Structural and functional insights into S-thiolation of

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1543.5523. 1. 28 D.CCAKQEPERNE.C + 2 Cysteinyl (C90, C91). 101 -. 119. 2410.1028. 2409.0955. 2409.1682. 2. 32 E.CFLQHKDDNPNLPRLVRPE.
Structural and functional insights into S-thiolation of human serum albumins Fumie Nakashima1, Takahiro Shibata1, 2, Kohei Kamiya1, Jun Yoshitake3, Ryosuke Kikuchi4, Tadashi Matsushita5, Isao Ishii6, Juan A. Giménez-Bastida7, Claus Schneider7 and Koji Uchida1, 8, * 1

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan 3 Institute for Innovation for Future Society, Nagoya University, Nagoya 464-8601, Japan 4 Department of Medical Technique, Nagoya University Hospital, Nagoya 466-8560, Japan. 5 Department of Clinical Laboratory and Blood Transfusion, Nagoya University Hospital, Nagoya 466-8560, Japan. 6 Department of Health Chemistry, Showa Pharmaceutical University, Tokyo 194-8543, Japan 7 Department of Pharmacology and Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232, U.S.A 8 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 1138657, Japan 2

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Supplemental Information Table of Contents Supplemental Method MALDI-TOF/TOF MS analysis for protein identification. Supplemental Tables (Table S1-S5) 1.

Identification of peak1 protein by MALDI-TOF/TOF MS analysis.

2.

Identification of peak2 protein by MALDI-TOF/TOF MS analysis.

3.

Masses of S-cysteinylated peptides detedcted with MALDI-TOF/TOF MS.

4.

Masses of S-homocysteinylated peptides detedcted with MALDI-TOF/TOF MS.

5.

Masses of cyseine-containing peptides detected with MALDI-TOF/TOF MS.

Supplemental Figures (Figure S1-S9) 1. Analysis of serum protein from hyperlipidemia patients. 2. Identification of peak 1 protein by MALDI-TOF/TOF MS analysis. 3. Identification of peak 2 protein by MALDI-TOF/TOF MS analysis. 4. Quantification of HSA-bound glutathione. 5. Mass spectra of S-cysteinylated peptides detedcted with MALDI-TOF/TOF MS. 6. Mass spectra of S-homocysteinylated peptides detedcted with MALDI-TOF/TOF MS. 7. Quantification of serum total thiols from normal subjects and hyperlipidemia patients. 8. Relationship between serum total and HSA-bound thiol. 9. HPLC chromatograph of modified rHSAs.

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Supplemental Method MALDI-TOF/TOF MS analysis for protein identification. After reduction and alkylation of the cysteine residues with acrylamide, protein was separated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) under reducing conditions. The separated protein band was processed for tryptic digestion using sequence grade modified trypsin (Promega) in 50 mM NH4HCO3 buffer overnight at 37 °C. The recovered peptides were then resolved by DiNa Nano-flow LC system (KYA Technologies Corporation), then directly fractionated onto a MALDI target plate with matrix 4-CHCA. The MALDI plates were analyzed using a Triple TOF 5800 System (AB SCIEX), a hybrid triple quadrupole time-of-flight mass spectrometer equipped with an ESI source, and the mass range was set at m/z 800-4000.

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Supplemental Tables (Table S1-S5) Supplemental Table 1. Identification of peak1 protein by MALDI-TOF/TOF MS analysis. Masses of tryptic peptides of peak 1 detected with MALDI-TOF/TOF MS. Position 1 5 11 13 21 42 52 65 65 74 82 82 94 94 99 99 99 99 99 107 115 115 115 115 115 137 138 145 146 163 182 182 200 213 234 241 241 241 263 263 275 287 287 287 318 318 324 324 337 338 352 360 360 360 373 390 390 390 390 390 403 414 415 415 437 445 467 476 476 485 485 501 501 501 501 525 526 546 546

