UVic-Genome BC Proteomics Centre

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1 QC or Bust: Performance Assessment for Quantitative Plasma Proteomics by MRM Andrew Percy UVic-Genome BC Proteomics Centre UVic-Genome BC Proteomics Centre US HUPO (Seattle, WA) April 7, 2014

2 Quantitation in Proteomics Need for QC Benchmarking Evolving trend to verify/validate candidate disease biomarkers in complex biofluids, such as blood plasma. Powerful protein quant technique involves MRM, or PRM, with isotopically labeled standards. Quantitative data must be precise and accurate. Assays must be transferable between labs and platforms. Accelerate biomarker pipeline and aid translation. Carr S.A. et al. Mol. Cell. Proteomics, 13, 2014, 907. QC kits would help improve reproducibility and transferability of MS-based quantitative proteomic assays.

3 Protein Quant Workflow MRM with Stable Isotope-labeled Standards (SIS) Matrix deplete, enrich, or neither 13 C/ 15 N Labeled Proteins reduce, alkylate, tryptically digest OR Proteolytic Digest 13 C/ 15 N Labeled Peptides Matrix Digest with Labeled Peptides solid phase extraction Concentrated and Desalted Peptides low ph RPLC- MRM/MS OR high ph RPLC Peptide Fractions low ph RPLC- MRM/MS Protein Quantitation

4 Importance of Standardization Case Example from Our Research 2D LC-MRM/MS for Plasma Protein Quant Peptide fractionation via alkaline RPLC and acidic RPLC. High ph eluent initially composed of 10 mm NH 4 COOH. QC stds revealed deterating column (Zorbax Eclipse) integrity. Performance recovered with new column, 2 nd SPE, or 10 mm NH 4 OH. Fractionation: Agilent 1260 LC-UV LC-MRM: Agilent 1290 with 6490 QqQ Column: Zorbax Eclipse Plus C 18 RRHD (2.5 x 150 mm, 1.8 µm) Percy A.J. et al. J. Proteomics, 2014, conditionally accepted.

5 Our Standardization Approach MRM Quantitative Plasma Proteomics Instrument Platform Assessment Daily Platform QC (Kit #1) Lyophilized SIS stds (in buffer and plasma digest) and 0.1% FA. XIC inspection and performance tracking. Monthly Platform QC (Kit #2) Lyophilized SIS stds (in buffer and plasma digests) and 0.1% FA. Generate curves and assess accuracy. Complete Analytical Workflow Evaluation Monthly Workflow QC (Kit #3) Plasma, trypsin, and SIS mix supplied. Generate curves and assess accuracy.

6 Solution and Sample Prep. Deoxycholate-catalyzed Denaturation Workflow

7 Protocol Utility Quantitative Plasma Proteomics Serum Albumin (P02768, 31 mg/ml) 1D LC-MRM/MS on proteins quantified in control plasma Myeloblastin (P24158, 44 ng/ml) Percy A.J. et al. Biochim. Biophys. Acta, 2013, in press.

8 QC Kit Protein Panel Afamin Ceruloplasmin Heparin cofactor II Alpha-1-antichymotrypsin Clusterin Inter-alpha-trypsin inhibitor heavy chain Alpha-1B-glycoprotein Coagulation factor XIIa light chain Kininogen-1 Alpha-2-antiplasmin Complement C3 L-selectin Angiotensinogen Complement C4 gamma chain Plasminogen Antithrombin-III Complement component C9 Prothrombin Apolipoprotein A1 Complement factor B Retinol-binding protein 4 Apolipoprotein A-II Complement factor H Serum Albumin Apolipoprotein A-IV Fibrinogen alpha chain Serum amyloid P-compenent Apolipoprotein B-100 Fibrinogen beta chain Transferrin Apolipoprotein C-I Fibrinogen gamma chain Transthyretin Apolipoprotein C-III Gelsolin Vitamin D-binding protein Apolipoprotein E Haptoglobin Vitronectin Beta-2-glycoprotein I Hemopexin Reported Concentration Range: 42 mg/ml (Albumin) to 1.6 µg/ml (L-selectin) Protein MW: 7 kda (Apolipoprotein C-I) to 513 kda (Apolipoprotein B-100)

9 QC Kit Target Peptides Natural (NAT) and Stable Isotope-labeled Standards (SIS) General Details SIS Peptides Synthesis 41 tryptic peptides C-terminal, 13 C/ 15 N-labels Fmoc chemistry at 5 µmol scale VGYVSGWGR (Haptoglobin, P00738) Purification RP-HPLC-MS MALDI-TOF-MS Characterization AAA CZE 95% average purity

10 Kit #1 Analysis Procedure and Representative XIC Rehydate Inject Transfer Std-flow LC-MRM/MS Apolipoprotein A-I Serum Albumin NAT peptides SIS peptides Fibrinogen γ chain Hemopexin 10 µg plasma digest 100 fmol balanced SIS mix

11 Kit #1 Repeatability LC-MS Performance Retention Time Stability Relative Signal Stability Peak Width Stability Standard-flow LC-MRM/MS Platform (6490). 1 analysis/day over 5 days. Minimal variation indicates maintenance of LC-MS performance throughout other sample analyses.

