Use of stable isotope labeled standard peptides in MRM for precise quantitation

Christoph Borchers UVic-Genome BC Proteomics Centre, Victoria, Canada McGill University, Montreal, Canada Targeted Quantitative Proteomics

Multiple Reaction Monitoring (MRM) Nature Method of the Year for 2012 Multiple Reaction Monitoring (MRM)

Q1 Q2 Q3

PEPTIDE

PEPTIDE PTIDEEPTIDETIDEIDEDE PEPTIDE

PEPTIDE

Collision chamber ∙ tandem MS (MS/MS) scan mode ∙ low noise & high sensitivity ∙ unique to QqQ ∙ broadlinear range (103 - 104) ∙ well established in small molecule quantitation Targeted Quantitative Proteomics

4 Improved accuracy of MRM assays by stable isotope standard (SIS) peptides ∙ Absolute specificity ∙ 4 molecular specific criteria (retention time, mass of peptide, masses of peptide fragments, mass ratios of the peptide fragments from SIS to endogenous peptide )

∙ “Absolute”/precise quantitation ∙ Amount and purity of peptide standard is accurately assessed

∙ Attomole sensitivity ∙ Optimized parameters

∙ High reproducibility ∙ <20% CV (analytical) SIS Peptides Reduce Variation

∙ Reproducibility of 45 plasma protein targets (n = 12) General Method Development Strategy and Tools

Robotic Peptide Synthesizer (Protein Technologies) High-Throughput Sample Prep.

Parallel sample handling 96 well format

denaturation

reduction

alkylation

trypsin digestion

SIS peptide addition Automated sample preparation • Hundreds of samples/day solid phase extraction • Highly reproducible (avg. 5% CV)

Lyophilized samples ready for LC-MRM/MS analysis Developed Quantitative Assays: Plasma

Google Images Quant Range from 1D LC-MRM/MS 192 Human Plasma (483 Interference-free Peptides)

Serum albumin (31 mg/mL)

Candidate Protein Category Markers CVD 81 Cancer 65 Apolipoprotein 16

Enrichment or LC fractionation required for deeper quant. Myeloblastin (19 ng/mL) Quant Range from 1D LC-MRM/MS 76 Mouse Plasma Proteins (99 Interference-free Peptides)

C57BL/6 strain VISLSGEHSIIGR (P08228)

40 CVD-linked Proteins in Panel R2 = 0.992

Superoxide dismutase (P08228; 57 mg/mL determined)

Parallel Evaluations (n = 3 process reps with quant via std curves) Tissue Type Qualified Heart Lung Brain Peptides 308 325 249 Proteins 212 227 168 2D LC-MRM/MS Toward Deeper Plasma Protein Quant

Fractionation Approach

1 2

etc.

high pH LC-UV 96-well Plate low pH LC-MRM/MS

Interference Screening with Human Plasma Human Plasma Protein Quant Comparison 2D vs. 1D LC-MRM/MS: 253 vs. 192 Proteins

Percy A. J. et al. J. Proteomics 106, 2014, 113. Developed Quantitative Assays: Human Dried Blood Spots

finger prick or dry under RT storage heel prick ambient conditions Dried Blood Spot Sampling

14

finger prick or dry under RT storage heel prick ambient conditions

Advantages of DBS samples • Less invasive sampling procedure • Simpler initial processing • Smaller sample volumes • Improved stability at room temperature • Reduced biohazard risks DBS Experimental Overview

Dried Blood Spot

Extraction, reduction, and denaturation

Proteins

Alkylate and digest (trypsin)

Peptides Tecan Freedom EVO

Add stable isotope-labeled standards

Peptides + Standards

SPE and UHPLC-MRM/MS Agilent Infinity 1290 LC Quantitative Data Agilent 6490 QQQ-MS Multiplexed DBS-MRM for 97 Proteins