-

10 10 20 20 41 51 64 81 81 81 93 93 106 106 106 114 114 114 114 114 136 136 137 137 137 144 144 159 159 174 186 190 205 218 240 257 262 262 274 274 286 313 313 313 323 336 336 336 348 348 359 372 372 372 389 402 402 410 410 410 410 428 428 428 444 466 475 484 484 500 500 519 519 521 521 534 534 557 557

Observed 1149.5907 698.3707 1226.5764 951.423 2504.3352 1149.6576 1526.5875 1874.9493 1945.9956 947.5121 1462.5458 1478.5504 1657.7419 1728.7906 1089.4939 1939.8612 1940.8407 2010.8983 2012.0114 940.44 2593.1797 2664.2209 2721.2783 2792.2959 2808.3008 1055.5667 927.475 1899.0016 1742.9054 1399.5779 645.3514 1074.535 720.3697 673.3677 789.4512 2128.8262 2556.072 2627.125 1386.5608 1386.5917 1574.7875 2988.375 2989.4785 3004.3772 695.3401 2300.1023 1623.8113 1639.7744 1467.8123 1311.7167 984.4741 1452.5543 1523.5923 1594.6331 2045.0439 1600.6633 1671.7059 2542.2466 2613.2869 2614.4141 960.5303 1639.91 1511.7891 1511.9017 986.4459 2702.3389 1002.5547 1095.4795 1166.5199 1853.8702 1924.8923 2273.9648 2275.1055 2489.1816 2559.1133 1128.6528 1000.5637 1342.6814 1358.5752

Mr(expt) 1148.5834 697.3634 1225.5691 950.4157 2503.3279 1148.6503 1525.5802 1873.942 1944.9883 946.5048 1461.5385 1477.5431 1656.7346 1727.7833 1088.4866 1938.8539 1939.8334 2009.891 2011.0041 939.4327 2592.1724 2663.2136 2720.271 2791.2886 2807.2935 1054.5594 926.4678 1897.9943 1741.8981 1398.5706 644.3441 1073.5277 719.3624 672.3605 788.444 2127.8189 2555.0647 2626.1177 1385.5535 1385.5844 1573.7802 2987.3677 2988.4712 3003.3699 694.3329 2299.095 1622.804 1638.7671 1466.805 1310.7094 983.4668 1451.547 1522.585 1593.6258 2044.0366 1599.656 1670.6986 2541.2393 2612.2796 2613.4068 959.523 1638.9027 1510.7818 1510.8944 985.4386 2701.3316 1001.5474 1094.4722 1165.5126 1852.8629 1923.885 2272.9575 2274.0982 2488.1743 2558.106 1127.6455 999.5564 1341.6741 1357.5679

Mr(calc) 1148.5686 697.3507 1225.5979 950.4345 2503.2933 1148.6077 1525.6024 1874.0084 1945.0455 946.527 1461.5574 1477.5523 1656.7678 1727.8049 1088.5073 1938.9006 1939.8846 2009.9377 2010.9217 939.441 2592.2352 2663.2724 2720.3302 2791.3673 2807.3622 1054.5811 926.4861 1897.9879 1741.8868 1398.5908 644.3493 1073.5353 719.3636 672.3707 788.4644 2127.8772 2555.1203 2626.1574 1385.6133 1385.6133 1573.8207 2987.3528 2988.3368 3003.3477 694.3286 2299.0983 1622.7803 1638.7752 1466.8358 1310.7347 983.4811 1451.5632 1522.6003 1593.6374 2044.0881 1599.7239 1670.761 2541.2686 2612.3057 2613.2897 959.5552 1638.9305 1510.8355 1510.8355 985.4473 2701.339 1001.5506 1094.4848 1165.522 1852.9029 1923.94 2273.031 2274.015 2488.1216 2558.1747 1127.6914 999.5964 1341.6275 1357.6224

Missed Cleavage 1 0 1 0 0 0 0 1 1 0 0 0 1 1 0 1 1 1 1 0 0 0 1 1 1 1 0 1 0 0 0 1 0 0 0 0 1 1 0 0 1 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 1 1 1 0 1 0 0 1 1 1 0 0 0 0 0 0 1 1 1 0 0 0

