Targeted MS quantitation assays for signal transduction protein pathways Paul Haney R&D Platform Manager Thermo Scientific Protein Research Products Rockford, IL Why Targeted Quantitative Proteomics via Mass Spec? . Measure protein abundance and protein isoforms (e.g. splice variants, PTMs) without the need for antibodies . Avoid immuno-based cross- reactivity during multiplexing. Validate relative quantitation data from discovery proteomic experiments 2 How Do You Select Peptides for Targeted MS Assays? List of Proteins Spectral library Discovery data repositories In silico prediction Pinpoint 1.2 Hypothesis Experimental List of target peptides and transitions 3 How Do You Select Peptides for Targeted MS Assays? List of Proteins Spectral library Discovery data repositories In silico prediction Pinpoint 1.2 Hypothesis Experimental Validation Assay results List of target peptides and transitions 4 Tools for Target Peptide Identification and Scheduling . Active-site probes for enzyme subclass enrichment . Rapid recombinant heavy protein expression using human cell-free extracts . Peptide retention time calibration mixture for chromatography QC and targeted method 100 80 60 acquisition scheduling Intensity 40 20 0 5 10 15 20 25 30 5 Tools for Target Peptide Identification and Scheduling . Active-site probes for enzyme subclass enrichment . Rapid recombinant heavy protein expression using human cell-free extracts • Stergachis, A. & MacCoss, M. (2011) Nature Methods (submitted) . Peptide retention time calibration mixture for 100 80 chromatography QC and targeted method 60 Intensity acquisition scheduling 40 20 0 5 10 15 20 25 30 6 How Do You Select Peptides for Targeted MS Assays? List of Proteins Discovery data Spectral library Repositories? In silico prediction? Reliable List of detectable target peptides and transitions? 7 ESPPredictor and SRM Concordance In silico prediction? Peptides ranked by ESPPredictor have very low correlation for good SRM signals 8 Number of Transcription Factor Spectra in NIST Database Spectral library Repositories? 9 In Vitro Protein Expression using Human IVT 2009 Products New Product Development SummaryDiscovery data Human vs Rabbit reticulocyte Human IVT Advantages: IVT Protein Expression . Rapid expression of full-length proteins (< 2 hr.) Human lysate . µg/ml Functional proteins with relevant post-translational modifications (phospho, glyco PTMs) Rabbit reticulocyte lysate . Ability to express pro-apoptotic or toxic proteins Luciferase . Amenable to modification of proteins through mutation and reaction supplements 10 High Throughput Transcription Factor Assay Development >100 transcription factor clones 11 IVT Expressed Transcription Factors are Pure & Full-length 12 Are These Peptides Useful for Targeted Assays in Lysate? IVT Extract Nuclear Extract c-Jun 13 Relative Abundance of Transcription Factors in Cell Lines Cell Line 14 Heavy Protein In Vitro Translation (IVT) Purification Purified Protein Expressed Protein Digestion Cells grown in standard suspension media Peptides Harvest Cells LC-MS Cell Lysate Reaction Mixture: IVT Data Analysis Vector - Stable isotope-labeled DNA amino acids - tRNA - mRNA stabilizers - RNA polymerase Expressed Protein 15 Heavy GFP Expression Timecourse Time