Table I. Target proteins and their signature peptides for MRM-MS assay development. Unlabeled and corresponding [13C] or [13C15N] labeled peptides were synthesized for optimization and employment of stable isotope dilution multiple reaction monitoring mass spectrometry.
| Protein | Signature Peptide | MH+ (mono) | z (Q1) | MRM Transitions |
|||
|---|---|---|---|---|---|---|---|
| Q1 | Q3 | ||||||
| AEBP1 | ILNPGEYRa | 961.51 | 2 | 481.26 | 524.25 | 621.3 | 735.34 |
| ILNPGEY[13C6]Ra | 967.53 | 2 | 484.27 | 530.27 | 627.32 | 741.36 | |
| NPFVLGANLNGGERa | 1457.75 | 2 | 729.38 | 887.43 | 1000.52 | 1099.59 | |
| NPFVLGANLNGGE[13C6]Ra | 1463.77 | 2 | 732.39 | 893.45 | 1006.54 | 1105.61 | |
| DTPVLSELPEPVVARa | 1621.88 | 2 | 811.44 | 767.44 | 880.53 | 1096.60 | |
| DTPVLSELPEPVVA[13C6]Ra | 1627.90 | 2 | 814.45 | 773.46 | 886.55 | 1102.62 | |
| VVNEECamcPTITRa | 1317.65 | 2 | 659.33 | 587.35 | 747.38 | 1119.51 | |
| VVNEECamcPTIT[13C6]Ra | 1323.67 | 2 | 662.34 | 593.37 | 753.40 | 1125.53 | |
| APR | AGLCamcQTFVYGGCamcRa,b | 1488.68 | 2 | 744.84 | 858.39 | 959.44 | 1087.50 |
| AGLCamcQTF[13C5]VYGGCamcRa,b | 1493.70 | 2 | 747.35 | 863.41 | 964.46 | 1092.52 | |
| BNP | MVQGSGCamcFGRa,b | 1098.49 | 2 | 549.74 | 683.29 | 740.31 | 868.37 |
| M[13C5]VQGSGCamcFGRa,b | 1103.51 | 2 | 552.25 | 683.29 | 740.31 | 868.37 | |
| MoxVQGSGCamcFGRa,b | 1114.48 | 2 | 557.74 | 683.29 | 740.31 | 868.37 | |
| Mox[13C5]VQGSGCamcFGRa,b | 1119.50 | 2 | 560.25 | 683.29 | 740.31 | 868.37 | |
| ISSSSGLGCamcKa,b | 995.49 | 2 | 498.24 | 708.33 | 795.37 | 882.40 | |
| ISSSSGLGCamc[13C6]Ka,b | 1001.51 | 2 | 501.26 | 714.35 | 801.39 | 888.42 | |
| CRP | ESDTSYVSLKb | 1128.50 | 2 | 564.77 | 609.36 | 696.39 | 797.44 |
| ESDTSYVSL[13C6]Kb | 1134.60 | 2 | 567.78 | 615.38 | 702.41 | 803.46 | |
| GYSIFSYATKb | 1136.60 | 2 | 568.78 | 716.36 | 829.45 | 916.48 | |
| GYSIFSYAT[13C6]Kb | 1142.60 | 2 | 571.79 | 722.38 | 835.47 | 922.50 | |
| FHL1 | AIVAGDQNVEYKa | 1306.66 | 2 | 653.84 | 952.44 | 1023.47 | 1122.54 |
| AIVAGDQNVEY[13C6]Ka | 1312.68 | 2 | 656.85 | 958.46 | 1029.49 | 1128.56 | |
| NPITGFGKa | 833.45 | 2 | 417.23 | 408.22 | 509.27 | 622.36 | |
| NPITGFG[13C6]Ka | 839.47 | 2 | 420.24 | 414.24 | 515.29 | 628.38 | |
| FCamcANTCamcVECamcRa | 1316.52 | 2 | 658.76 | 824.34 | 938.38 | 1009.42 | |
| FCamcANTCamcVECamc[13C6]Ra | 1322.54 | 2 | 661.77 | 830.36 | 944.40 | 1015.44 | |
| DCamcFTCamcSNCamcKa | 1191.42 | 2 | 596.22 | 508.22 | 668.25 | 769.30 | |
