Table 2. Phosphorylation sites, phosphopeptides, and site-discriminating ions identified in immunoprecipitated NaV1.5 proteins from sham and TAC mouse left ventricles using MS.
| Phosphorylation site(s) | Phosphopeptide sequence | m/z (charge) | −10lgP score | b Ion | Phospho b ion | y Ion | Phospho y ion | TAC/sham ratio |
|---|---|---|---|---|---|---|---|---|
| S12 | 9-GTS(pS)FRR | 560.279 (+2) | 21.6 | b3 (+1) | (-) | y3 | (-) | 1.2 (n = 1) |
| S36 | 35-G(pS)ATSQESR | 616.279 (+2) | 32.1 | b1 | (-) | y7 | (-) | 1.3 (n = 1) |
| S42 | 35-GSATSQE(pS)REGLPEEEAPRPQLDLQASK | 887.947 (+4) | 73.1 | b7 (+1) | (-) | y19 (+2) | y21 (+2) | 1.5 ± 0.03 (n = 3) |
| S39 + S42 | 35-GSAT(pS)QE(pS)REGLPEEEAPRPQLDLQASK | 907.937 (+4) | 68.4 | b4 (+1) | (-) | y19 (+2) | y21 | 1.2 ± 0.12 (n = 2) |
| S457 (+ S459 or S460) | 452-GVDTV(pS)RSSLEMSPLAPVTNHER | 957.782 (+3) | 77.1 | b5 (+1) | b7 (−98) (+2) | y14 (+1) | (-) | 1.1 ± 0.09 (n = 10) |
| S459 + S460 | 452-GVDTVSR(pS)(pS)LEMSPLAPVTNHER | 958.117 (+3) | 41.5 | b7 (+2) | b11 (+2) | y12 (+1) | (-) | 1.2 (n = 1) |
| S460 | 459-S(pS)LEMSPLAPVTNHER | 1039.003 (+2) | 61.2 | b1 (+1) | b4 (+1) | y14 (+1) | (-) | 1.0 ± 0.05 (n = 18) |
| S483 | 481-RL(pS)SGTEDGGDDRLPK | 561.038 (+4) | 52.4 | b2 | b3 | y13 (+2) | (-) | 1.0 ± 0.03 (n = 10) |
| S484 | 482-LS(pS)GTEDGGDDR | 759.319 (+2) | 45.7 | b2 | (-) | y9 (+1) | (-) | 1.2 ± 0.07 (n = 12) |
| T486 | 484-SG(pT)EDGGDDRLPK | 628.973 (+3) | 21.9 | b1 | b5 | y8 (+2) | (-) | 1.2 (n = 1) |
| S483 + S484 | 480-KRL(pS)(pS)GTEDGGDDRLPK | 536.476 (+5) | 63 | b3 | (-) | y12 (+2) | (-) | 1.3 ± 0.04 (n = 19) |
| S497 | 482-LSSGTEDGGDDRLPK(pS)DSEDGPR | 732.845 (+4) | 60.2 | b12 (+2) | (-) | y7 (+1) | y12 | 1.3 ± 0.07 (n = 7) |
| S483 + S497 | 481-RL(pS)SGTEDGGDDRLPK(pS)DSEDGPR | 791.862 (+4) | 40.1 | b2 | b3 (−98) | y6 (+1) | y12 (+2) | 0.7 (n = 1) |
| S484 + S497 | 482-LS(pS)GTEDGGDDRLPK(pS)DSEDGPR | 1003.448 (+3) | 54.8 | b2 (+1) | b3 | y7 (+1) | y12 (+2) | 1.7 ± 0.04 (n = 2) |
| S499 | 497-SD(pS)EDGPR | 586.245 (+2) | 42 | b2 | b5 | y4 (+1) | y6 | 1.4 ± 0.17 (n = 3) |
| S510 | 505-ALNQL(pS)LTHGLSR | 573.643 (+3) | 51.8 | b4 (+1) | (-) | y7 (+1) | (-) | 0.9 ± 0.03 (n = 3) |
| S516 | 505-ALNQLSLTHGL(pS)R | 573.644 (+3) | 46.7 | b9 | (-) | y1 (+1) | (-) | 0.9 (n = 1) |
