Selected reaction monitoring assays

Abstract

Provided herein are methods for developing selected reaction monitoring mass spectrometry (LC-SRM-MS) assays.

1 . A composition comprising isolated synthetic peptides said composition comprising ALQASALK (SEQ ID NO: 58), GFLLLASLR (SEQ ID NO: 1644), LGGPEAGLGEYLFER (SEQ ID NO: 596), YVSELHLTR (SEQ ID NO: 878), or YYIAASYVK (SEQ ID NO: 643). 
     
     
         2 . A composition comprising at least 10 isolated synthetic peptides said composition comprising at least 2 isolated synthetic peptides of at least 5 proteins selected from LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, KIT, GGH or GSLG1, and wherein the at least two isolated synthetic peptides are selected from ADDGRPFPQVIK (SEQ ID NO: 57), ALQASALK (SEQ ID NO: 58), QLLLTADDR (SEQ ID NO: 60), ADHHATNGVVHLIDK (SEQ ID NO: 136), DILATNGVIHYIDELLIPDSAK (SEQ ID NO: 137), LTLLAPLNSVFK (SEQ ID NO: 139), SPYQLVLQHSR (SEQ ID NO: 140), ADDILASPPR (SEQ ID NO: 331), AVGLAGTFR (SEQ ID NO: 332), TEAPSATGQASSLLGGR (SEQ ID NO: 335), GGSTSYGTGSETESPR (SEQ ID NO 554), NSLFEYQK (SEQ ID NO: 556), TVIGPDGHK (SEQ ID NO: 557), VQHIQLLQK (SEQ ID NO: 558), ALFQDIK (SEQ ID NO: 593), DDVALEGVSHFFR (SEQ ID NO: 594), KPAEDEWGK (SEQ ID NO: 595), LGGPEAGLGEYLFER (SEQ ID NO: 596), LNQALLDLHALGSAR (SEQ ID NO: 597), MGDHLTNLHR (SEQ ID NO: 598), LYGSAGPPPTGEEDTAEK (SEQ ID NO: 663), TWNDPSVQQDIK (SEQ ID NO: 664), VYEGERPLTK (SEQ ID NO: 665), IIIQESALDYR (SEQ ID NO: 666), LDPALQDK (SEQ ID NO: 667), LIAQDYK (SEQ ID NO: 668), NDINILK (SEQ ID NO: 669), VAELSSDDFHLDR (SEQ ID NO: 670), ALPGTPVASSQPR (SEQ ID NO: 839), EVPLLQSLWLAHNEIR (SEQ ID NO: 840), LPGLPEGAFR (SEQ ID NO: 841), TVAAGALASLSHLK (SEQ ID NO: 843), ASHEEVEGLVEK (SEQ ID NO: 938), STHTLDLSR (SEQ ID NO: 940), VEIFYR (SEQ ID NO: 941), YSSDYFQAPSDYR (SEQ ID NO: 942), AALSGANVLTLIEK (SEQ ID NO: 958), TVLWPNGLSLDIPAGR (SEQ ID NO: 959), VFFTDYGQIPK (SEQ ID NO: 960), ADEGISFR (SEQ ID NO: 1321), ATAVMPDGQFK (SEQ ID NO: 1322), DISLSDYK (SEQ ID NO: 1323), GLFIIDDK (SEQ ID NO: 1324), IGHPAPNFK (SEQ ID NO: 1325), LVQAFQFTDK (SEQ ID NO: 1326), QGGLGPMNIPLVSDPK (SEQ ID NO: 1327), QITVNDLPVGR (SEQ ID NO: 1328), SVDETLR (SEQ ID NO: 1329), TIAQDYGVLK (SEQ ID NO: 1330), GGTLSTPQTGSENDALYEYLR (SEQ ID NO: 1588), LDTLAQEVALLK (SEQ ID NO: 1589), NWETEITAQPDGGK (SEQ ID NO: 1590), FQDLVDAVR (SEQ ID NO: 1641), FTGSQPFGQGVEHATANK (SEQ ID NO: 1642), FVFGTIPEDILR (SEQ ID NO: 1643), GFLLLASLR (SEQ ID NO: 1644), YVSELHLTR (SEQ ID NO: 878), YYIAASYVK (SEQ ID NO: 643) or GTSQNDPNWVVR (SEQ ID NO: 1645). 
     
