Acknowledgements…….. Vagisha Sharma MacCoss Lab Don Marsh Mike MacCoss Brian Pratt Brendan Maclean Kaipo Temura Rich Johnson Max Gelb Jarrett Egertson Hoofnagle Lab Genn Merrihew 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
Sele lection of Protein Targets for DBS Welln llness Assay • • • • • • • • • • Acid Phosphatase Acid Phosphatase Biotinidase Biotinidase FSH FSH Lactate Dehydrogenase (heart) Lactate Dehydrogenase (heart) Pseudocholinesterase Pseudocholinesterase glutamyl transferase (I/V) glutamyl transferase (I/V) • • • • • • • • • • Alanine Aminotransferase Alanine Aminotransferase Cancer antigen 125 Cancer antigen 125 Lactoferrin Lactoferrin Pyruvate kinase Pyruvate kinase light chains Light Chains • • • • • • • • • • Albumin Albumin Cancer antigen 15-3 Cancer antigen 15-3 Haptoglobin Haptoglobin Renin Renin • • • • • • β -hCG β -hCG • • • • Aldolase Aldolase Human epididymis protein 4 Human epididymis protein 4 Lipase Lipase Retinol binding protein Retinol binding protein • • • • • • • • • • Alkaline Phosphatase Alkaline Phosphatase Carcinoembryonic antigen (CEA) Carcinoembryonic antigen (CEA) Hemoglobin A1C (HBB) Hemoglobin A1C (HBB) Lp(a) Lp(a) Mesothelin-related peptide Mesothelin-related peptide • • • • • • • • • • α -1-acid glycoprotein α -1-acid glycoprotein Ceruloplasmin Ceruloplasmin Hemopexin Hemopexin LP-PLA2 LP-PLA2 Sorbital dehydrogenase Sorbital dehydrogenase • • • • • • • • • • α -1-antitrypsin (SERPINA1) α -1-antitrypsin (SERPINA1) Cholinesterase Cholinesterase Her-2/neu Her-2/neu Leutinizing hormone Leutinizing hormone Thyroglobulin Thyroglobulin • • • • • • • • • • α -1-antichymotrypsin α -1-antichymotrypsin Complement C1 (C1R/C1S) Complement C1 (C1R/C1S) Human growth hormone Human growth hormone Lysozyme Lysozyme TSH TSH (SERPINA3)* (SERPINA3)* • • • • • • • • Complement C1 inhib. Complement C1 inhib. Human placental lactogen Human placental lactogen Myeloperoxidase Myeloperoxidase Thyroxine binding globulin Thyroxine binding globulin • • α -1-antiplasmin (SERPINF2) α -1-antiplasmin (SERPINF2) • • • • • • • • Complement C1Q Complement C1Q IgA IgA Myoglobin Myoglobin Tissue plasminogen activator Tissue plasminogen activator • • α -2-HS-glycoprotein (FetuinA) α -2-HS-glycoprotein (FetuinA) • • • • • • • • Complement C3 Complement C3 IgD IgD Osteocalcin Osteocalcin Transferrin Transferrin • • α – fetoprotein (AFP) α – fetoprotein (AFP) • • • • • • • • Complement C4 Complement C4 IgE IgE Parathyroid hormone Parathyroid hormone Troponin T (TnT) Troponin T (TnT) • • Amylase Amylase • • • • • • • • Complement C5 Complement C5 IgG IgG Phosphohexose isomerase (GPI) Phosphohexose isomerase (GPI) Troponin I (TnI) Troponin I (TnI) • • ACE ACE • • • • • • • • C-reactive Protein C-reactive Protein IgM IgM Plasminogen Plasminogen Trypsin Trypsin • • Antithrombin III (SERPINC1) Antithrombin III (SERPINC1) • • • • • • • • Creatine Kinase-BB Creatine Kinase-BB Inhibin-A Inhibin-A Plasminogen activator inhib. Plasminogen activator inhib. Urokinase Urokinase • • Apolipoprotein A1 Apolipoprotein A1 • • • • • • • • Creatine Kinase-MM Creatine Kinase-MM Insulin Insulin Prealbumin (transthyretin) Prealbumin (transthyretin) Vitamin D binding protein* Vitamin D binding protein* • • Apolipoprotein