Score 79 31 66 60 108 85 111 74 138 53 102 110 66 83 43 60 53 121 47 59 55 71 88 110 59 70 49 79 112 78 34 57 37 40 53 84 26 63 29 70 46 220 86 120 40 148 108 67 76 90 54 61 65 79 131 48 97 125 149 110 63 122 128 106 43 65 27 51 59 68 86 136 28 48 177 94 71 84 103

Peptide DAHKSEVAHR.F K.SEVAHR.F R.FKDLGEENFK.A K.DLGEENFK.A K.ALVLIAFAQYLQQCPFEDHVK.L + Propionamide (C) K.LVNEVTEFAK.T K.TCVADESAENCDK.S + 2 Propionamide (C) K.SLHTLFGDKLCTVATLR.E K.SLHTLFGDKLCTVATLR.E + Propionamide (C) K.LCTVATLR.E + Propionamide (C) R.ETYGEMADCCAK.Q + 2 Propionamide (C) R.ETYGEMADCCAK.Q + Oxidation (M); 2 Propionamide (C) K.QEPERNECFLQHK.D K.QEPERNECFLQHK.D + Propionamide (C) R.NECFLQHK.D + Propionamide (C) R.NECFLQHKDDNPNLPR.L R.NECFLQHKDDNPNLPR.L + Deamidated (NQ) R.NECFLQHKDDNPNLPR.L + Propionamide (C) R.NECFLQHKDDNPNLPR.L + Deamidated (NQ); Propionamide (C) K.DDNPNLPR.L R.LVRPEVDVMCTAFHDNEETFLK.K R.LVRPEVDVMCTAFHDNEETFLK.K + Propionamide (C) R.LVRPEVDVMCTAFHDNEETFLKK.Y R.LVRPEVDVMCTAFHDNEETFLKK.Y + Propionamide (C) R.LVRPEVDVMCTAFHDNEETFLKK.Y + Oxidation (M); Propionamide (C) K.KYLYEIAR.R K.YLYEIAR.R R.RHPYFYAPELLFFAK.R R.HPYFYAPELLFFAK.R K.AAFTECCQAADK.A + 2 Propionamide (C) K.LDELR.D K.LDELRDEGK.A K.CASLQK.F + Propionamide (C) K.AWAVAR.L K.LVTDLTK.V K.VHTECCHGDLLECADDR.A + 3 Propionamide (C) K.VHTECCHGDLLECADDRADLAK.Y + 2 Propionamide (C) K.VHTECCHGDLLECADDRADLAK.Y + 3 Propionamide (C) K.YICENQDSISSK.L K.YICENQDSISSK.L K.LKECCEKPLLEK.S + 2 Propionamide (C) K.SHCIAEVENDEMPADLPSLAADFVESK.D + Propionamide (C) K.SHCIAEVENDEMPADLPSLAADFVESK.D + Deamidated (NQ); Propionamide (C) K.SHCIAEVENDEMPADLPSLAADFVESK.D + Oxidation (M); Propionamide (C) K.NYAEAK.D K.NYAEAKDVFLGMFLYEYAR.R K.DVFLGMFLYEYAR.R K.DVFLGMFLYEYAR.R + Oxidation (M) R.RHPDYSVVLLLR.L R.HPDYSVVLLLR.L K.TYETTLEK.C K.CCAAADPHECYAK.V + Propionamide (C) K.CCAAADPHECYAK.V + 2 Propionamide (C) K.CCAAADPHECYAK.V + 3 Propionamide (C) K.VFDEFKPLVEEPQNLIK.Q K.QNCELFEQLGEYK.F K.QNCELFEQLGEYK.F + Propionamide (C) K.QNCELFEQLGEYKFQNALLVR.Y K.QNCELFEQLGEYKFQNALLVR.Y + Propionamide (C) K.QNCELFEQLGEYKFQNALLVR.Y + Deamidated (NQ); Propionamide (C) K.FQNALLVR.Y K.KVPQVSTPTLVEVSR.N K.VPQVSTPTLVEVSR.N K.VPQVSTPTLVEVSR.N K.CCKHPEAK.R + Propionamide (C) K.RMPCAEDYLSVVLNQLCVLHEK.T + 2 Propionamide (C) K.TPVSDRVTK.C K.CCTESLVNR.R + Propionamide (C) K.CCTESLVNR.R + 2 Propionamide (C) R.RPCFSALEVDETYVPK.E R.RPCFSALEVDETYVPK.E + Propionamide (C) K.EFNAETFTFHADICTLSEK.E + Propionamide (C) K.EFNAETFTFHADICTLSEK.E + Deamidated (NQ); Propionamide (C) K.EFNAETFTFHADICTLSEKER.Q + Deamidated (NQ) K.EFNAETFTFHADICTLSEKER.Q + Propionamide (C) K.KQTALVELVK.H K.QTALVELVK.H K.AVMDDFAAFVEK.C K.AVMDDFAAFVEK.C + Oxidation (M)

