Biomolecule analysis using Mass Spectrometry. ELECTROPHORESIS / ISOELECTRIC FOCUSING Ettan IPGphor I/II IEF (GE Amersham) Dalt II Separation Unit (GE.

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Presentation transcript:

Biomolecule analysis using Mass Spectrometry

ELECTROPHORESIS / ISOELECTRIC FOCUSING Ettan IPGphor I/II IEF (GE Amersham) Dalt II Separation Unit (GE Amersham) Protean Plus DodecaCell (Bio-Rad) Micro-Rotofor Cell (Bio-Rad) IMAGING PharosFX (Bio-Rad) EXQuest automated spotcutter (Bio-Rad) PDQuest (Bio-Rad) HPLC/UPLC 1100 Cap/NanoHPLC (Agilent Technologies) 3000 Ultimate nanoUPLC (Dioinex) MASS SPECTROMETRY MAPII 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

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