B Apolipoprotein B • • • • • • • • Cystatin C Cystatin C IGF-1 IGF-1 BNP BNP Vitronectin Vitronectin • • Apolipoprotein C2* Apolipoprotein C2* • • • • • • • • Erythopoeitin Erythopoeitin IGF-2 IGF-2 Procalcitonin Procalcitonin Von Willebrand factor Von Willebrand factor • • Apolipoprotein C3* Apolipoprotein C3* • • • • • • • • Factor IX antigen IGFBP-1 IGFBP-1 Prolactin Zinc- α -2-glycoprotein* Factor IX antigen Prolactin Zinc- α -2-glycoprotein • • Apolipoprotein E* Apolipoprotein E* • • • • • • Factor X Factor X IGFBP-2 IGFBP-2 Properidin Factor B (CFB) Properidin Factor B (CFB) • • Apolipoprotein H* Apolipoprotein H* • • • • • • Factor XIII Factor XIII Interleukin-2 receptor Interleukin-2 receptor Prostatic acid phosphatase Prostatic acid phosphatase • • Apolipoprotein J* Apolipoprotein J* • • • • • • Ferritin Ferritin Isocitric dehydrogenase Isocitric dehydrogenase PSA PSA • • Aspartate Aminotransferase Aspartate Aminotransferase κ light chains κ light chains • • • • • • Fibrinogen Fibrinogen Protein C Protein C • • β -2-microglobulin β -2-microglobulin • • • • • • Fibronectin Fibronectin Kininogen 1* Kininogen 1* Protein S Protein S • • β -Thromboglobulin β -Thromboglobulin Adapted from: Tables 1&2 - N. Leigh Anderson. Clin. Chem. (2010), 56, 177-185.
Selection of Protein in Targets for DBS Well llness Assay N. Leigh Anderson & Norman G. Anderson Mol Cell Proteomics ( 2002), 1, 845-867
Sele lection of Protein Targets for DBS Welln llness Assay TALDO ZA2G VTDB ALDOA MDHC IgG KAD1 TTHY CRP APOB CP CFAB C1R FIB α / 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 SRM SIGNAL INTENSITY SRM Peak Area ESPPREDICTOR SCORE 0.7 4.0E+06 0.6 0.5 3.0E+06 0.4 2.0E+06 0.3 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.
Prakash, A. et al. J Proteome Res. 2009 8(6): 2733 – 2739. SRM Signal Intensity Spectrum Counts vs. SRM Signal Intensity: Apolipoprotein B100 0.0E+00 1.0E+06 2.0E+06 3.0E+06 4.0E+06 5.0E+06 6.0E+06 TGISPLALIK FIIPGLK AQIPILR LAIPEGK NIILPVYDK ATFQTPDFIVPLTDLR EIFNMAR IGVELTGR GVISIPR EVYGFNPEGK ENFAGEATLQR SPAFTDLHLR VELEVPQLCSFILK LDFSSQADLR LVELAHQYK AVSMPSFSILGSDVR independent DDA runs # of +2 charge state spectra in 12 SRM Peak Area SEYQADYESLR QSWSVCK LGNNPVSK VPQTDMTFR LHVAGNLK NFATSNK SQAIATK IISDYHQQFR SISAALEHK VTQEFHMK LQDFSDQLSDYYEK ESDEETQIK LPQQANDYLNSFNWER AGHIAWTSSGK DAVEKPQEFTIVAFVK AASGTTGTYQEWK TQFNNNEYSQDLDAYNTK LAPGELTIIL NTFTLSYDGSLR QTVNLQLQPYSLVTTLNSDLK LLLQMDSSATAYGSTVSK DEPTYILNIK AQNLYQELLTQEGQASFQGLK FNSSYLQGTNQITGR INDILEHVK DFSAEYEEDGK VPSYTLILPSLELPVLHVPR QELNGNTK NLQDLLQFIFQLIEDNIK NFVASHIANILNSEELDIQDLK QVLFLDTVYGNCSTHFTVK IDFLNNYALFLSPSAQQASW… 0 2 4 6 8 10 12 #of Spectra
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 R.YEDGTLSLTSTSDLQSGIIK.N [1536, 1555] K.TTLTAFGFASADLIEIGLEGK.G [674, 694] K.ALVEQGFTVPEIK.T [2578, 2590] R.TGISPLALIK.G [219, 228] Apolipoprotein B100 Peptides 241 191
Round 2 nanoLC MS/MS : iRT Calibration with 15 N-APOA1 12 Measured Retention Time 10 40 Measured Retention Time 35 8 30 6 25 4 20 15 2 10 0 5 y = 0.1586x + 16.885 -50 0 50 100 150 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 Standard Peptides Standard Standard 72hrs in A/S 241 72hrs in A/S 191 55
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