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Supplemental Table 2. Identification of peak2 protein by MALDI-TOF/TOF MS analysis. Masses of tryptic peptides of peak 2 detected with MALDI-TOF/TOF MS. Position 1 5 11 13 21 21 21 42 65 65 65 74 82 82 82 94 94 94 99 99 99 99 99 107 115 115 115 115 115 137 138 145 146 163 175 182 200 213 219 234 241 241 241 263 263 275 287 287 318 318 318 324 324 337 338 352 360 360 373 390 390 390 390 390 390 403 414 415 445 467 476 476 476 485 485 501 501 501 501 501 501 525 526 546 546 546

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

10 10 20 20 41 41 41 51 73 81 81 81 93 93 93 106 106 106 106 114 114 114 114 114 136 136 137 137 137 144 144 159 159 174 181 190 205 218 225 240 257 262 262 274 274 286 313 313 323 336 336 336 336 348 348 359 372 372 389 402 402 410 410 410 410 410 428 428 466 472 484 484 484 500 500 519 519 519 521 521 521 534 534 557 557 557

Observed 1149.6279 698.387 1226.6522 951.4334 2434.2598 2504.3069 2505.3181 1149.5868 1017.5012 1874.9221 1945.9714 947.5287 1391.4935 1462.5707 1478.6016 1657.7701 1728.8123 1729.7687 1089.5018 1939.8688 1940.8682 2010.9178 2012.0138 940.4733 2593.1545 2664.1797 2721.2466 2792.2712 2808.2727 1055.636 927.4799 1898.9993 1742.8868 1399.6169 786.4415 1074.5723 720.3903 673.3762 875.5167 789.4596 2128.8599 2556.0796 2627.1257 1386.5618 1457.6744 1574.8185 2988.3418 3004.3215 695.3604 2300.1016 2301.1069 1623.7968 1639.7778 1467.783 1311.6967 984.4985 1523.642 1594.6838 2045.0261 1600.6648 1671.6873 2542.2207 2613.2551 2613.2961 2614.2957 960.5396 1639.9016 1511.8065 2702.3391 674.3772 1024.4467 1095.4441 1166.5387 1853.8573 1924.8708 2202.9299 2203.9517 2273.9424 2489.0874 2489.137 2559.0745 1128.6578 1000.5624 1342.577 1342.6844 1358.6757

Mr(expt) 1148.6206 697.3797 1225.6449 950.4261 2433.2525 2503.2996 2504.3108 1148.5795 1016.4939 1873.9148 1944.9641 946.5215 1390.4862 1461.5634 1477.5943 1656.7628 1727.805 1728.7614 1088.4945 1938.8615 1939.8609 2009.9105 2011.0065 939.4661 2592.1472 2663.1724 2720.2393 2791.2639 2807.2654 1054.6287 926.4726 1897.992 1741.8795 1398.6096 785.4342 1073.565 719.383 672.3689 874.5094 788.4523 2127.8526 2555.0723 2626.1184 1385.5545 1456.6671 1573.8112 2987.3345 3003.3142 694.3531 2299.0943 2300.0996 1622.7895 1638.7705 1466.7757 1310.6894 983.4912 1522.6347 1593.6765 2044.0188 1599.6575 1670.68 2541.2134 2612.2478 2612.2888 2613.2884 959.5323 1638.8943 1510.7992 2701.3318 673.3699 1023.4394 1094.4368 1165.5314 1852.85 1923.8635 2201.9226 2202.9444 2272.9351 2488.0801 2488.1297 2558.0672 1127.6505 999.5551 1341.5697 1341.6771 1357.6684