     
         3 . The composition of  claim 2 , wherein at least one of the isolated synthetic peptides is isotopically labeled. 
     
     
         4 . The composition of  claim 2 , wherein the measured quantity of each of the at least 2 isolated synthetic peptides of at least 5 proteins is known. 
     
     
         5 . The composition of  claim 2 , further comprising one or more polar solvents. 
     
     
         6 . The composition of  claim 2 , further comprising an additional five isolated synthetic peptides of at least one protein selected from LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, KIT, GGH or GSLG1. 
     
     
         7 . The composition of  claim 2 , further comprising an additional five isolated synthetic peptides of at least one protein selected from APOE, BASP1, CD14, FOXA2 or HSPB1. 
     
     
         8 . A composition comprising at least 25 isolated synthetic peptides said composition comprising at least five isolated synthetic peptides of at least five proteins selected from LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, GSLG1, KIT or GGH wherein the at least five isolated synthetic peptides are selected from ADDGRPFPQVIK (SEQ ID NO: 57), ALQASALK (SEQ ID NO: 58), QLLLTADDR (SEQ ID NO: 60), ADHHATNGVVHLIDK (SEQ ID NO: 136), DILATNGVIHYIDELLIPDSAK (SEQ ID NO: 137), LTLLAPLNSVFK (SEQ ID NO: 139), SPYQLVLQHSR (SEQ ID NO: 140), ADDILASPPR (SEQ ID NO: 331), AVGLAGTFR (SEQ ID NO: 332), TEAPSATGQASSLLGGR (SEQ ID NO: 335), GGSTSYGTGSETESPR (SEQ ID NO 554), NSLFEYQK (SEQ ID NO: 556), TVIGPDGHK (SEQ ID NO: 557), VQHIQLLQK (SEQ ID NO: 558), ALFQDIK (SEQ ID NO: 593), DDVALEGVSHFFR (SEQ ID NO: 594), KPAEDEWGK (SEQ ID NO: 595), LGGPEAGLGEYLFER (SEQ ID NO: 596), LNQALLDLHALGSAR (SEQ ID NO: 597), MGDHLTNLHR (SEQ ID NO: 598), LYGSAGPPPTGEEDTAEK (SEQ ID NO: 663), TWNDPSVQQDIK (SEQ ID NO: 664), VYEGERPLTK (SEQ ID NO: 665), IIIQESALDYR (SEQ ID NO: 666), LDPALQDK (SEQ ID NO: 667), LIAQDYK (SEQ ID NO: 668), NDINILK (SEQ ID NO: 669), VAELSSDDFHLDR (SEQ ID NO: 670), ALPGTPVASSQPR (SEQ ID NO: 839), EVPLLQSLWLAHNEIR (SEQ ID NO: 840), LPGLPEGAFR (SEQ ID NO: 841), TVAAGALASLSHLK (SEQ ID NO: 843), ASHEEVEGLVEK (SEQ ID NO: 938), STHTLDLSR (SEQ ID NO: 940), VEIFYR (SEQ ID NO: 941), YSSDYFQAPSDYR (SEQ ID NO: 942), AALSGANVLTLIEK (SEQ ID NO: 958), TVLWPNGLSLDIPAGR (SEQ ID NO: 959), VFFTDYGQIPK (SEQ ID NO: 960), ADEGISFR (SEQ ID NO: 1321), ATAVMPDGQFK (SEQ ID NO: 1322), DISLSDYK (SEQ ID NO: 1323), GLFIIDDK (SEQ ID NO: 1324), IGHPAPNFK (SEQ ID NO: 1325), LVQAFQFTDK (SEQ ID NO: 1326), QGGLGPMNIPLVSDPK (SEQ ID NO: 1327), QITVNDLPVGR (SEQ ID NO: 1328), SVDETLR (SEQ ID NO: 1329), TIAQDYGVLK (SEQ ID NO: 1330), GGTLSTPQTGSENDALYEYLR (SEQ ID NO: 1588), LDTLAQEVALLK (SEQ ID NO: 1589), NWETEITAQPDGGK (SEQ ID NO: 1590), FQDLVDAVR (SEQ ID NO: 1641), FTGSQPFGQGVEHATANK (SEQ ID NO: 1642), FVFGTIPEDILR (SEQ ID NO: 1643), GFLLLASLR (SEQ ID NO: 1644), YYIAASYVK (SEQ ID NO: 643), YVSELHLTR (SEQ ID NO: 878), or GTSQNDPNWVVR (SEQ ID NO: 1645). 
     
