93 1 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDH KPSNTK

7700.7644 102-171 3 CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR

7661.7808 31-101 3 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK

7533.6859 31-100 2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER

7346.5629 101-167 3 KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK

7218.4679 102-167 2 CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK

7136.5274 135-196 3 TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK

7048.4261 31-96 1 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK

6706.2358 31-93 0 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK

6177.9708 128-180 3 DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR

6150.8609 235-288 3 EEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSK

6088.9146 240-293 3 NQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK

5532.5926 240-288 2 NQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSK

5531.6409 224-271 3 EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYK

5430.5768 272-318 3 TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

5361.5545 135-180 2 TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR

5246.4575 250-295 3 GFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR

5039.4665 128-171 2 DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR

5003.3243 250-293 2 GFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK

4557.1700 128-167 1 DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK

4447.0023 250-288 1 GFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSK

4313.1342 289-326 3 LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

4263.9849 235-271 2 EEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYK

4223.0501 135-171 1 TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR


59

Table 8 - Chain Immunoglobulin heavy constant gamma 2 at positions <1-326 (continued)


4110.0869 97-134 3 TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR

3834.9925 168-199 3 TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK

3756.8122 294-326 2 SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3740.7536 135-167 0 TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK

3645.7166 240-271 1 NQVSLTCLVKGFYPSDISVEWESNGQPENNYK

3635.8608 94-127 3 VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPK

3624.8271 101-134 2 KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR

3583.8002 172-201 3 EEQFNSTFRVVSVLTVVHQDWLNGKEYKCK

3543.7008 289-318 2 LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

3513.6790 296-326 1 WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3496.7321 102-134 1 CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR

