viii

Index

Acknowledgement ..................................................................................................................... i

Summary ................................................................................................................................... ii

Zussamenfassung ..................................................................................................................... v

Index ...................................................................................................................................... viii

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

1.1 Proteomics: an overview ........................................................................................... 1

1.1.1 Proteome .................................................................................................................. 1

1.1.2 Significance of post-translational modification (PTM) ........................................... 3

Sulfenation as PTM......................................................................................................... 4

1.2 Ionization techniques in mass spectrometry for proteomics ................................. 6

1.2.1 ESI (Electrospray Ionization) .................................................................................. 6

1.2.2 NSI (Nanospray Ionization) ..................................................................................... 7

1.2.3 MALDI (Matrix Assisted Laser Desorption Ionization) ......................................... 8

1.2.4 ICP (Inductively Coupled Plasma) .......................................................................... 8

1.3 Fragmentation of peptides and proteins using mass spectrometry .................... 10

1.3.1 CID (Collision-induced dissociation) .................................................................... 11

1.3.2 IRMPD (Infrared multiphoton dissociation) ......................................................... 11

1.3.3 HCD (Higher energy collisional dissociation) ...................................................... 12

1.4 Quantification of peptides and proteins using mass spectrometry ..................... 12

1.4.1 Label-free quantitative proteomics ........................................................................ 15

1.4.2 Quantitative proteomics using stable isotope labeling .......................................... 15

Metabolic labeling ........................................................................................................ 15

Enzymatic labeling........................................................................................................ 17

Chemical labeling ......................................................................................................... 17

1.4.3 Quantitative proteomics using bi-functional chelating agents as chemical labels

and lanthanides as mass markers ...................................................................................... 23

ECAT (Element Coded affinity Tag) ............................................................................ 24

MECT (Metal Element Chelate Tag) ............................................................................ 25

1.5 Quantitative proteomics via natural element tags and chemical labels using

inductively coupled plasma mass spectrometry (ICP-MS) ............................................ 25

1.5.1 Via naturally occurring heteroatom tags ............................................................... 26

Sulfur............................................................................................................................. 26

Phosphorus .................................................................................................................... 27

Selenium ....................................................................................................................... 27

1.5.2 Via “artificial” elemental labeling ......................................................................... 28

Iodine ............................................................................................................................ 28

Iron ................................................................................................................................ 28

Mercury ......................................................................................................................... 29

ix

MeCAT (Metal Coded Affinity Tag) using lanthanides ............................................... 29

1.6 Multi-elemental labeling of peptides and proteins ............................................... 31

Polymer-Based Elemental Tags .................................................................................... 31

Nanoparticles ................................................................................................................ 32

2 Aim and Scope of Work ................................................................................................. 34

3 Results and Discussion ................................................................................................... 35

3.1 Dual labeling of peptides using MeCAT and DOTA derivatives ........................ 35

Advantages of cysteinyl-peptide enrichment on Thiopropyl Sepharose 6B resin ........ 38

Optimization of capture/wash/release of cysteinyl-peptide from Thiopropyl Sepharose

6B resin ......................................................................................................................... 39

Optimization of on-resin amino labeling ...................................................................... 40

Evaluation of on-resin amino labeling .......................................................................... 42

Peptide elution from the resin ....................................................................................... 46

Effect of excess TCEP on MeCAT-IA labeling ........................................................... 47

Removal of excess TCEP.............................................................................................. 48

Estimation of losses ...................................................................................................... 49

Thiol labeling and demonstration of dual labeling ....................................................... 50

Methodological scope for quantitative applications using standard addition technique

....................................................................................................................................... 57

Conclusion and Outlook ............................................................................................... 58

3.2 Labeling of proteins and peptides using gold ....................................................... 60

Monomaleimido-Undecagold ....................................................................................... 60

Labeling of protein with Monomaleimido-Undecagold ............................................... 61

C3 LC-ESI-FTICR-MS and LC-ICP-MS analysis of proteins labeled with

Monomaleimido-Undecagold ....................................................................................... 62

SDS-PAGE separation of proteins labeled with Monomaleimido-Undecagold ........... 62

OFFGEL electrophoresis of proteins labeled with Monomaleimido-Undecagold ....... 63

Labeling of peptides with Monomaleimido-Undecagold ............................................. 65

Conclusion and Outlook ............................................................................................... 67

3.3 Quantification of sulfenic acid modifications in peptides and proteins using

molecular and elemental mass spectrometry ................................................................... 68

Synthesis of Ln-DOTA-Dimedone ............................................................................... 71