course for GFP Expression 100 100 80 80 60 60 Concentration % Heavy (µg/mL) 40 40 20 20 0 0 0 2 4 6 Incubation time (hours) 16 Heavy Protein Expression of Phosphorylated BAD Schematic of BAD Signaling Pathway Heavy BAD Protein Expression Akt1 BAD BAD BAD P P + 14-3-3σ 14-3-3 Bcl-2 Bax BAD Cell survival Cell death 17 Heavy Protein Expression of Phosphorylated BAD 110 HSSYPAGTEDDEGmGEEPSPFr 105 807.9967 t acted o \ ya \Ju y 0 \ ea y e by ad3 3 # 0 6 6 R=47700 z=3 100 ITMS, CID, z=+3, Mono m/z=807.66321 Da, MH+=2420.97507 Da, Match Tol.=0.8 Da 95 807.6632 y₅⁺ R=47605 z=3 90 12 613.35 b₁₇²⁺ 85 904.84 10 ) 3 80 ^ 0 b₁₆²⁺ 1 ( >95% isotope ] 8 840.59 808.3307 s 75 t n R=47704 u y₆⁺ b₁₉²⁺ o c z=3 [ 70 742.37 y 996.92 t 6 i s n incorporation e 65 t n b₂₁²⁺ I y₃⁺ b₁₃⁺ 4 1119.32 1346.44 60 429.28 y₄⁺ b₁₂⁺ 55 2 y₅²⁺ 516.36 1289.59 b₁₆⁺ b₁₇⁺ 307.34 Relative Abundance Relative 1808.76 50 1679.59 0 45 500 1000 1500 2000 m/z 40 808.6644 R=47504 35 z=3 30 25 20 808.9987 R=48004 15 z=3 10 804.6613 807.3299 809.3320 804.3274 804.9958 803.6577 R=45000 805.8584 806.3579 806.9905 R=44104 R=48204 809.6719 5 R=43705 R=46704 R=33704 z=3 R=42604 R=48504 R=43104 z=? z=3 R=50604 z=3 z=3 z=? z=? z=? z=? z=3 0 803.0 803.5 804.0 804.5 805.0 805.5 806.0 806.5 807.0 807.5 808.0 808.5 809.0 809.5 810.0 m/z Light Heavy 18 Summary of IVT-SRM Results . IVT-SRM is a scalable strategy for targeted peptide selection . Empirical peptide strategy is more successful than in silico prediction • Saves money on synthetic peptides • Improves sensitivity by identifying best peptides • Identifies peptides likely to be modified in cells . Human proteins expressed using Heavy IVT system show > 90% heavy isotope incorporation . Protein phosphorylation and heterodimers known to interact with BAD protein were identified demonstrating PTM and proper folding 19 Tools for Target Peptide Identification and Scheduling . Active-site probes for enzyme subclass enrichment . Rapid recombinant protein expression using human cell-free extracts . Peptide retention time calibration mixture for chromatography QC and targeted method 100 80 60 acquisition scheduling Intensity 40 20 0 5 10 15 20 25 30 20 Peptide Retention Time Calibration Mixture # Observed Mass Hydrophobicity Sequence ( Z=2) Factor (HF) HF 1 SSAAPPPPPR* 493.77 7.57 2 GISNEGQNASIK* 613.32 15.50 3 HVLTSIGEK* 499.28 15.52 4 DIPVPKPK* 451.28 17.65 5 IGDYAGIK* 422.73 19.15 6 TASEFDSAIAQDK* 695.83 25.88 7 SAAGAFGPELSR* 586.80 25.24 8 ELGQSGVDTYLQTK* 773.89 28.37 9 GLILVGGYGTR* 558.32 32.18 10 GILFVGSGVSGGEEGAR* 801.41 34.52 11 SFANQPLEVVYSK* 745.39 34.96 12 LTILEELR* 498.80 37.30 13 NGFILDGFPR* 573.30 40.42 14 ELASGLSFPVGFK* 679.37 41.18 15 LSSEAPALFQFDLK* 787.42 46.66 21 Evaluate and Monitor System Performance Thermo Scientific LTQ Orbitrap XL Gradient: 0.25 % per minute; 300 nL per minute Column: C18 (Michrom Magic C18; 3 µm; 200 Å ; 0.075 mm x 20 cm) 0 60.73 NL: 1.13E8 741.39 Base Peak F: FTMS 100 8 + p NSI Full ms 2 3 50.41 10 11 [400.00-1600.00] 20.63 6 769.89 59.92 80 609.31 21.62 5 40.79 12 MS 31.63 796.41 68.69 492.28 691.83 neattrainersetpointper 418.73 7 493.80 centc 60 43.71 1373.51 76.7514 31.90 581.80 58.02 568.30 676.37 418.73 553.32 40 9 77.00 15.321 676.37 Relative Abundance 488.77 20 28.234 1583.38 22.92 34.40 