12 Rigorous Performance QC Kits #2 & 3 Standardization Kits Rationale behind SIS Balancing Concentration-balanced Equimolar Raw Peak Area L1 L2 L3 L4 L5 L6 L7 ~1:1 of SIS:NAT Workflow and LC-MS platform assessment kits use balanced Balanced SIS mixes give lower variation. mixes. Kuzyk M.A. et al. Mol. Cell. Proteomics, 8, 2009, 1860.

13 Kit Measurement Reproducibility Lot-to-Lot Variability (Kit #3) Storage Stability (Kit #2) Complement factor H (SPDVINGSPISQK) Inter-alpha-trypsin inhibitor HC (AAISGENAGLVR ) 24 µg/ml (Day 2) 23 µg/ml (Day 90)

14 Kits Help Reveal Errors or Deficits Intra-lab Evaluation of Kit #3 log (Protein Concentration, Tester 3) y = 0.44x R 2 = log (Protein Concentration, Reference Values) log (Protein Concentration, Tester 4) y = 0.95x R 2 = log (Protein Concentration, Reference Values) # of Proteins Reference Tester 3 Tester 4 Tester 3 (1 st yr. undergrad) procedural errors (e.g., SIS peptide dilution series) Tester 4 (MSc. student) deficient instrument sensitivity 0 < Linear dynamic range Percy A. J. et al. J. Proteome Res., 12, 2013, 222.

15 Inter-lab Study No. 1 MRM Plasma Protein Quant Kit analysis on similar and dissimilar platforms: std-flow LC with 6490 std-flow LC with QTRAP 4000 nanolc with QTRAP 4000 nanolc with TSQ Vantage nanolc with Q Exactive nanolc with Xevo TQ 3 internal and 3 external instruments Parameter optimization and interference screening conducted with supplied standards prior to protein quant. The reproducibility of protein concentrations between instruments was evaluated.

16 Inter-lab Quant Comparison TSQ Vantage vs. Reference Values on 6490 Concentrations of 37 common plasma proteins compared: Protein classification by efficiency: Proc JL et al. J. Proteome Res., 9, 2010, 5422.

17 Protein Conc Comparison 22 Common Proteins Quantified Across 6 Platforms Albumin_serum Vitronectin Alpha-1-antichymotrypsin Vitamin D-binding protein Apolipoprotein A-I Transthyretin Plasminogen Kininogen-1 Apolipoprotein B-100 Apolipoprotein E Clusterin Inter-alpha-trypsin inhibitor HC Heparin cofactor II Hemopexin Coagulation factor XII a LC Complement C3 Complement C4 gamma chain Haptoglobin Complement factor B Fibrinogen beta chain Complement factor H Fibrinogen alpha chain std-flow LC-MRM/MS (6490) std-flow LC-MRM/MS (QTRAP 4000) nanolc-mrm/ms (QTRAP 4000) nanolc-mrm/ms (Xevo TQ) nanolc-mrm/ms (Q Exactive) nanolc-mrm/ms (TSQ Vantage) Percy A. J. et al. J. Proteomics, 95, 2013, 66.

18 Inter-lab Study No. 2 (In Progress) Kit Investigations across 16 Labs Multicentric study to determine reproducibility and transferability of kits across platforms and labs. 4% 23% 73% Evaluation criteria includes: - Kits ability to pinpoint procedural errors/instrument deficits. - Precision within and between kits, instruments, and labs. - Accuracy of quantitation.

19 Standardization Kit for 6490 Quantitative Plasma Proteomics Kit Contents Standards or key components to prepare the standards. Detailed SOPs. LC-MS conditions and parameters. Reference values. Performance quality guide. Commercialized through MRM Proteomics for Agilent s 6490.

20 Kit Requirements Agilent 1290 LC (standard-flow) with 6490 QqQ After retention time scheduling, run the SIS in plasma digest standards: 1 std for LC-MS assessment. 7 stds in triplicate for workflow evaluation. 30 min gradient with 4 min post-run equilibration for standard-flow LC-MRM/MS analysis. ~35 min performance check/day for platform assessment. ~16 h processing time for workflow evaluation. Use UVic standard curve generator tool with processed data to rapidly obtain performance values.

21 Data Analysis Software Tool Curve Generation and Data Extraction Automated extraction of attributes and performance metrics from processed data in seconds.

22 Summary and Outlook Standardization will help ensure high quality quantitative data can be reproduced by clinical reserach laboratories across different MS platforms. We are developing standardization kits for performance evaluation in MRM-based quantitative plasma proteomics. Daily/monthly assessment of workflow and/or instrument platform. LC-MRM/MS (Agilent 6490) commercialized (MRM Proteomics) Large-scale, multi-site study currently in progress. Standard curve generator tool developed to aid analysis. Alternative kits for standardization in development.

23 Acknowledgements UVic-Genome BC Proteomics Centre (Victoria, BC, Canada) Jessica Tamura-Wells Juncong Yang Andrew Chambers Yassene Mohammed Joel Nyberg Christoph Borchers Collaborators (Study No. 1) Dominik Domanski Funding Agencies Genome Canada Genome BC Julia Burkhart Western Economic Diversification of Canada Albert Sickmann