Purification of Ln-DOTA-Dimedone ........................................................................... 73

In vitro oxidation of cysteine residues and reactivity with dimedone .......................... 76

In vitro oxidation of peptides and labeling with Ln-DOTA-Dimedone ....................... 78

Parallel labeling of peptides with Ln-DOTA-Dimedone and Ln-MeCAT-IA ............. 82

Consecutive labeling of peptides with Ln-DOTA-Dimedone and Ln-MeCAT-IA ...... 83

Optimization of in vitro oxidation of proteins and labeling with Ln-DOTA-Dimedone

....................................................................................................................................... 84

Parallel labeling of proteins with Ln-DOTA-Dimedone and Ln-MeCAT-IA .............. 85

Consecutive labeling of proteins with Ln-DOTA-Dimedone and Ln-MeCAT-IA ...... 86

ESI-MS/MS based relative quantification of sulfenic acid modifications.................... 87

x

Conclusion and Outlook ............................................................................................... 90

4 Materials and Methods .................................................................................................. 92

4.1 Dual labeling of peptides using MeCAT and DOTA derivatives ........................ 92

Chemicals and materials ............................................................................................... 92

Synthesis of Ln-MeCAT-IA ......................................................................................... 92

Proteins and peptides .................................................................................................... 93

Selection of peptides ..................................................................................................... 93

Protein digestion ........................................................................................................... 94

Cysteinyl-peptide enrichment on the Thiopropyl Sepharose 6B resin ......................... 94

On-resin amino labeling ................................................................................................ 94

Peptide elution from the resin and removal of excess TCEP........................................ 95

Cysteine labeling with Ln-MeCAT-IA ......................................................................... 95

C18 nanoLC-ESI-FTICR-MS analysis of labeled peptides ........................................... 95

C18 LC-ESI-FTICR-MS and LC-ICP-MS of labeled peptides ..................................... 96

4.2 Labeling of proteins and peptides using gold ....................................................... 97

Chemicals and materials ............................................................................................... 97

Proteins and peptides .................................................................................................... 97

Labeling of protein with Monomaleimido-Undecagold ............................................... 97

C3 LC-ESI-FTICR-MS and LC-ICP-MS analysis of proteins labeled with

Monomaleimido-Undecagold ....................................................................................... 97

SDS-PAGE separation of proteins labeled with Monomaleimido-Undecagold ........... 98

OFFGEL electrophoresis of proteins labeled with Monomaleimido-Undecagold ....... 98

ICP-MS analysis of OFFGEL fractions ........................................................................ 99

Labeling of peptides with Monomaleimido-Undecagold ............................................. 99

C18 LC-ESI-FTICR-MS and LC-ICP-MS analysis of peptides labeled with

Monomaleimido-Undecagold ....................................................................................... 99

4.3 Quantification of sulfenic acid modifications in peptides and proteins using

molecular and elemental mass spectrometry ................................................................. 100

Chemicals and materials ............................................................................................. 100

Synthesis of Ln-DOTA-Dimedone ............................................................................. 100

LC-UV-MS analysis of the synthesized Ln-DOTA-Dimedone.................................. 101

Purification of the synthesized Ln-DOTA-Dimedone ................................................ 101

Proteins and peptides .................................................................................................. 101

In vitro oxidation of peptides and labeling with dimedone ........................................ 101

In vitro oxidation of peptides and labeling with Ln-DOTA-Dimedone ..................... 101

Labeling of peptides with Ln-MeCAT-IA .................................................................. 102

C18 nanoLC-ESI-FTICR-MS analysis of peptides labeled with dimedone, Ln-DOTA-

Dimedone or Ln-MeCAT-IA ...................................................................................... 102

SCX LC-ICP-MS analysis of p1 labeled with Tb-DOTA-Dimedone or Ho-MeCAT-IA

..................................................................................................................................... 102

Oxidation of proteins and labeling with Ln-DOTA-Dimedone .................................. 103

Labeling of proteins with Ln-MeCAT-IA .................................................................. 103

SDS-PAGE separation of proteins .............................................................................. 103

xi

ICP-MS analysis of protein bands in SDS-PAGE ...................................................... 103

Parallel labeling of peptides and proteins ................................................................... 104

Consecutive labeling of peptides and proteins............................................................ 104

Instrumental parameters for IRMPD and HCD fragmentation ................................... 104

5 References ...................................................................................................................... 106

6 Abbreviations ................................................................................................................ 118

Lebenslauf ............................................................................................................................. 120

Publikationsliste ................................................................................................................... 122