3428.7664 220-249 3 GQPREPQVYTLPPSREEMTKNQVSLTCLVK

3414.7916 168-196 2 TKPREEQFNSTFRVVSVLTVVHQDWLNGK

3352.6960 172-199 2 EEQFNSTFRVVSVLTVVHQDWLNGKEYK

3293.6705 97-127 2 TVERKCCVECPPCPAPPVAGPSVFLFPPKPK

2990.5325 224-249 2 EPQVYTLPPSREEMTKNQVSLTCLVK

2987.3788 294-318 1 SRWQQGNVFSCSVMHEALHNHYTQK

2965.5121 1-30 2 ASTKGPSVFPLAPCSRSTSESTAALGCLVK

2932.4951 172-196 1 EEQFNSTFRVVSVLTVVHQDWLNGK

2873.5342 181-205 3 VVSVLTVVHQDWLNGKEYKCKVSNK

2808.4107 101-127 1 KCCVECPPCPAPPVAGPSVFLFPPKPK

2744.2456 296-318 0 WQQGNVFSCSVMHEALHNHYTQK

2705.3490 272-295 2 TTPPMLDSDGSFFLYSKLTVDKSR

2680.3158 102-127 0 CCVECPPCPAPPVAGPSVFLFPPKPK

2578.3003 5-30 1 GPSVFPLAPCSRSTSESTAALGCLVK

2572.3188 218-239 3 TKGQPREPQVYTLPPSREEMTK

2560.1262 250-271 0 GFYPSDISVEWESNGQPENNYK

2462.2159 272-293 1 TTPPMLDSDGSFFLYSKLTVDK

2445.2958 181-201 2 VVSVLTVVHQDWLNGKEYKCK

2383.3092 214-234 3 TISKTKGQPREPQVYTLPPSR

2343.1761 220-239 2 GQPREPQVYTLPPSREEMTK

2214.1917 181-199 1 VVSVLTVVHQDWLNGKEYK

1954.0504 218-234 2 TKGQPREPQVYTLPPSR

1921.1229 206-223 3 GLPAPIEKTISKTKGQPR


60



1913.0888 200-217 3 CKVSNKGLPAPIEKTISK

1911.1273 202-219 3 VSNKGLPAPIEKTISKTK

1905.8939 272-288 0 TTPPMLDSDGSFFLYSK

1904.9422 224-239 1 EPQVYTLPPSREEMTK

1904.0309 197-213 3 EYKCKVSNKGLPAPIEK

1793.9908 181-196 0 VVSVLTVVHQDWLNGK


1722.8764 235-249 1 EEMTKNQVSLTCLVK

1681.9846 202-217 2 VSNKGLPAPIEKTISK

1639.8187 168-180 1 TKPREEQFNSTFR

1617.8417 1-16 1 ASTKGPSVFPLAPCSR

1483.8301 200-213 2 CKVSNKGLPAPIEK

1482.8890 206-219 2 GLPAPIEKTISKTK

1366.6882 17-30 0 STSESTAALGCLVK

1286.6739 224-234 0 EPQVYTLPPSR

1253.7463 206-217 1 GLPAPIEKTISK

1252.7259 202-213 1 VSNKGLPAPIEK

1230.6299 5-16 0 GPSVFPLAPCSR

1157.5222 172-180 0 EEQFNSTFR

1115.6531 214-223 2 TISKTKGQPR

1104.6081 240-249 0 NQVSLTCLVK

1098.5612 197-205 2 EYKCKVSNK

974.5629 94-101 2 VDKTVERK

846.4679 94-100 1 VDKTVER

835.4342 128-134 0 DTLMISR

824.4876 206-213 0 GLPAPIEK

818.4730 289-295 1 LTVDKSR

788.4512 319-326 0 SLSLSPGK

686.3944 218-223 1 TKGQPR

678.3603 200-205 1 CKVSNK

677.4192 214-219 1 TISKTK

670.3229 197-201 1 EYKCK

637.2861 235-239 0 EEMTK

632.3726 97-101 1 TVERK

61



575.3399 289-293 0 LTVDK

504.2776 97-100 0 TVER

501.3143 168-171 0 TKPR

457.2517 220-223 0 GQPR

448.2766 214-217 0 TISK

447.2562 202-205 0 VSNK

439.2187 197-199 0 EYK

406.2296 1-4 0 ASTK

361.2081 94-96 0 VDK

262.1510 294-295 0 SR

250.1220 200-201 0 CK

248.1605 218-219 0 TK

147.1128 101-101 0 K

62

Table 9 - Immunoglobulin heavy constant gamma 3 at positions 1-377


9488.7191 1-93 3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYTCNVNHKPSNTK

9443.6976 5-96 3 GPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYTCNVNHKPSNTKVDK

9101.5073 5-93 2 GPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYTCNVNHKPSNTK

8388.1866 17-97 3 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNH KPSNTKVDKR

8232.0855 17-96 2 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNH KPSNTKVDK

7889.8952 17-93 1 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNH KPSNTK

7611.8380 31-101 3 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELK

7427.4207 301-365 3 GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNR

7361.4591 275-339 3 EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSK

7142.5479 31-97 2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKR

6986.4468 31-96 1 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDK

6650.1251 286-344 3 EEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDK

6646.3498 159-218 3 CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAK

6644.2565 31-93 0 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTK

6302.1142 149-206 3 SCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFK

6274.9899 291-346 3 NQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSR

6093.8030 286-339 2 EEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSK

6031.8568 291-344 2 NQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDK

5475.5347 291-339 1 NQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSK

5391.6014 186-231 3 TPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFR

5248.6770 159-206 2 CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFK

5189.3996 301-346 2 GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSR

5053.5185 179-222 3 DTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPR

4946.2665 301-344 1 GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDK

4842.4750 207-247 3 WYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGK

4755.2722 134-178 3 SCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPK

4571.2220 179-218 2 DTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAK

4518.2514 144-185 3 CPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISR

4389.9444 301-339 0 GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSK

4237.1022 186-222 2 TPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPR

3963.9991 149-185 2 SCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISR

3865.0031 219-250 3 TKPREEQYNSTFRVVSVLTVLHQDWLNGKEYK


63

Table 9 - Immunoglobulin heavy constant gamma 3 at positions 1-377 (continued)