16 Actin, alpha cardiac muscle 1 Beta-2-glycoprotein 1 Complement C3 Actin, aortic smooth muscle Beta-2-microglobulin Complement C4-B Afamin C4b-binding protein alpha chain Complement C5 Alpha-1-antichymotrypsin Carbonic anhydrase 1 Complement component C8 alpha chain Alpha-1-antitrypsin Carbonic anhydrase 2 Complement component C8 beta chain Alpha-2-antiplasmin Carboxypeptidase N subunit 2 Complement component C9 Alpha-2-HS-glycoprotein Cathelicidin antimicrobial peptide Complement factor B Alpha-2-macroglobulin CD5 antigen-like Complement factor H Alpha-enolase Ceruloplasmin Complement factor I Angiotensinogen Chloride intracellular channel protein 1 Coronin-1A -III Cholinesterase Fermitin family homolog 3 Apolipoprotein A-I Clusterin Fetuin-B Apolipoprotein A-IV alpha chain Apolipoprotein B-100 Coagulation factor XII Fibrinogen beta chain Apolipoprotein C-I Coagulation factor XIII A chain Apolipoprotein C-II Complement C1q subcomponent subunit A Fibronectin Apolipoprotein D Complement C1q subcomponent subunit B Filamin-A Apolipoprotein E Complement C1q subcomponent subunit C Galectin-3 Apolipoprotein L1 Complement C1r subcomponent Galectin-3-binding protein Apolipoprotein(a) Complement C1s subcomponent Gelsolin Continued Multiplexed DBS-MRM for 97 Proteins

17 Hemopexin Pigment epithelium-derived factor Heparin cofactor 2 Plasma protease C1 inhibitor Histidine triad nucleotide-binding protein 1 Protein AMBP Histidine-rich glycoprotein Protein DJ-1 Histone H4 Protein S100-A8 Hyaluronan-binding protein 2 Protein S100-A9 Ig mu chain C region Serotransferrin Ig mu heavy chain disease protein Serum albumin Insulin-like growth factor-binding protein complex acid labile subunit Serum amyloid A-4 protein Integrin alpha-IIb Serum paraoxonase/arylesterase 1 Inter-alpha-trypsin inhibitor heavy chain H1 Talin-1 Inter-alpha-trypsin inhibitor heavy chain H2 Thrombospondin-1 -1 Thyroxine-binding globulin Leucine-rich alpha-2-glycoprotein Transthyretin Lipopolysaccharide-binding protein Triosephosphate isomerase Lumican Vitamin D-binding protein Lysozyme C Vitamin K-dependent Neutrophil gelatinase-associated lipocalin Zinc-alpha-2-glycoprotein Peroxiredoxin-2

Largest DBS-MRM assay for proteins Extracted Ion Chromatograms

18

(169 peptides, 97 proteins)

Highly-multiplexed analysis (97 proteins measured in 43 min analysis) Protein Quantitation

19

Peptides Per Protein

Wide dynamic range (Almost 5 orders of magnitude) DBS-MRM Analytical Precision

20

Highly repeatable and reproducible results (n=3) Volumetric DBS Sampling - MicroSafe

21 MicroSafe DBS Collection • Fills by capillary action (Click image to play video) • Accurate volumes • Disposable • Low cost (~ $0.41 each)

SafeTec MicroSafe DBS spotting for patient sampling at home Volumetric Blood Spotting for DBS

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Device Median CV Biohit Pipette 5.0% Intra-assay CV for 169 peptides (97 proteins) Drummond Microcaps 4.7% quantified in DBS by LC-MRM-MS. (n=3) SafeTec MicroSafe 6.2% High volumetric spotting precision Stability in DBS Samples

23 (Stable peptide concentration values within 20%)

Most targets are stable in DBS samples Biomarker Assessment and Quality Control Kits for Quantitative Proteomics BAK-76 Components and Strategy

Supplied Prep. Components ∙ 2 lyophilized SIS peptide mixtures. ∙ Plasma and trypsin can be provided upon request.

Kit Contents ∙ Detailed operating procedure. ∙ MassHunter LC-MS acquisition method and worklist. ∙ MassHunter analysis method. ∙ Data analysis software – Qualis-SIS – and input file templates.

Kit Strategy ∙ Post-digestion addition of SIS mix to control and user- supplied experimental plasma samples (n = 20 or 50). Precise Protein Quant 76 Plasma Proteins (101 Interference-free Peptides)

42 CVD-linked Beta-2-glycoprotein 1 (P02749) Proteins in Panel

n = 5 process replicates (prepared and processed on different days) Application to Patient Samples 20 De-identified Plasma Samples Partner BAK with QC Kits Helps Ensure Consistent Performance Maintained

LC-MS Platform Assessment (#1) Additionally Supplied ∙ Daily Use (#1a) • SOPs and narrative video.

