Increasing Sensitivity In LC-MS. Simplification and prefractionation of complex protein samples

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1 Increasing Sensitivity In LC-MS Simplification and prefractionation of complex protein samples

2 Protein Analysis Workflow Simplify Fractionate Separate Identify High Abundant Protein Removal reduction of complexity, enrichment pi-based fractionation Fractionation, desalting Multiple Affinity Removal OFFGEL mrp LC MS Data Quality Control CE Bioanalyzer

3 Sample Preparation For Biofluids MARS A polyclonal antibody based system to rapidly deplete multiple high abundant proteins in serum/plasma/csf. H H L H L L Launched in August 2003 H L L H Individual Ab materials are mixed in selected HL H H L H percentages and packed into a column format Agilent continues to innovate and lead this market Apply Crude Human Serum HH H H HH H H Unbound Fraction (low abundance proteins) Bound Fraction (high abundance proteins) Total Run Time (30 min) Buffer A Buffer B L L L L L L H H H H HH H H Low-Abundance Proteins Free from Interferences High-Abundance Proteins

4 Multiple Affinity Removal System Columns & spin cartridges Removes top 6, 7 or 14 high abundant proteins (HAP) from serum/plasma etc. at 95-99% levels each Depletes HAP found in serum/plasma, urine, BALF, CSF, tissue and other biological samples Removal of HAP has the benefit of: Unmasking the low abundant proteins (LAP) of interest e.g. potential biomarker, Increasing dynamic range of the analysis Page 4 July 23, 2009

5 MARS Column Configurations Hu-6 + Hu-6C / Hu-7 / Hu-14 Ms-3 Two new configurations available soon: HSA & HSA/IgG Anti Albumin Anti IgG Anti Transferrin and the same configurations in spin cartridge format

6 Multiple Affinity Removal System 14 MARS Anti HSA Anti IgG Anti Transferrin Anti Haptoglobin Anti Alpha1-antitrypsin Anti IgA Anti Fibrinogen Anti Alpha2-Macroglobulin Anti Alpha1-Acid Glycoprotein Anti IgM Anti Apolipoprotein AI Anti Apolipoprotein AII Anti Complement C3 Anti Transthyretin Removes 92-94% of total protein Proteomic Sample High Abundance Protein Low Abundance Protein Proteomic Sample with Low Abundance Proteins In Column Flow-through

7 mrp-c18 Protein Fractionation Column mrp mrp (macroporous reverse-phase) What is it? Reverse Phase column for protein separation and fractionation. The silica based particles and recommended LC methods have been optimized for: Highest recoveries of protein samples (95% - 99% of loaded sample) Highest resolution separations Reproducibility High sample loading capacity (3X higher than most standard RP columns) Lifetime Can be used for desalting with quick gradient

8 mrp-c18 Recovery using Serum mrp Immunodepleted serum Blank Protein Conc. Protein Conc. Protein Conc. % recovery Pre-mRP* No column mrp recovery g 49.3 g 99%

9 NEW Complex Proteomics Standard Proteins extracted from genetically distant archaeon Pyrococcus furiosus Complex sample for evaluation of LC/MS protein identification workflow Over 1500 proteins, spanning wide ranges of size, pi and hydrophobicity For benchmarking and validation of proteomic workflow s over time, across instruments, methods or labs

10 OFFGEL Fractionation OFFGEL separates proteins or peptides by pi based on the resolving power of immobilized phgradient (IPG) gels provides analytes in solution compatible with LC-MS and upfront sample preparation techniques Low ph Low ph High ph High ph Low ph High ph

11 3100 Features OFFGEL Protein/peptide fractionation with pi resolution µg to mg load capacity (50 g 5 mg per sample) Possible to run conventional in-gel IEF as well as OFFGEL fractionation Diagnostics: Online current control for each individual sample allow to check fractionation quality & progress of each individual sample

12 Protein or Peptide Fractionation Protein-based OFFGEL Peptide-based 4-fold increase of detected proteins due to OFFGEL fractionation

13 OFFGEL Protein Fractionation 4 times better sensitivity with OFFGEL E.coli lysate OFFGEL ph 4-7, 24 fractions digest HPLC/MS

14 OFFGEL Combination of IEF with SDS-PAGE Visualizing protein expression differences in complex samples Bioanalyzer New without OFFGEL

15 Sensitivity: Silver Staining vs. Bioanalyzer OFFGEL Bioanalyzer Size / kda Size / kda ng of total protein loaded per gel lane or chip well Silver stained PA-gel (4-12% BisTris minigel) LM 2100 Pseudo gel view

16 Why Use Microfluidics For Nanospray LC/MS? Integrate functional components onto a reusable, biocompatible chip enrichment and analytical nanocolumns nanospray emitter fittings and connection capillaries directly on a reusable biocompatible polymer chip.