| DCamcFTCamcSNCamc[13C6]Ka | 1197.44 | 2 | 599.22 | 514.24 | 674.27 | 775.32 | |
| HRP | DTIVNELRa,b | 959.52 | 2 | 480.26 | 531.29 | 630.36 | 743.44 |
| DTIVNE[13C6]LRa,b | 965.54 | 2 | 483.27 | 537.31 | 636.38 | 749.46 | |
| IL-33 | DFWLHANNKa,b | 763.38 | 3 | 382.19 | 441.73 | 583.29 | 696.38 |
| DFW[13C6]LHANNKa,b | 767.39 | 3 | 384.20 | 444.74 | 583.29 | 702.40 | |
| DNHLALIKa,b | 923.54 | 2 | 462.27 | 480.22 | 551.26 | 664.34 | |
| DNHLA[13C6]LIKa,b | 929.56 | 2 | 465.28 | 480.22 | 551.26 | 670.36 | |
| TDPGVFIGVKa,b | 1032.58 | 2 | 516.79 | 662.42 | 719.45 | 816.50 | |
| TDPGVFIGV[13C6]Ka,b | 1038.60 | 2 | 519.80 | 668.44 | 725.47 | 822.52 | |
| LEP | INDISHTQSVSAKa,b | 1399.73 | 3 | 467.24 | 543.28 | 586.80 | 643.82 |
| INDISHTQS[13C6]VSAKa,b | 1404.75 | 3 | 468.92 | 543.28 | 589.31 | 646.33 | |
| MBP | HGFLPRa | 726.41 | 2 | 363.71 | 385.26 | 532.32 | 589.35 |
| HGF[13C6]LPRa | 732.43 | 2 | 366.72 | 391.28 | 538.34 | 595.37 | |
| YLASASTMDHARa,b | 1322.63 | 3 | 441.54 | 488.22 | 523.74 | 817.36 | |
| Y[13C6]LASASTMDHARa,b | 1328.65 | 3 | 443.55 | 488.22 | 523.74 | 817.36 | |
| MPO | IPCamcFLAGDTRa,b | 1149.58 | 2 | 575.29 | 632.34 | 779.40 | 939.44 |
| IPCamcF[13C6]LAGDTRa,b | 1155.60 | 2 | 578.30 | 638.36 | 785.42 | 945.46 | |
| IANVFTNAFRa,b | 1152.62 | 2 | 576.81 | 755.38 | 854.45 | 968.49 | |
| IANVFTNAF[13C6]Ra,b | 1158.64 | 2 | 579.82 | 761.40 | 860.47 | 974.51 | |
| MYO | LFTGHPETLEKa | 1271.68 | 3 | 424.56 | 506.26 | 579.79 | 716.38 |
| LFTGHPET[13C6]LEKa | 1277.70 | 3 | 426.57 | 509.27 | 582.80 | 722.40 | |
| MYL3 | AAPAPAPPPEPERPKa | 1524.82 | 2 | 762.91 | 626.36 | 1046.56 | 1214.65 |
| AAPAPAPPPEPERP[13C6]Ka | 1530.84 | 2 | 765.92 | 632.38 | 1032.58 | 1220.67 | |
| ALGQNPTQAEVLRa | 1396.75 | 2 | 698.88 | 715.41 | 913.51 | 1027.55 | |
| ALGQNPTQAEVL[13C6]Ra | 1402.77 | 2 | 701.89 | 721.43 | 919.53 | 1033.57 | |
| EVEFDASKa | 924.43 | 2 | 462.72 | 420.21 | 567.28 | 696.32 | |
| EVEFDAS[13C6]Ka | 930.45 | 2 | 465.73 | 426.23 | 573.3 | 702.34 | |
| HVLATLGERa | 995.56 | 2 | 498.29 | 646.35 | 759.44 | 858.50 | |
| HVLATLGE[13C6]Ra | 1001.58 | 2 | 501.30 | 652.37 | 765.46 | 864.52 | |
| NT-proBNP | MoxVLYTLRa,b | 911.51 | 2 | 456.25 | 552.31 | 665.40 | 764.47 |
| MoxVLYT[13C6]LRa,b | 917.53 | 2 | 459.26 | 558.33 | 671.42 | 770.49 | |
| MVLYTLRa,b | 895.51 | 2 | 448.26 | 552.31 | 665.40 | 764.47 | |