∙ Lyophilized SIS in pre-digested plasma.

∙ XIC inspection and performance tracking.

∙ Monthly Use (#1b)

∙ Lyophilized SIS in pre-digested plasma. • LC-MS conditions and ∙ Generate curves and assess accuracy. parameters. • Reference values. Complete Workflow Evaluation (#2) • Performance quality guide. ∙ Monthly Use • Data analysis software (Qualis-SIS). ∙ Raw plasma, trypsin, and SIS mix.

∙ Generate curves and assess accuracy. QC Performance Kit Panel 35 Proteins with 1 Peptide per Protein

Afamin (P43652) Beta-2-glycoprotein I (P02749) Haptoglobin (P00738) Alpha-1-antichymotrypsin (P01011) Ceruloplasmin (P00450) Hemopexin (P02790) Alpha-1B-glycoprotein (P04217) Clusterin (P10909) Heparin cofactor II (P05546) Alpha-2-antiplasmin (P08697) Coagulation factor XII (P00748) Inter-alpha-trypsin inhibitor heavy chain (P19827) Angiotensinogen (P01019) Complement C3 (P01024) Kininogen-1 (P01042) Antithrombin-III (P01008) Complement C4-B (P0C0L5) Plasminogen (P00747) Complement component C9 Apolipoprotein A-I (P02647) Retinol-binding protein 4 (P02753) (P02748) Apolipoprotein A-II (P02652) Complement factor B (P00751) Serum albumin (P02768) Apolipoprotein A-IV (P06727) Complement factor H (P08603) Transthyretin (P02766) Apolipoprotein B-100 (P04114) (P02671) Vitamin D-binding protein (P02774) Apolipoprotein C-I (P02654) Fibrinogen beta chain (P02675) Vitronectin (P04004) Apolipoprotein E (P02649) Gelsolin (P06396)

Concentration Range: 4 orders of magnitude Protein MW: 7 kDa (Apolipoprotein C-I) to 513 kDa (Apolipoprotein B-100) QC Kit Utility Demonstrated Intra- and Inter-laboratory Evaluation

∙ Kits can reveal procedural errors or instrument deficits.

Percy A. J. et al. J. Proteomics 95, 2013, 66. Percy A. J. et al. J. Prot. Res. 12, 2013, 222.

Example from Recent Inter-lab Study (16 Participants, 19 Platforms)

Technician Error Error-free Analysis with Kit 1b Precision/Accuracy Errors with Kit 2 Pinpointed (Site 6) Determined Conc. = 7.6 µg/mL (Ref Conc. = 7.2 µg/mL)

TGAQELLR R2 = 0.981 (Gelsolin; P06396) Summary

∙ Developed quantitative MRM assays for human proteins:

1D LC-MRM/MS 2D LC-MRM/MS Plasma CSF Urine DBS Plasma Peptides 483 311 213 169 625 Proteins 192 130 136 97 253 31 mg/mL to 118 μg/mL to 8 μg/mL to 18 mg/mL to 15 mg/mL to 450 Conc Range 19 ng/mL 550 pg/mL 25 pg/mL 190 ng/mL pg/mL

∙ Quantified proteins have putative association to NCDs. ∙ Developed kits: ∙ QC Performance Evaluation → standardization

∙ Biomarker Assessment → discovery and verification ∙ Qualis-SIS software aids rapid data analysis and results interpretation. Online MRM Assay Design

Christoph Borchers Yassene Mohammed Linking Knowledge Bases and MRM Experimental Data

Experiments Custom MRM Assay Design

∙ Browse 2500+ peptides and add to assay ∙ Search and sort based on protein name, accession number, peptide sequence or species (currently human and mouse) Peptide Tracker: a Knowledge Base for Validated MRM Peptides

∙ 2500+ peptides covering: ∙ More than 900 human proteins ∙ More than 800 mouse proteins ∙ More than 500 rat proteins ∙ More than 300 pig proteins ∙ … ∙ The peptides are mapped to reference proteome from other organisms periodically Qualis-MRM-Designer Optimizing MRM Assay LC Gradient

∙ Optimization of the LC gradient according to selected peptides ∙ Export of gradient table and predicted new retention time ∙ (The algorithm is being validated based on empirical data) Qualis-MADpipe: MRM Assay Design Pipeline

