Acknowledgements……..

MacCoss Lab Vagisha Sharma Mike MacCoss Don Marsh Brendan Maclean Brian Pratt Rich Johnson Kaipo Temura Jarrett Egertson Max Gelb Genn Merrihew Hoofnagle Lab Sonia Ting Andy Hoofnagle Han-Yin Yang Clark Henderson Scott Goulding Jennifer Wallace Brook Nunn Jess Becker Emma Timmons-Schiff Funding Nick Shulman NIH/NRSA Research Training Grant #T32HG00003

Outline

• Selection of candidate proteins for the multiplex analysis of DBS via targeted proteomics • The currently employed strategies for the selection of candidate peptides for targeted proteomics • An empirical refinement process for the selection of optimal peptides and their respective MS/MS transitions

Selection of Protein Targets for DBS Wellness Assay

• Acid Phosphatase • Biotinidase • FSH • Lactate Dehydrogenase (heart) • Pseudocholinesterase

• Alanine Aminotransferase • Cancer antigen 125 •  glutamyl transferase (I/V) • Lactoferrin • Pyruvate kinase

• Albumin • Cancer antigen 15-3 • Haptoglobin •  lightLight chains Chains • Renin

• Aldolase • Human epididymis protein 4 • β-hCG • Lipase • Retinol binding protein

• Alkaline Phosphatase • Carcinoembryonic antigen (CEA) • Hemoglobin A1C (HBB) • Lp(a) • Mesothelin-related peptide

• α-1-acid glycoprotein • Ceruloplasmin • Hemopexin • LP-PLA2 • Sorbital dehydrogenase

• α -1-antitrypsin (SERPINA1) • Cholinesterase • Her-2/neu • Leutinizing hormone • Thyroglobulin

• α -1-antichymotrypsin • Complement C1 (C1R/C1S)(C1R/C1S) • Human growth hormone • Lysozyme • TSH (SERPINA3)* • Complement C1 inhib. • Human placental lactogen • Myeloperoxidase • Thyroxine binding globulin • α -1-antiplasmin (SERPINF2) • Complement C1Q • IgA • Myoglobin • Tissue plasminogen activator • α -2-HS-glycoprotein (FetuinA) • Complement C3 • IgD • Osteocalcin • Transferrin • α –fetoprotein (AFP) • Complement C4 • IgE • Parathyroid hormone • Troponin T (TnT) • Amylase • Complement C5 • IgG • Phosphohexose isomerase (GPI) • Troponin I (TnI) • ACE • C-reactive Protein • IgM • Plasminogen • Trypsin • Antithrombin III (SERPINC1) • Creatine Kinase-BB • Inhibin-A • Plasminogen activator inhib. • Urokinase • Apolipoprotein A1 • Creatine Kinase-MM • Insulin • Prealbumin (transthyretin) • Vitamin D binding protein* • Apolipoprotein B • Cystatin C • IGF-1 • BNP • Vitronectin • Apolipoprotein C2* • Erythopoeitin • IGF-2 • Procalcitonin • Von Willebrand factor • Apolipoprotein C3* • Factor IX antigen • IGFBP-1 • Prolactin • Zinc- α -2-glycoprotein*glycoprotein • Apolipoprotein E* • Factor X • IGFBP-2 • Properidin Factor B (CFB) • Apolipoprotein H* • Factor XIII • Interleukin-2 receptor • Prostatic acid phosphatase • Apolipoprotein J* • Ferritin • Isocitric dehydrogenase • PSA • Aspartate Aminotransferase • Fibrinogen • κ light chains • Protein C • β -2-microglobulin • Fibronectin • Kininogen 1* • Protein S • β -Thromboglobulin Adapted from: Tables 1&2 - N. Leigh Anderson. Clin. Chem. (2010), 56, 177-185.

Selection of Protein Targets for DBS Wellness Assay

N. Leigh Anderson & Norman G. Anderson Mol Cell Proteomics (2002), 1, 845-867

Selection of Protein Targets for DBS Wellness Assay TALDO ZA2G VTDB ALDOA MDHC IgG KAD1 TTHY

CRP APOB CP CFAB C1R FIBα/β FINC HBB/A1c LDHB SYUA Adapted from: Vaisar, T. et al., J. Clin. Inves. 2007 117(3); 746–756 How Does One Select an Optimal Set of Peptides for a Targeted Proteomic Experiment?

Should one use peptides identified in previous discovery experiments?

Should one use empirical refinement of analytical standards?

Apolipoprotein B (P04114) – 516 kDa, Serum reference range (~0.5-2 g/L)

Walldius, G. et al. J Intern Med. 2006, 259; 493-519 Walldius, G. et al. Lancet, 2001, 358(9298); 2026-33 ESPPredictor Score vs. SRM Signal Intensity: Apolipoprotein B100

1 6.0E+06

0.9 5.0E+06

0.8 ESPPredictor

0.7 SRM Peak Area 4.0E+06 0.6

0.5 3.0E+06

0.4 2.0E+06

ESPPREDICTOR SCORE ESPPREDICTOR 0.3 SRM SIGNAL INTENSITY SIGNAL SRM

0.2 1.0E+06 0.1

0 0.0E+00

Mallick, P. & Aebersold, R. et al. Nature Biotechnology(2007) 25 (1): 125-131 Fusaro, V.A. & Carr, S.A. et al. Nature Biotechnology (2009) 27:190-198.