| S525 | 524-S(pS)RGSIFTFR | 489.584 (+3) | 40.7 | b1 (+1) | (-) | y8 (+2) | (-) | 0.8 ± 0.09 (n = 3) |
| S539 | 536-DQG(pS)EADFADDENSTAGESESHR | 921.701 (+3) | 70.9 | b3 (+1) | b5 (+1) | y14 (+1) | (-) | 1.5 ± 0.09 (n = 9) |
| S549 | 534-RRDQGSEADFADDEN(pS)TAGESESHR | 769.579 (+4) | 55.3 | b15 (+2) | (-) | y9 (+1) | (-) | 3.2 (n = 1) |
| S560 | 559-T(pS)LLVPWPLR | 745.921 (+2) | 30.2 | b1 | b4 | y8 (+1) | (-) | 1.0 (n = 1) |
| S571 | 569-RP(pS)TQGQPGFGTSAPGHVLNGK | 911.466 (+3) | 55.2 | b1 (+1) | b7 (+1), b7 (+2) | y19 | (-) | 0.8 ± 0.03 (n = 19) |
| S581 | 569-RPSTQGQPGFGT(pS)APGHVLNGK | 683.856 (+4) | 48.5 | b12 (+2) | (-) | y8 (+2) | (-) | 0.9 (n = 1) |
| S593 | 591-RN(pS)TVDCNGVVSLLGAGDAEATSPGSHLLR | 841.660 (+4) | 71.7 | b2 | b3 | y16 (+1) | (-) | 0.8 ± 0.05 (n = 4) |
| S664 | 662-AL(pS)AVSVLTSALEELEESHRK | 936.506 (+3) | 60.6 | b2 (+1) | b5 (+1) | y18 (+2) | (-) | 1.0 ± 0.04 (n = 28) |
| S667 | 662-ALSAV(pS)VLTSALEELEESHR | 817.416 (+3) | 64.1 | b5 (+1) | b7 (+1) | y13 (+1) | (-) | 0.9 ± 0.06 (n = 12) |
| T670 | 662-ALSAVSVL(pT)SALEELEESHRK | 702.629 (+4) | 54.3 | b7 (+1) | (-) | y12 (+2) | (-) | 1.1 ± 0.1 (n = 4) |
| S671 | 662-ALSAVSVLT(pS)ALEELEESHRK | 702.629 (+4) | 63.2 | b9 (+2) | b10 (+2) | y11 (+2) | (-) | 1.7 ± 0.17 (n = 12)*** |
| S664 + S667 | 662-AL(pS)AV(pS)VLTSALEELEESHRK | 886.771 (+3) | 58.8 | b2 | b3 | y15 (+2) | y18 | 1.1 ± 0.05 (n = 18) |
| S664 + T670 | 662-AL(pS)AVSVL(pT)SALEELEESHR | 844.072 (+3) | 59.3 | b2 (+1) | b5 (+1), b8 (+1) | y11 (+1) | (-) | 1.0 ± 0.14 (n = 3) |
| S664 + S671 | 662-AL(pS)AVSVLT(pS)ALEELEESHR | 844.072 (+3) | 58.3 | b2 (+1) | b5 (+1), b9 (+2) | y10 (+1) | (-) | 1.8 ± 0.13 (n = 9)*** |
| S667 + T670 | 662-ALSAV(pS)VL(pT)SALEELEESHRK | 722.621 (+4) | 54.1 | b5 (+1) | b8 | y12 (+2) | (-) | 1.2 ± 0.37 (n = 4) |
| S667 + S671 | 662-ALSAV(pS)VLT(pS)ALEELEESHR | 844.073 (+3) | 60.5 | b5 (+1) | b8 (+1), b9 | y10 (+1) | (-) | 1.4 ± 0.17 (n = 7)** |
| T999 | 993-KPAALA(pT)HSQLPSCIAAPR | 824.114 (+3) | 48.8 | b6 (+1) | (-) | y12 (+1) | (-) | 0.8 (n = 1) |
| S1001 | 1001-(pS)QLPSCIAAPR | 503.591 (+3) | 32.6 | (-) | b2 (+1) | y8 (+1) | (-) | 1.0 ± 0.11 (n = 4) |
| S1005 | 993-KPAALATHSQLP(pS)CIAAPRSPPPPEVEKAPPAR | 702.389 (+6) | 71.4 | b11 (+2) | (-) | y18 (+2) | y21 (+2) | 0.8 ± 0.16 (n = 2) |
| S1012 | 1012-(pS)PPPPEVEK | 759.405 (+2) | 42.3 | (-) | b1 (+1) | y8 (+1) | (-) | 0.9 ± 0.03 (n = 17) |