     
         9 . The composition of  claim 8 , wherein the at least five isolated synthetic peptides are selected from GFLLLASLR (SEQ ID NO: 1644), AVGLAGTFR (SEQ ID NO: 332), ALPGTPVASSQPR (SEQ ID NO: 839), LDTLAQEVALLK (SEQ ID NO: 1589), LGGPEAGLGEYLFER (SEQ ID NO: 596), TWNDPSVQQDIK (SEQ ID NO: 664), ALQASALK (SEQ ID NO: 58), LTLLAPLNSVFK (SEQ ID NO: 139), VEIFYR (SEQ ID NO: 941), TVLWPNGLSLDIPAGR (SEQ ID NO: 959), NSLFEYQK (SEQ ID NO: 556), QITVNDLPVGR (SEQ ID NO: 1328), YYIAASYVK (SEQ ID NO: 643), YVSELHLTR (SEQ ID NO: 878), and IIIQESALDYR (SEQ ID NO: 666). 
     
     
         10 . A composition comprising at least 5 transition ion pairs said composition comprising at least one transition ion pair of at least 5 proteins selected from ALDOA, TSP1, FRIL, KIT, and GGH, wherein the at least one transition ion pair consists of a precursor ion with a corresponding m/z and a fragment ion with a corresponding ion m/z, and wherein the transition ion pairs are selected from precursor ALQASALK (SEQ ID NO: 58) transition pair 401.25-260.2, 401.25-331.2, 401.25-489.3, or 401.25-617.4; precursor GFLLLASLR (SEQ ID NO: 1644) transition pair 495.31-318.2, 495.31-375.2, 495.31-446.3, or 495.31-559.4; precursor YYIAASYVK (SEQ ID NO: 643) transition pair 539.28-567.3, 539.28-638.40, 539.28-751.4, or 539.28-914.5; precursor LGGPEAGLGEYLFER (SEQ ID NO: 596) transition pair 804.41-451.2, 804.41-525.3, 804.41-564.3, or 804.41-913.4; and precursor YVSELHLTR (SEQ ID NO: 878) transition pair 373.21-263.1, 373.21-276.2, 373.21-526.3, or 373.21-639.4, wherein each of the precursor ions has a double or triple charge. 
     
     
         11 . The composition of  claim 10 , further comprising an additional five transition ion pairs comprising one transition ion pair of at least one protein selected from LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, KIT, GGH or GSLG1. 
     