17 HPLC-Chip Microfluidics Technology High Performance Nano-separations made Routine Fluidic, Sample Ports Enrichment Column Analytical Column 75 x 50 µm cross section C18, C8 NEW 300A, 80A NEW Laser Ablated ESI Tip

18 Chip Technology for Proteomics & Biomarker Discovery/Validation AVAILABLE NOW! Protein ID Chips GPC Chip Intact Protein Chip Infusion Chip Calibration Chip Custom Chip High Capacity Chip Intact Protein Analysis Quantitative Proteomics Peptide/protein digests Biomarker Discovery Phosphopeptide Chip COMING SOON! GlycoPro Chip Glycoprotein Analysis Phosphoprotein Analysis

19 Agilent 7100 Capillary Electrophoresis System Agilent Technologies introduces the Next Generation Capillary Electrophoresis System

20 Capillary Electrophoresis at Agilent Former G1600 HP 3 D-system Agilent has more than 15 years of CE experience and CE is one of Agilent s core technologies (4000 systems installed) Agilent pioneered the proprietary bubble cell capillaries & high sensitivity cell providing highest CE sensitivity levels in UV detection

21 Agilent 7100 Capillary Electrophoresis System Evolution of the proven 1600 CE Technology Unprecedented sensitivity and handling New! Backward-compatible with previous generation Sample throughput with Agilent Replenishment-system Fully flexible on detectors (DAD-UV, MS, LIF, CCD,..) Agilent ChemStation control and data analysis

22 Agilent 7100 Capillary Electrophoresis System Using External Detectors Agilent s CE-ESI-MS systems provide Single vendor solution: Plug and spray Full portfolio, MSD, Triple-Quad, ToF, Q-ToF Most robust and reliable ESI-Interface for CE Capillary exit on ground no compromises!

23 Agilent 7100 Capillary Electrophoresis The CE-MS advantage CE-Electrospray is a powerful method for high resolution separation and identification for a wide variety of molecules. Additionally it is an orthogonal separation technique to HPLC The Agilent interface combines the advantage of a grounded CE capillary end with a robust and easy to use spray needle that fits into the whole portfolio of Agilent MS instruments Widest choice of detctors DAD, LIF, CCD, Single-Q, IT, ToF, QQQ, QToF, ICP-MS Only vendor for plug & spray ESI-MS complete CE-MS portfolio with a single interface solution Easy change from LC-MS to CE-MS and back quick and robust ESI by MS-adaptor & spray needle kit using Agilent Chemstation software Combined detection option UV-DAD plus MS detection, LIF plus MS detection Grounded CE capillary end No compromises between separation- or ESI-spray voltage

24 Summary: Use in Protein Biomarker Applications Sample Prep Simplify/Deplete Extraction of unique proteins from samples and simplify sample for analysis Extract protein from tissue or blood with FFPE Protein Extraction Solution or PPS Silent Surfactant Blood serum/plasma samples (or CSF, urine) deplete highabundant proteins with Multiple Affinity Removal System Fractionate Separate/LC Prep Identify Reduction of complexity and enrichment Quality Control 2100 Bioanalyzer Fractionate, concentrate, and desalt proteins with mrp-c OFFGEL Digest with Proteomics Grade Trypsin Agilent 1200 Series Liquid Chromatography New Complex Proteomics Standard Agilent 6000 Series Mass Spectrometry w/ HPLC-Chip Agilent Restricted

25 Summary Sensitivity and reproducibility can be greatly enhanced by sample prefractionation Immunodepletion, reverse phase and pi-based fractionation can be employed as tools for reducing sample complexity Agilent s prefractionation tools are compatible and can be used in various combinations and sequences For more information visit