Mr(calc) 1148.5686 697.3507 1225.5979 950.4345 2433.2402 2503.2933 2504.2773 1148.6077 1016.5291 1874.0084 1945.0455 946.527 1390.5203 1461.5574 1477.5523 1656.7678 1727.8049 1728.7889 1088.5073 1938.9006 1939.8846 2009.9377 2010.9217 939.441 2592.2352 2663.2724 2720.3302 2791.3673 2807.3622 1054.5811 926.4861 1897.9879 1741.8868 1398.5908 785.4469 1073.5353 719.3636 672.3707 874.5025 788.4644 2127.8772 2555.1203 2626.1574 1385.6133 1456.6504 1573.8207 2987.3528 3003.3477 694.3286 2299.0983 2300.0823 1622.7803 1638.7752 1466.8358 1310.7347 983.4811 1522.6003 1593.6374 2044.0881 1599.7239 1670.761 2541.2686 2612.3057 2612.3057 2613.2897 959.5552 1638.9305 1510.8355 2701.339 673.3395 1023.4477 1094.4848 1165.522 1852.9029 1923.94 2201.9939 2202.9779 2273.031 2488.1216 2488.1216 2558.1747 1127.6914 999.5964 1341.6275 1341.6275 1357.6224

Missed Cleavage 1 0 1 0 0 0 0 0 0 1 1 0 0 0 0 1 1 1 0 1 1 1 1 0 0 0 1 1 1 1 0 1 0 0 0 1 0 0 1 0 0 1 1 0 0 1 0 0 0 1 1 0 0 1 0 0 0 0 0 0 0 1 1 1 1 0 1 0 1 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0

Score 82 30 65 62 38 108 47 72 57 88 143 52 49 96 111 72 82 30 54 84 32 98 21 59 32 77 67 102 56 63 44 78 100 64 33 57 40 40 33 54 109 37 92 59 56 72 227 155 45 142 82 100 58 79 83 73 48 73 119 72 103 132 155 54 89 59 126 119 77 43 48 57 62 98 96 125 25 151 55 73 187 84 71 44 96 84