     
         12 . The composition of  claim 11 , further comprising an additional five transition ion pairs comprising one transition ion pair of at least one protein selected from APOE, BASP1, CD14, FOXA2 or HSPB1, wherein the additional one transition ion pair consists of a precursor ion with a corresponding m/z and a fragment ion with a corresponding m/z, and wherein the transition ion pairs are selected from precursor AATVGSLAGQPLQER (SEQ ID NO: 82) transition pair 479.4-600.3, 479.4-642.4, 479.4-827.4, or 479.4-898.5; precursor ALMDETMK (SEQ ID NO: 83) transition pair 469.72-508.2, 469.72-469.72, 469.72-508.2, 469.72-623.3, 469.72-754.3, or 469.72-867.4; precursor GEVQAMLGQSTEELR (SEQ ID NO: 85) transition pair 824.4-616.3, 824.4-729.4, 824.4-734.4, or 824.4-919.4; precursor LAVYQAGAR (SEQ ID NO: 86) transition pair 474.77-502.3, 474.77-665.3, 474.77-764.4, or 474.77-835.4; precursor LGPLVEQGR (SEQ ID NO: 87) transition pair 484.78-588.3, 484.78-701.4, 484.78-798.4, or 484.78-855.5; precursor SELEEQLTPVAEETR (SEQ ID NO: 88) transition pair 865.93-459.2, 865.93-801.4, 865.93-902.5, or 865.93-930.4; precursor SWFEPLVEDMQR (SEQ ID NO: 89) transition pair 768.86-274.1, 768.86-421.2, 768.86-678.3, or 768.86-987.5; precursor WELALGR (SEQ ID NO: 90) transition pair 422.74-316.1, 422.74-345.2, 422.74-529.3, or 422.74-658.4; precursor AAEAAAAPAESAAPAAGEEPSK (SEQ ID NO: 118) transition pair 983.97-414.2, 983.97-485.2, 983.97-556.3, or 983.97-885.4; precursor AEGAATEEEGTPK (SEQ ID NO: 119) transition pair 645.3-258.1, 645.3-789.4, 645.3-890.4, or 645.3-961.4; precursor APEQEQAAPGPAAGGEAPK (SEQ ID NO: 120) transition pair 592.62-629.3, 592.62-754.3, 592.62-825.4, or 592.62-951.5; precursor EKPDQDAEGK (SEQ ID NO: 122) transition pair 558.76-404.2, 558.76-647.3, 558.76-859.4, or 558.76-913.4; precursor ESEPQAAAEPAEAK (SEQ ID NO: 123) transition pair 714.34-515.3, 714.34-644.3, 714.34-786.4, or 714.34-857.4; precursor ETPAATEAPSSTPK (SEQ ID NO: 124) transition pair 693.84-616.3, 693.84-816.4, 693.84-917.5, or 693.84-988.5; precursor GYNVNDEK (SEQ ID NO: 125) transition pair 469.71-276.2, 469.71-505.2, 469.71-604.3, or 469.71-718.3; precursor SDGAPASDSKPGSSEAAPSSK (SEQ ID NO: 126) transition pair 644.96-260.1, 644.96-331.1, 644.96-418.2, or 644.96-586.2; precursor TEAPAAPAAQETK (SEQ ID NO: 127) transition pair 642.83-541.3, 642.83-744.4, 642.83-815.4, or 642.83-983.5; precursor AFPALTSLDLSDNPGLGER (SEQ ID NO: 206) transition pair 987-500.3, 987-628.3, 987-742.4, or 987-944.4; precursor ATVNPSAPR (SEQ ID NO: 207) transition pair 456.75-386.2, 456.75-430.2, 456.75-527.3, or 456.75-740.4; precursor ELTLEDLK (SEQ ID NO: 208) transition pair 480.77-260.2, 480.77-504.3, 480.77-617.4, or 480.77-718.4; precursor FPAIQNLALR (SEQ ID NO: 209) transition pair 571.84-714.4, 571.84-827.5, 571.84-898.5, or 571.84-995.6; precursor LTVGAAQVPAQLLVGALR (SEQ ID NO: 211) transition pair 593.03-416.3, 593.03-515.3, 593.03-628.4, or 593.03-740.4; precursor NVSWATGR (SEQ ID NO: 212) transition pair 445.73-301.2, 445.73-404.2, 5, 445.73-590.3, or 445.73-677.3; precursor STLSVGVSGTLVLLQGAR (SEQ ID NO: 213) transition pair 879.51-431.2, 879.51-544.3, 879.51-657.4, or 879.51-970.6; precursor SWLAELQQWLKPGLK (SEQ ID NO: 214) transition pair 599.67-274.1, 599.67-414.3, 599.67-542.4, or 599.67-841.5; precursor VDADADPR (SEQ ID NO: 215) transition pair 429.7-272.2, 429.7-573.3, 429.7-644.3, or 429.7-759.3; precursor AYEQVMHYPGYGSPMPGSLAMGPVTNK (SEQ ID NO: 577) transition pair 961.45-558.3, 961.45-615.3, 961.45-817.4, or 961.45-930.5; precursor EAAGAAGSGK (SEQ ID NO: 578) transition pair 409.7-348.2, 409.7-490.3, 409.7-547.3, or 409.7-618.3; precursor MHSASSMLGAVK (SEQ ID NO: 579) transition pair 406.87-374.2, 406.87-487.3, 406.87-601.2, or 406.87-732.3; precursor TGLDASPLAADTSYYQGVYSRPIMNSS (SEQ ID NO: 582) transition pair 955.45-387.2, 955.45-545.3, 955.45-648.3, or 955.45-826.4; precursor AQLGGPEAAK (SEQ ID NO: 731) transition pair 471.26-313.2, 471.26-427.2, 471.26-629.3, or 471.26-742.4; precursor DGVVEITGK (SEQ ID NO: 732) transition pair 459.25-305.2, 459.25-418.3, 459.25-547.3, or 459.25-646.4; precursor GPSWDPFR (SEQ ID NO: 733) transition pair 481.23-419.2, 481.23-534.3, 481.23-720.3, or 481.23-807.4; precursor LATQSNEITIPVTFESR (SEQ ID NO: 734) transition pair 953.5-639.3, 953.5-744.4, 953.5-835.4, or 953.5-857.4; precursor LFDQAFGLPR (SEQ ID NO: 735) transition pair 582.31-272.2; 582.31-442.3, 582.31-660.4, or 582.31-903.5; precursor QDEHGYISR (SEQ ID NO: 736) transition pair 368.84-262.2, 368.84-538.3, 368.84-595.3, 368.84-730.3, 368.84-504.3, 368.84-660.4, 368.84-747.4, or 368.84-834.4; precursor VPFSLLR (SEQ ID NO: 738) transition pair 416.26-488.3, 416.26-488.3, 416.26-635.4, or 416.26-732.4; or precursor VSLDVNHFAPDELTVK (SEQ ID NO: 739) transition pair 595.31-347.2, 595.31-460.3, 595.31-801.4, or 595.31-872.5, wherein each of the precursor ions has a double or triple charge. 
     