53.15 62.99 78.29 14.25 19.95 469.32 39.88 48.06 64.56 70.64 783.42 89.39 429.09 565.80 609.80 418.73 654.98 453.35 770.38 760.89 741.88 544.32 559.30 667.36 0 20.50 60.64 NL: 1.14E8 741.39 68.55 Base Peak F: FTMS 100 609.31 59.69 493.80 + p NSI Full ms 21.68 796.41 [400.00-1600.00] 492.28 31.37 MS 80 40.69 50.17 418.73 neattrainersetpointper 691.83 769.89 57.79 73.44 centd 60 43.60 553.32 568.30 581.80 76.63 676.37 40 15.13 488.77 44.02 20 27.87 581.80 83.27 14.27 20.24 22.88 469.32 32.81 39.67 52.30 53.56 62.94 64.51 70.72 78.04 783.42 88.97 429.09 552.29 609.80 418.73 655.31 770.38 851.43 760.89 741.88 544.32 559.30 429.09 0 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Time (min) Thermo Scientific TSQ Vantage Gradient: 0.25 % per minute; 180 µL per minute Column: C18 (Thermo Hypersil Gold C18; 3 µm; 100 Å ;1.0 mm x 15 cm) 610.29 2.57E4 100 5 Base Peak 9 66.45 MS 80 28.54 772.46 point25perc 666.34 12 entrtcm15_ 1 7 3 60 5.94 660.38 40.29 10 11 2 19.823 658.35 57.48 13 40 6 8 17.23 747.42 934.52 4 70.67 717.39 37.61 46.82 Relative Abundance 25.36 704.37 20 994.48 15 6.49 665.43 1111.56 14 1.14 660.38 15.56 21.74 30.55 32.42 40.87 49.12 61.25 73.46 80.32 83.98 87.77 553.27 910.50 805.42 666.34 645.36 658.35 910.50 376.22 781.42 1078.59 561.26 522.29 0 53.81 NL: 610.29 66.42 2.54E4 100 28.67 772.46 Base Peak 666.34 MS 80 point25perc entrtcm15_ 5.94 4 60 660.38 40.22 57.32 19.88 805.42 934.52 40 747.42 17.33 70.64 717.39 37.73 46.69 704.37 20 25.23 461.24 1111.56 6.49 80.79 0.27 665.43 40.94 47.45 73.59 84.55 660.38 16.71 30.68 37.07 57.97 63.37 77.36 1004.52 89.15 553.27 717.39 666.34 388.26 805.42 660.38 934.52 659.37 781.42 547.32 546.29 553.27 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (min) 22 Retention Time as a Function of Hydrophobicity 23 Predicted Retention Time for Targeting HeLa Peptides Tryptic Digest of HeLa Protein Extract 24 Scheduling Enables Targeted Quantitation and Mutiplexing RT: 8.54 - 9.39 11.66 NL: 100 9.18 1.28E5NL: scheduled 100 9.17E4 90 90 TIC MSTIC MS 9.17 transferrin_ 80 10.19 9.20 transferrin_ 80 9.86 scheduled_scheduled_ 9.18 70 11.03 101113061 70 101113061331 60 11.63 331 9.17 9.15 60 9.20 50 8.71 8.72 11.06 9.21 9.84 409.88 8.74 11.69 50 8.69 30 9.13 Relative Abundance 10.22 12.47 12.85 9.23 40 8.72 9.15 8.75 11.48 9.10 20 8.68 9.21 12.829.07 9.09 30 8.74 8.66 8.77 11.88 9.25 Relative Abundance 8.69 10 11.00 8.79 9.04 12.87 9.13 8.55 10.468.65 8.82 8.87 8.88 8.99 9.02 9.26 9.28 9.35 20 9.23 0 11.91 10.49 11.42 12.89 9.18 NL: 8.68 9.09 9.81 100 5.58E4 10 9.25 11.94 12.96 8.30 90 10.52 TIC MS 8.25 9.79 13.29 13.54 13.59 transferrin_ 0 80 8.71 11.65 NL: unschedule d_1011130 100 70 9.24 1.01E5 10.24 70445 90 60 TIC MS 50 transferrin_ 80 unscheduled 9.8940 unschedule 70 11.06 d_1011130 30 8.77 9.12 70445 60 9.18 20 8.71 10 8.65 9.06 50 8.54 8.65 8.67 8.75 8.8311.548.83 8.89 8.94 8.98 9.05 9.09 9.16 9.19 9.28 9.30 9.36 8.71 0 10.48 12.49 40 9.24 8.6 8.7 8.8 8.9 9.0 12.849.1 9.2 9.3 Time (min) 30 11.00 11.47 12.89 13.36 20 8.77 11.91 12.55 12.91 9.83 10.18 10 8.30 10.77 11.41 12.00 13.42 8.18 9.06 9.30 9.71 13.60 0 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 Time (min) 25 PRTC Summary Thermo Scientific Pierce Peptide Retention Time Calibration Mixture (PRTC) uses: .