3773.8751 345-377 3 SRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

3754.8057 186-218 1 TPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAK

3701.8350 144-178 2 CPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPK

3613.8107 223-252 3 EEQYNSTFRVVSVLTVLHQDWLNGKEYKCK

3560.7637 340-369 3 LTVDKSRWQQGNIFSCSVMHEALHNRFTQK

3530.7419 347-377 2 WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

3460.5574 102-133 3 TPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPK

3444.8022 219-247 2 TKPREEQYNSTFRVVSVLTVLHQDWLNGK

3428.7664 271-300 3 GQPREPQVYTLPPSREEMTKNQVSLTCLVK

3382.7066 223-250 2 EEQYNSTFRVVSVLTVLHQDWLNGKEYK

3375.5952 98-128 3 VELKTPLGDTTHTCPRCPEPKSCDTPPPCPR

3234.3967 114-143 3 CPEPKSCDTPPPCPRCPEPKSCDTPPPCPR

3234.3967 119-148 3 SCDTPPPCPRCPEPKSCDTPPPCPRCPEPK

3234.3967 129-158 3 CPEPKSCDTPPPCPRCPEPKSCDTPPPCPR

3173.5493 179-206 1 DTLMISRTPEVTCVVVDVSHEDPEVQFK

3147.5827 149-178 1 SCDTPPPCPRCPAPELLGGPSVFLFPPKPK

3056.4941 340-365 2 LTVDKSRWQQGNIFSCSVMHEALHNR

3053.4864 207-231 2 WYVDGVEVHNAKTKPREEQYNSTFR

3004.4417 345-369 2 SRWQQGNIFSCSVMHEALHNRFTQK

2990.5325 275-300 2 EPQVYTLPPSREEMTKNQVSLTCLVK

2962.5057 223-247 1 EEQYNSTFRVVSVLTVLHQDWLNGK

2910.5619 159-185 1 CPAPELLGGPSVFLFPPKPKDTLMISR

2906.3052 102-128 2 TPLGDTTHTCPRCPEPKSCDTPPPCPR


2863.4804 1-30 2 ASTKGPSVFPLAPCSRSTSGGTAALGCLVK

2761.3086 347-369 1 WQQGNIFSCSVMHEALHNRFTQK

2680.1444 119-143 2 SCDTPPPCPRCPEPKSCDTPPPCPR

2680.1444 134-158 2 SCDTPPPCPRCPEPKSCDTPPPCPR

2572.3188 269-290 3 TKGQPREPQVYTLPPSREEMTK

2500.1721 345-365 1 SRWQQGNIFSCSVMHEALHNR

2478.2591 97-118 3 RVELKTPLGDTTHTCPRCPEPK

2476.2686 5-30 1 GPSVFPLAPCSRSTSGGTAALGCLVK


2383.3092 265-285 3 TISKTKGQPREPQVYTLPPSR

2357.1329 186-206 0 TPEVTCVVVDVSHEDPEVQFK

2343.1761 271-290 2 GQPREPQVYTLPPSREEMTK

2322.1580 98-118 2 VELKTPLGDTTHTCPRCPEPK

2266.1972 94-113 3 VDKRVELKTPLGDTTHTCPR


64



2257.0389 347-365 0 WQQGNIFSCSVMHEALHNR


2180.9595 114-133 2 CPEPKSCDTPPPCPRCPEPK

2180.9595 129-148 2 CPEPKSCDTPPPCPRCPEPK

2094.1456 159-178 0 CPAPELLGGPSVFLFPPKPK

1954.0504 269-285 2 TKGQPREPQVYTLPPSR

1935.1385 257-274 3 ALPAPIEKTISKTKGQPR

1927.1044 251-268 3 CKVSNKALPAPIEKTISK

1925.1429 253-270 3 VSNKALPAPIEKTISKTK

1924.0069 97-113 2 RVELKTPLGDTTHTCPR

1918.0466 248-264 3 EYKCKVSNKALPAPIEK

1904.9422 275-290 1 EPQVYTLPPSREEMTK

1898.9871 207-222 1 WYVDGVEVHNAKTKPR

1852.8680 102-118 1 TPLGDTTHTCPRCPEPK


1767.9058 98-113 1 VELKTPLGDTTHTCPR


1722.8764 286-300 1 EEMTKNQVSLTCLVK

1696.0003 253-268 2 VSNKALPAPIEKTISK

1655.8136 219-231 1 TKPREEQYNSTFR

1626.7073 114-128 1 CPEPKSCDTPPPCPR

1626.7073 119-133 1 SCDTPPPCPRCPEPK


1626.7073 134-148 1 SCDTPPPCPRCPEPK



1497.8457 251-264 2 CKVSNKALPAPIEK

1496.9046 257-270 2 ALPAPIEKTISKTK

1416.6906 207-218 0 WYVDGVEVHNAK

1298.6157 102-113 0 TPLGDTTHTCPR

1292.7208 366-377 1 FTQKSLSLSPGK

1286.6739 275-285 0 EPQVYTLPPSR

1267.7620 257-268 1 ALPAPIEKTISK

1266.7416 253-264 1 VSNKALPAPIEK

1264.6565 17-30 0 STSGGTAALGCLVK


1173.5171 223-231 0 EEQYNSTFR

1115.6531 265-274 2 TISKTKGQPR


65



1104.6081 291-300 0 NQVSLTCLVK

1098.5612 248-256 2 EYKCKVSNK

1072.4550 119-128 0 SCDTPPPCPR

1072.4550 134-143 0 SCDTPPPCPR

1072.4550 149-158 0 SCDTPPPCPR

986.5993 94-101 2 VDKRVELK

838.5032 257-264 0 ALPAPIEK

835.4342 179-185 0 DTLMISR

818.4730 340-346 1 LTVDKSR