Spectrum Counts vs. SRM Signal Intensity: Apolipoprotein B100

6.0E+06 12

SRM Peak Area 5.0E+06 10 # of +2 charge state spectra in 12 independent DDA runs

4.0E+06 8

3.0E+06 6 #of Spectra #of

SRM Signal Intensity Signal SRM 2.0E+06 4

1.0E+06 2

0.0E+00 0

FIIPGLK

GVISIPR

AQIPILR

LAIPEGK

SQAIATK

NFATSNK

EIFNMAR

IGVELTGR

QSWSVCK

NIILPVYDK

SISAALEHK

INDILEHVK

LGNNPVSK LHVAGNLK

LAPGELTIIL

ESDEETQIK

QELNGNTK

TGISPLALIK

VTQEFHMK

DEPTYILNIK

LVELAHQYK

IISDYHQQFR

SPAFTDLHLR

VPQTDMTFR

LDFSSQADLR

EVYGFNPEGK

SEYQADYESLR

DFSAEYEEDGK

ENFAGEATLQR

AGHIAWTSSGK

NTFTLSYDGSLR

VELEVPQLCSFILK

LQDFSDQLSDYYEK

AASGTTGTYQEWK

AVSMPSFSILGSDVR

FNSSYLQGTNQITGR

ATFQTPDFIVPLTDLR

DAVEKPQEFTIVAFVK

NLQDLLQFIFQLIEDNIK

LPQQANDYLNSFNWER

LLLQMDSSATAYGSTVSK

VPSYTLILPSLELPVLHVPR

TQFNNNEYSQDLDAYNTK

QVLFLDTVYGNCSTHFTVK

IDFLNNYALFLSPSAQQASW…

QTVNLQLQPYSLVTTLNSDLK NFVASHIANILNSEELDIQDLK

Prakash, A. et al. J Proteome Res. 2009 8(6): 2733–2739. AQNLYQELLTQEGQASFQGLK Our Approach………….. Workup of Recombinant/Native Protein Standard

Expand, Purify, In Vitro Enrichment via & Sequence Transcription Glutathione- cDNA clone and Translation Sepharose Resin

http://dnasu.org

Gene Product of Interest GST

IEAIPQIDK + IEAIPQIDK

~25-500 femtomoles/25uL IVT < $20.00/Protein SRM Method Refinement Cycle

Insert Hypothesis Here

Build SRM Method

Peptide Refinement Criterion: . Intensity . Detectability in . Product Ion Matrix Distribution . Digestion Run SRM Method . Stability Kinetics . iRT Calibration . Precision . Accuracy

Evaluate SRM Results Round 1 nanoLC MS/MS: SRM Signal Intensity Rank

All tryptic peptides 7-25 amino acids in length Round 1 nanoLC MS/MS: Relative Product Ion Distribution

K.TTLTAFGFASADLIEIGLEGK.GR.YEDGTLSLTSTSDLQSGIIK.NK.ALVEQGFTVPEIK.T [2578, [1536,2590][674, 694] 1555] R.TGISPLALIK.G [219, 228]

Apolipoprotein B100 Peptides 241 191 Round 2 nanoLC MS/MS: iRT Calibration with 15N-APOA1

12

10 40

8 35 30 6 25 4 20 2 15

Measured Measured Retention Time 10 0 y = 0.1586x + 16.885 5

-50 0 50 100 150 Measured Retention Time R² = 0.9979 0 Relative Hydrophobicity -20 0 20 40 60 80 100 120 Relative Hydrophobicity Round 2 nanoLC MS/MS: Peptide Stability Analysis

K.DNVFDGLVR.V [4168, 4176] K.TTLTAFGFASADLIEIGLEGK.G [674, 694]

Apolipoprotein B100 Standard Peptides Standard Standard 72hrsStandard in A/S 241 72hrs in A/S 191 55 Round 3 nanoLC MS/MS: Validation of Peptides/Transitions in Matrix

iRT Calibration

Panorama Chromatogram Library

Apolipoprotein B100 Peptides 241 191 55 32 Round 4 nanoLC MS/MS: Digestion Time Course

K.ALVEQGFTVPEIK.T [2578, 2590]

Apolipoprotein B100 Peptides 241 191 55 32 19 Method Refinement Considerations

Empirical Peptide Refinement: Apolipoprotein B100 Peptides Round 1 -> peptides with non-existent or ambiguous chromatograms Round 2 -> peptides that fail to meet minimum stability requirements 241 Round 3 -> peptides that were not observed in matrix 191 Round 4 -> peptides that gave a sub-optimal digestion characteristics 55 32 19

Normalization? Calibration? APOA1 Global Internal Standard (15N Protein)1 Single Point Calibrator2 - Assign concentration with clinical immunoassay & measure in triplicate in each batch 2) Cox, B. et al. Clin. Chem. 2014, 60(3); 541-8.

1) Hoofnagle, A.N. et al. Clin. Chem. 2012, 58(4); 777-781. Scheduled SRM Method for Analysis of DBS

55 Proteins, 1406 transitions, 281 Peptides Interested in developing a similar targeted assay?

• Let the MacCoss lab help! • Services and consulting offered for development and application of targeted proteomics assays: – DIA – PRM – SRM • Website: http://services.maccosslab.org • Email: [email protected] • In Person: Jarrett Egertson or Mike MacCoss