| MVLYT[13C6]LRa,b | 901.53 | 2 | 451.27 | 558.33 | 671.42 | 770.49 | |
| EVATEGIRa,b | 874.47 | 2 | 437.74 | 474.27 | 575.31 | 646.35 | |
| EVATEG[13C6]IRa,b | 880.49 | 2 | 440.75 | 480.29 | 581.33 | 652.37 | |
| PSA | LSEPAELTDAVKa,b | 1272.68 | 2 | 636.84 | 775.42 | 846.46 | 943.51 |
| LSEPAELTDAV[13C6]Ka,b | 1278.70 | 2 | 639.85 | 781.44 | 852.48 | 949.53 | |
| IVGGWECamcEKa,b | 1077.51 | 2 | 539.26 | 808.33 | 865.35 | 964.42 | |
| IVGGWECamcE[13C6]Ka,b | 1083.53 | 2 | 542.27 | 814.35 | 871.37 | 970.44 | |
| sCD40L | SLSLLNCamcEEIKa,b | 1305.68 | 2 | 653.34 | 792.36 | 905.44 | 1105.56 |
| SLSLLNCamcEE[13C6]IKa,b | 1311.70 | 2 | 656.35 | 798.38 | 911.46 | 1111.58 | |
| SQFEGFVKa,b | 941.48 | 2 | 471.24 | 579.31 | 726.38 | 854.44 | |
| SQFEGF[13C6]VKa,b | 946.50 | 2 | 473.75 | 584.33 | 731.40 | 859.46 | |
| TTSVLQWAEKa,b | 1162.62 | 2 | 581.81 | 661.33 | 774.41 | 960.51 | |
| TTSV[13C6]LQWAEKa,b | 1168.64 | 2 | 584.82 | 661.33 | 780.43 | 966.54 | |
| EASSQAPFIASLCamcLKa,b | 1621.83 | 2 | 811.42 | 691.38 | 1048.59 | 1119.62 | |
| EASSQAPFIASLCamc[13C6]LKa,b | 1627.85 | 2 | 814.43 | 697.40 | 1054.61 | 1125.64 | |
| Tropomyosin 1 | QLEDELVSLQKa,b | 1301.69 | 2 | 651.35 | 687.44 | 816.48 | 931.51 |
| QLEDELVSLQ[13C615N2]Ka,b | 1309.71 | 2 | 655.36 | 695.45 | 824.5 | 939.52 | |
| LVIIESDLERa,b | 1186.67 | 2 | 593.84 | 748.35 | 861.43 | 974.52 | |
| LVIIESDLE[13C615N4]Ra,b | 1196.68 | 2 | 598.84 | 758.36 | 871.44 | 984.52 | |
| Troponin I | NIDALSGMEGRa,b | 1162.56 | 2 | 581.78 | 636.28 | 749.36 | 935.43 |
| NIDA[13C6]LSGMEGRa,b | 1168.58 | 2 | 584.79 | 636.28 | 755.38 | 941.45 | |
| TLLLQIAKa,b | 899.60 | 2 | 450.30 | 459.29 | 572.38 | 685.46 | |
| TLLLQIA[13C6]Ka,b | 905.62 | 2 | 453.31 | 465.31 | 578.40 | 691.48 | |
| NITEIADLTQKa,b | 1245.68 | 2 | 623.34 | 675.37 | 788.45 | 1018.54 | |
| NITEIADLTQ[13C6]Ka,b | 1251.70 | 2 | 626.35 | 681.39 | 794.47 | 1024.56 | |
| Troponin T | VLAIDHLNEDQLRa,b | 1535.82 | 3 | 512.61 | 662.34 | 718.88 | 774.37 |
| VLAIDHLNEDQ[13C6]LRa,b | 1541.84 | 3 | 514.62 | 665.35 | 721.89 | 780.39 | |
| YEINVLRa,b | 906.50 | 2 | 453.76 | 501.31 | 614.4 | 743.44 | |
| YEINV[13C6]LRa,b | 912.52 | 2 | 456.77 | 507.33 | 620.42 | 749.46 | |
Notes: Uniformly labeled amino acids are indicated in bold. Camc, carbamidomethyl cysteines; Mox, methionine sulfoxide.
a Peptides used in the main LOD/LOQ comparison and background studies.
b Peptides used in all of the chromatography optimization experiments.