788.4512 370-377 0 SLSLSPGK

686.3944 269-274 1 TKGQPR

678.3603 251-256 1 CKVSNK

677.4192 265-270 1 TISKTK

670.3229 248-252 1 EYKCK

644.4090 97-101 1 RVELK

637.2861 286-290 0 EEMTK

575.3399 340-344 0 LTVDK

573.2701 114-118 0 CPEPK

573.2701 129-133 0 CPEPK

573.2701 144-148 0 CPEPK

523.2875 366-369 0 FTQK

517.3092 94-97 1 VDKR

501.3143 219-222 0 TKPR

488.3078 98-101 0 VELK

457.2517 271-274 0 GQPR

448.2766 265-268 0 TISK

447.2562 253-256 0 VSNK

439.2187 248-250 0 EYK

406.2296 1-4 0 ASTK

361.2081 94-96 0 VDK

262.1510 345-346 0 SR

250.1220 251-252 0 CK

248.1605 269-270 0 TK

175.1189 97-97 0 R


66

Table 10 -– Immunoglobulin heavy constant gamma 4 at positions <1-327


9204.5594 5-93 3 GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTK

8335.1376 17-96 3 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDK

8003.0408 1-79 3 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTK

7992.9473 17-93 2 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTK

7840.8626 102-172 3 YGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR

7801.8040 98-168 3 VESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK

7615.8290 5-79 2 GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTK

7370.5136 225-289 3 EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR

7358.5660 102-168 2 YGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK

7157.5377 136-197 3 TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK

7143.5683 31-97 3 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR

6987.4672 31-96 2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK

6645.2769 31-93 1 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK

6404.2169 17-79 1 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK

6184.9654 129-181 3 DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR

6068.9538 241-294 3 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK

5925.8374 221-272 3 GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

5512.6317 241-289 2 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR

5487.6035 225-272 2 EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

5427.5949 273-319 3 TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK

5368.5491 136-181 2 TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR

5226.4966 251-296 3 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR

5056.5465 31-79 0 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK

5030.4662 129-172 2 DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR

4983.3635 251-294 2 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK

4548.1697 129-168 1 DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK

4427.0414 251-289 1 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR

4330.1495 290-327 3 LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

4245.1697 97-135 3 RVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISR

4214.0498 136-172 1 TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR

4089.0686 98-135 2 VESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISR

3865.0031 169-200 3 TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK

3773.8275 295-327 2 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK


67

Table 10 -– Immunoglobulin heavy constant gamma 4 at positions <1-327 (continued)