Peptide Peptide optimization Synthesis

Peptides available Picker Library Peptide Picker

Tracker Workflow input ports min_peptide_lengthmax_peptide_lengthunique_peptides no_sss only_mature_pep no_internal_R_K in_all_iso no_mc_Keil no_mc_only_P no_pp no_w no_mod organism AccNr output_file

Create_file

Workflow_control

Workflow input ports

AccNr reportTxt

Split_AccNr

test_in_file seperator_value

Remove_String_DuplicatesMerge_String_List_to_a_String

Workflow output ports

leftAccNr previouslyMeasuredleftAccNrList

Find_Candidate_Peptides_in_Protein

outputPath

outputPathTimed

UniProt_RetrieveXML

Workflow input ports

extract_features find_isoforms find_mod_pos min_peptide_lengthmax_peptide_lengthproteinAccNr unique_peptides no_sss only_mature_pep no_internal_R_K in_all_iso no_mc_Keil outputFile no_mc_only_P no_pp no_w no_mod organism

List of list_AccNr regex_value string_value Peptides UniProt_RetrieveFasta Split Peptides exctract_canseq Peptidecutter pathToHtml2xhtmlExe html2xhtml eff_value

find_peptides

merg_peptides_tables

mark_mod

intersect_peptides

mark_features

filter1 choose_organism

test_in_GPMDB proteins Workflow input ports unavailable test_unique seq species peptideTrackerTxt merge_filter_unique condition_checker unavailable testPRIDE Find_peptides No_peptides Workflow input ports Workflow input ports Workflow input ports

test_in_Tracker test_inPeptideAtlas sequence species seq species sentinel sentinel seq species

REST_Service REST_Service No_peptide_logic

Workflow output ports

JsonPath seperator_value Parse_REST_1 observedInSpeciesgpmObserved totObs

REST_Service_2 Merge_String_List_to_a_String

JsonPath_2 REST_Service_2seperator_value_2

Flatten_List Split_string_into_string_list_by_regular_expression

test_pride_logic Parse_REST_2

Workflow output ports Workflow output ports

responseBody responseBody_2 nodelist_2 out1 outputlist nodelist observedInSpeciesgpmObserved totObs

merge_information

add_pss Flatten_List Flatten_List_2 Flatten_List_3

add_ess Merge_String_List_to_a_StringMerge_String_List_to_a_String_2Merge_String_List_to_a_String_3

Workflow output ports

add_het observedInSpeciesgpmObserved totObs

extract_name filter2

protein_name separator_value add_vscore

append_report_to_file write_html_table

Workflow output ports

protein_name canseq_fasta all_criteria_features_tableall_passed_mand_tableall_sorted_html_tablevscore_sorted_html_tableall_vscore all_vscore_table all_sorted_table all_passed_mand_test_tablein_pride

Workflow output ports

protein_name canseq_fasta all_criteria_features_tableall_passed_mand_tableall_sorted_html_tablevscore_sorted_html_tableall_vscore all_vscore_table all_vscore_sorted_tablereport all_passed_mand_test_tablein_pride leftAccNr output_file concatenate_reports Peptides available

MRM Designer

Optimized assay Qualis-MASA: MRM Assay Statistical Analysis

Outliers Comparison with Time series analysis removal reference values

MADpipe Quality Control – Daily

∙ 35 heavy labeled peptides in digested plasma ∙ Qualis-QC: visualizing time traces and percentage change of responses, RT and FWHM

• Instrument passed vendor tests • But not all peptides restored previous levels (wider spread) Quality Control – Monthly

∙ Using standard curves of 35 heavy labeled peptides in digested plasma ∙ Qualis-Monitor: monitor LLOQ change and concentration of endogenous peptides over time ∙ Can also be used to compare instruments/platforms Acknowledgements

UVic-Genome BC Proteomics Centre Quantitative Proteomics Bioinformatics MRM Proteomics SIS Peptide Production Andrew Percy, PhD Yassene Mohammed, PhD Suping Zhang, PhD

Andrew Chambers, Operations Manager PhD Andrea Palmer Derek Smith

Juncong Yang Management

Gary Kruppa, PhD Funding Agencies Agilent Technologies Christine Miller Steve Fischer