Biomolecule analysis using Mass Spectrometry

ELECTROPHORESIS / ISOELECTRIC FOCUSINGEttan IPGphor I/II IEF (GE Amersham)Dalt II Separation Unit (GE Amersham)Protean Plus DodecaCell (Bio-Rad)Micro-Rotofor Cell (Bio-Rad)

IMAGINGPharosFX (Bio-Rad)EXQuest automated spotcutter (Bio-Rad)PDQuest (Bio-Rad)

HPLC/UPLC1100 Cap/NanoHPLC (Agilent Technologies)3000 Ultimate nanoUPLC (Dioinex)

MASS SPECTROMETRYMAPII preparative MALDI robot (Bruker Daltonics)AutoFlex I MALDI-ToF (Bruker Daltonics)QSTAR Elite (ESI / MALDI) (AB Sciex)4000 QTRAP (ESI) (AB Sciex)

EQUIPMENT:

Application Overview

• Discovery Proteomics:– Gel-Based– Solution-Based

• Targeted Proteomics

• Biosimilars characterization using Intact Protein Mass analysis

Discovery Proteomics 1: Gel Based Protein Expression analysis

QSTAR

QTRAP

Single spot/band ID from 2D / 1D gels Profiling of tomato, potato, wheat and proteases expressed in Yarrowia lipolytica and Tobacco using SDS-PAGE LCMSMS workflow

300 – 1000 proteins per lane Labour Intensive + Large/Small/Acidic/Basic proteins are problematic

Discovery Proteomics 2: Solution-based Protein Expression Analysis

QSTAR

RP RP

Table 1: Efficiency of large-scale quantitative proteomic approaches to study changes in malarial parasite P. falciparum

Method 2-DE i SILAC & MudPIT ii iTRAQ & Micro-Rotofor / LC

No. P. falciparum IDs 125 1253 205

1. Method step

2. Number of fractions produced / analysed at each step

3. Approximate time (h) per step

1 2 3 1 2 3 1 2 3 IPG strip rehydration In-gel IEF SDS-PAGE Gel fixing Gel staining In-gel protein digest MALDI spotting and analysis*

n/a n/a n/a n/a n/a n/a n/a

10 15 1.5 18 24 18 10.5iii

Protein offline WAX, pH elution Protein offline WAX, salt elution Protein precipitation In-solution protein digest Peptide online SCX/RP, soluble proteins Peptide online SCX/RP, insoluble proteins

3 3 n/a n/a 18 36

1 1 1 18 36iv 72iv

In-solution protein digest iTRAQ labelling Peptide in-solution IEF Peptide online RP

n/a n/a 10 10

18 2 1.5 25 v

MS analysis time (h) per sample

10.5 108 25

MS analysis time (h) if using SILAC / iTRAQ vi

n/a 54 6.2 (4plex iTRAQ) 3.1 (8plex iTRAQ)

MS analysis time (min) per protein ID vii

5 2.5 1.8 (4plex iTRAQ) 0.9 (8plex iTRAQ)

Total analysis time (h) per sample

80 129 46.5

Total analysis time (h) if using SILAC / iTRAQ vi

n/a 64.5 11.6 (4plex iTRAQ) 5.8 (8plex iTRAQ)

Total analysis time (min) per protein ID vii

38 3 3.4 (4plex iTRAQ) 1.7 (8plex iTRAQ)

i: Smit et al., 2010 ii: Prieto et al., 2008 iii: MALDI spotting and analysis time = Data acquisition time per spot (5 min) x Number of spots analysed (125) iv: Peptide online SCX/RP analysis time = Number of fractions (18 or 36) x SCX/RP run time (2h) v: Peptide online RP analysis time = Number of fractions (10) x RP run time (2.5h) vi: MS or Total analysis time if using SILAC / iTRAQ = MS or Total analysis time per sample ÷ 2 (SILAC) or 4 (4plex iTRAQ) or 8 (8plex iTRAQ) vii: MS or Total analysis time per protein ID = (MS or Total analysis time per sample x 60) ÷ No. P. falciparum IDs

pH Salt

• Complementary to Shotgun proteomic experiments• Verify and Validate data from Exploratory Proteomics experiments• More accurate quantitation, reproducibility and sensitivity with less analysis time

• Test hypothesis

• Absolute Protein quantification from crude cell lysates

Targeted Proteomics: Protein Verification, Validation and Absolute Quantitation

QTRAP

Targeted Proteomics: MRM, MIDAS and AQUA for protein Verification, Validation and Quantitation

MRM Relative Quant

MRM Absolute Quant

25,680 Da

Biosimilars Characterization

QSTAR

Future applications / workflows….

• Bioinformatics solutions for complex sample analysis

• Trypsin immobilization for faster digestions

• Use of fast flow columns for rapid peptide and protein separation

• Automated 2D LCMSMS

• Label Free Relative Quantitation

• DNA / RNA analysis

• Glycan profiling

• Lipid profiling

• Protein structural characterization using mass spec

• Collaborations…

• Dr. Dalu Macama• Dr. Tsepo Tsekoa• Dr. Tharina van Brummelen• Dr. John Becker• Mrs. Sindisiwe Buthelezi• Mr. Sipho Mamputha