| S1056 and/or T1058 | 1030-FEEDKRPGQGTPGDTEPVCVPIAVAESDTDDQEEDEENSLGTEEEESSK (1 Phospho) | 1230.559 (+5) | 57.3 | b24 (+2) | (-) | y11 (+1) | (-) | 1.8 ± 0.04 (n = 5) |
| T1105 | 1097-AWSQVSET(pT)SSEAEASTSQADWQQER | 1070.132 (+3) | 64.3 | b8 (+1) | b9 | y12 (+1) | (-) | 1.1 (n = 1) |
| S1107 | 1097-AWSQVSETTS(pS)EAEASTSQADWQQER | 1070.135 (+3) | 69.9 | b10 (+1) | b15 (+1) | y14 (+1) | (-) | 1.2 (n = 1) |
| S1138 | 1136-ED(pS)YSEGSTADMTNTADLLEQIPDLGEDVKDPEDCFTEGCVR | 1312.582 (+4) | 44.7 | (-) | b3 (+1) | y23 (+2) | (-) | 1.3 ± 0.01 (n = 2) |
| T1809 | 1809-(pT)QFIEYLALSDFADALSEPLR | 903.451 (+3) | 54.8 | (-) | b1, b2 (+1) | y14 (+1) | (-) | 0.8 (n = 1) |
| S1937 | 1933-QQAG(pS)SGLSDEDAPER | 978.430 (+2) | 60.1 | b4 (+1) | (-) | y11 (+1) | (-) | 1.3 (n = 1) |
| S1964 | 1964-(pS)GPLSSSSISSTSFPPSYDSVTR | 885.752 (+3) | 50.3 | (-) | b1 (+1), b4 (+1) | y14 (+1) | (-) | 1.2 ± 0.11 (n = 3) |
| S1969 | 1964-SGPLS(pS)SSISSTSFPPSYDSVTR | 885.753 (+3) | 56.9 | b5 | b6 (+2) | y14 (+1) | (-) | 1.0 ± 0.04 (n = 3) |
| S1971 | 1963-RSGPLSSS(pS)ISSTSFPPSYDSVTR | 937.787 (+3) | 48.5 | b8 (+1) | (-) | y14 (+1) | (-) | 0.9 ± 0.09 (n = 3) |
| S1973 | 1964-SGPLSSSSI(pS)STSFPPSYDSVTR | 885.750 (+3) | 61.8 | b9 (+1) | b10 (+2) | y13 | (-) | 0.9 ± 0.02 (n = 6) |
| S1974 | 1964-SGPLSSSSIS(pS)TSFPPSYDSVTR | 885.750 (+3) | 61.8 | b10 | (-) | y12 (+1) | (-) | 0.9 ± 0.03 (n = 2) |
| S1989 | 1987-AT(pS)DNLPVR | 641.324 (+2) | 37.7 | b2 | b5 | y6 (+1) | (-) | 0.8 ± 0.07 (n = 2) |
| S2011 | 2002-SEDLADFPP(pS)PDRDR | 675.975 (+3) | 38.8 | b7 (+1) | (-) | y5 (+2) | y8 | 1.0 ± 0.05 (n = 15) |
The site-discriminating ions observed in MS/MS spectra of each annotated NaV1.5 (UniProt reference sequence K3W4N7) phosphopeptide support the assignment of the indicated phosphorylation site(s). The −10lgP scores attest to the quality of peptide identification. The manually verified charge state of unphosphorylated and phosphorylated site-discriminating b and y ions is reported in parentheses. (-) indicates that the ion was not detected. Mean ± SEM phosphopeptide abundance ratios in TAC left ventricles (n = 5) versus sham left ventricles (n = 4) mαNaVPAN-IPs were calculated from n phosphopeptide(s). **, P < 0.01; ***, P < 0.001; Mann-Whitney test. m/z, mass-to-charge ratio.