3770.9436 94-128 3 VDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPK

3731.7533 136-168 0 TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK

3645.8306 102-135 1 YGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISR

3629.7216 241-272 1 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

3613.8107 173-202 3 EEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

3599.8560 219-250 3 AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

3544.6848 290-319 2 LTVDKSRWQEGNVFSCSVMHEALHNHYTQK

3530.6943 297-327 1 WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

3444.8022 169-197 2 TKPREEQFNSTYRVVSVLTVLHQDWLNGK

3428.7533 97-128 2 RVESKYGPPCPSCPAPEFLGGPSVFLFPPKPK

3400.7239 221-250 2 GQPREPQVYTLPPSQEEMTKNQVSLTCLVK

3382.7066 173-200 2 EEQFNSTYRVVSVLTVLHQDWLNGKEYK

3272.6522 98-128 1 VESKYGPPCPSCPAPEFLGGPSVFLFPPKPK

2988.3628 295-319 1 SRWQEGNVFSCSVMHEALHNHYTQK

2965.5121 1-30 2 ASTKGPSVFPLAPCSRSTSESTAALGCLVK

2962.5057 173-197 1 EEQFNSTYRVVSVLTVLHQDWLNGK

2962.4900 225-250 1 EPQVYTLPPSQEEMTKNQVSLTCLVK

2943.5244 215-240 3 TISKAKGQPREPQVYTLPPSQEEMTK


2829.4142 102-128 0 YGPPCPSCPAPEFLGGPSVFLFPPKPK

2745.2296 297-319 0 WQEGNVFSCSVMHEALHNHYTQK

2701.3831 273-296 2 TTPPVLDSDGSFFLYSRLTVDKSR

2578.3003 5-30 1 GPSVFPLAPCSRSTSESTAALGCLVK

2549.2776 80-101 3 TYTCNVDHKPSNTKVDKRVESK

2544.1313 251-272 0 GFYPSDIAVEWESNGQPENNYK

2514.2657 219-240 2 AKGQPREPQVYTLPPSQEEMTK


2458.2500 273-294 1 TTPPVLDSDGSFFLYSRLTVDK

2315.1336 221-240 1 GQPREPQVYTLPPSQEEMTK


2106.0396 80-97 2 TYTCNVDHKPSNTKVDKR

1949.9385 80-96 1 TYTCNVDHKPSNTKVDK

1919.0630 201-218 3 CKVSNKGLPSSIEKTISK

1910.0051 198-214 3 EYKCKVSNKGLPSSIEK

1901.9279 273-289 0 TTPPVLDSDGSFFLYSR

1897.0865 207-224 3 GLPSSIEKTISKAKGQPR

1887.0909 203-220 3 VSNKGLPSSIEKTISKAK

1876.8997 225-240 0 EPQVYTLPPSQEEMTK



68

Table 10 -– Immunoglobulin heavy constant gamma 4 at positions <1-327 (continued)


1687.9588 203-218 2 VSNKGLPSSIEKTISK

1655.8136 169-181 1 TKPREEQFNSTYR


1607.7482 80-93 0 TYTCNVDHKPSNTK

1489.8043 201-214 2 CKVSNKGLPSSIEK

1458.8526 207-220 2 GLPSSIEKTISKAK

1366.6882 17-30 0 STSESTAALGCLVK

1259.7205 207-218 1 GLPSSIEKTISK

1258.7001 203-214 1 VSNKGLPSSIEK


1173.5171 173-181 0 EEQFNSTYR

1104.6081 241-250 0 NQVSLTCLVK

1098.5612 198-206 2 EYKCKVSNK

1085.6425 215-224 2 TISKAKGQPR

960.5472 94-101 2 VDKRVESK

835.4342 129-135 0 DTLMISR

830.4618 207-214 0 GLPSSIEK

818.4730 290-296 1 LTVDKSR

804.4825 320-327 0 SLSLSLGK

678.3603 201-206 1 CKVSNK

670.3229 198-202 1 EYKCK

656.3838 219-224 1 AKGQPR

647.4086 215-220 1 TISKAK

618.3569 97-101 1 RVESK

575.3399 290-294 0 LTVDK

517.3092 94-97 1 VDKR

501.3143 169-172 0 TKPR

462.2558 98-101 VESK

457.2517 221-224 0 GQPR

448.2766 215-218 0 TISK

447.2562 203-206 0 VSNK

439.2187 198-200 0 EYK

406.2296 1-4 0 ASTK

361.2081 94-96 0 VDK

262.1510 295-296 0 SR

250.1220 201-202 0 CK

218.1499 219-220 0 AK

175.1189 97-97 0 R


69

C. Biotools Analysis

Figure 35 – Results obtained from Biotools database search for MS analysis of IgG 1

(database: Swiss-Prot)



70



Figure 38 - Results obtained from Biotools database search for MS analysis of IgG 4


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Analysis of trypsin digested IgG using capillary ......Analysis of trypsin digested IgG using capillary electrophoresis and mass spectrometry Ana Isabel Fernandes de Carvalho Thesis