     
         13 . The composition of  claim 11 , wherein the at least five transition ion pairs are selected from precursor GFLLLASLR (SEQ ID NO: 1644) transition pair 495.31-559.40, precursor LGGPEAGLGEYLFER (SEQ ID NO: 596) transition pair 804.40-913.40, precursor ALQASALK (SEQ ID NO: 58) transition pair 401.25-617.40, YYIAASYVK (SEQ ID NO: 643) transition pair 539.28-638.40, precursor LGGPEAGLGEYLFER (SEQ ID NO: 596) transition pair 804.41-451.2, 804.41-525.3, 804.41-564.3, 804.41-913.4, and precursor YVSELHLTR (SEQ ID NO: 878) transition pair 373.21-263.1, 373.21-276.2, 373.21-526.3, 373.21-639.4. 
     
     
         14 . A method comprising analyzing the composition of  claim 8  using mass spectrometry. 
     
     
         15 . The method of  claim 14 , wherein the method uses selected reaction monitoring mass spectrometry. 
     
     
         16 . A method comprising analyzing the composition of  claim 8  using Enzyme-Linked Immunosorbent Assay (ELISA). 
     
     
         17 . A multiplexed LC-SRM-MS assay for the measurement of a plurality of proteins in a single sample comprising:
 a) generating a set of peptides and corresponding transitions for each protein to be monitored;   b) optimizing the collision energy for each transition such that interference among the transitions is avoided;   c) selecting a set of transitions that have the greatest peak areas for each protein, and wherein the selected transitions do not interfere with the ions in the sample;   d) measuring a plurality of proteins in a sample.   
     
     
         18 . The assay according to  claim 17  wherein the two best peptides per protein and the two best transitions per peptide are selected based on experimental data resulting from LC-SRM-MS analysis of one or more of the following experimental samples: a biological disease sample, a biological control sample, and a mixture of synthetic peptides. 
     
     
         19 . The assay according to  claim 18  wherein the biological disease and biological control samples are processed using an immunodepletion method prior to LC-SRM-MS analysis. 
     
     
         20 . The assay according to  claim 19 , wherein the experimental samples contain internal standard peptides. 
     
     
         21 . The assay according to  claim 19 , wherein the LC-SRM-MS analysis method specifies a maximum of 7000 transitions, including transitions of the internal standard peptides and transitions. 
     
     
         22 . The Assay according to  claim 17 , wherein the top two transitions per peptide are selected according to one or more of the following criteria:
 (1) the transitions exhibit the largest peak areas measured in either of the two biological samples;   (2) the transitions are not interfered with by other ions;   (3) the transitions do not exhibit an elution profile that visually differs from those of other transitions of the same peptide;   (4) the transitions are not beyond the detection limit of both of the two biological experimental samples; and   (5) the transitions do not exhibit interferences.   
     
     
         23 . The assay according to  claim 17 , wherein the top two peptides per protein are selected according to one or more of the following criteria:
 (1) one or more peptides exhibit two transitions according to  claim 20  and represent the largest combined peak areas of the two transitions according to  claim 20 ; and   (2) one or more peptides exhibit one transition according to  claim 20  and represent the largest combined peak areas of the two transitions according to  claim 20 .   
     
     
         24 . An assay developed according to  claim 17 .