Peptide DAHKSEVAHR.F K.SEVAHR.F R.FKDLGEENFK.A K.DLGEENFK.A K.ALVLIAFAQYLQQCPFEDHVK.L + Deamidated (NQ) K.ALVLIAFAQYLQQCPFEDHVK.L + Propionamide (C) K.ALVLIAFAQYLQQCPFEDHVK.L + Deamidated (NQ); Propionamide (C) K.LVNEVTEFAK.T K.SLHTLFGDK.L K.SLHTLFGDKLCTVATLR.E K.SLHTLFGDKLCTVATLR.E + Propionamide (C) K.LCTVATLR.E + Propionamide (C) R.ETYGEMADCCAK.Q + Propionamide (C) R.ETYGEMADCCAK.Q + 2 Propionamide (C) R.ETYGEMADCCAK.Q + Oxidation (M); 2 Propionamide (C) K.QEPERNECFLQHK.D K.QEPERNECFLQHK.D + Propionamide (C) K.QEPERNECFLQHK.D + Deamidated (NQ); Propionamide (C) R.NECFLQHK.D + Propionamide (C) R.NECFLQHKDDNPNLPR.L R.NECFLQHKDDNPNLPR.L + Deamidated (NQ) R.NECFLQHKDDNPNLPR.L + Propionamide (C) R.NECFLQHKDDNPNLPR.L + Deamidated (NQ); Propionamide (C) K.DDNPNLPR.L R.LVRPEVDVMCTAFHDNEETFLK.K R.LVRPEVDVMCTAFHDNEETFLK.K + Propionamide (C) R.LVRPEVDVMCTAFHDNEETFLKK.Y R.LVRPEVDVMCTAFHDNEETFLKK.Y + Propionamide (C) R.LVRPEVDVMCTAFHDNEETFLKK.Y + Oxidation (M); Propionamide (C) K.KYLYEIAR.R K.YLYEIAR.R R.RHPYFYAPELLFFAK.R R.HPYFYAPELLFFAK.R K.AAFTECCQAADK.A + 2 Propionamide (C) K.AACLLPK.L + Propionamide (C) K.LDELRDEGK.A K.CASLQK.F + Propionamide (C) K.AWAVAR.L R.LSQRFPK.A K.LVTDLTK.V K.VHTECCHGDLLECADDR.A + 3 Propionamide (C) K.VHTECCHGDLLECADDRADLAK.Y + 2 Propionamide (C) K.VHTECCHGDLLECADDRADLAK.Y + 3 Propionamide (C) K.YICENQDSISSK.L K.YICENQDSISSK.L + Propionamide (C) K.LKECCEKPLLEK.S + 2 Propionamide (C) K.SHCIAEVENDEMPADLPSLAADFVESK.D + Propionamide (C) K.SHCIAEVENDEMPADLPSLAADFVESK.D + Oxidation (M); Propionamide (C) K.NYAEAK.D K.NYAEAKDVFLGMFLYEYAR.R K.NYAEAKDVFLGMFLYEYAR.R + Deamidated (NQ) K.DVFLGMFLYEYAR.R K.DVFLGMFLYEYAR.R + Oxidation (M) R.RHPDYSVVLLLR.L R.HPDYSVVLLLR.L K.TYETTLEK.C K.CCAAADPHECYAK.V + 2 Propionamide (C) K.CCAAADPHECYAK.V + 3 Propionamide (C) K.VFDEFKPLVEEPQNLIK.Q K.QNCELFEQLGEYK.F K.QNCELFEQLGEYK.F + Propionamide (C) K.QNCELFEQLGEYKFQNALLVR.Y K.QNCELFEQLGEYKFQNALLVR.Y + Propionamide (C) K.QNCELFEQLGEYKFQNALLVR.Y + Propionamide (C) K.QNCELFEQLGEYKFQNALLVR.Y + Deamidated (NQ); Propionamide (C) K.FQNALLVR.Y K.KVPQVSTPTLVEVSR.N K.VPQVSTPTLVEVSR.N K.RMPCAEDYLSVVLNQLCVLHEK.T + 2 Propionamide (C) K.TPVSDR.V K.CCTESLVNR.R K.CCTESLVNR.R + Propionamide (C) K.CCTESLVNR.R + 2 Propionamide (C) R.RPCFSALEVDETYVPK.E R.RPCFSALEVDETYVPK.E + Propionamide (C) K.EFNAETFTFHADICTLSEK.E K.EFNAETFTFHADICTLSEK.E + Deamidated (NQ) K.EFNAETFTFHADICTLSEK.E + Propionamide (C) K.EFNAETFTFHADICTLSEKER.Q + Deamidated (NQ) K.EFNAETFTFHADICTLSEKER.Q + Deamidated (NQ) K.EFNAETFTFHADICTLSEKER.Q + Propionamide (C) K.KQTALVELVK.H K.QTALVELVK.H K.AVMDDFAAFVEK.C K.AVMDDFAAFVEK.C K.AVMDDFAAFVEK.C + Oxidation (M)

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Supplemental Table 3. Masses of S-cysteinylated peptides detedcted with MALDITOF/TOF MS. Masses of Trypsin digested (A) and V8 protease digested (B) peptides detected with MALDI-TOF/TOF MS.

A Position 21 94 390 485

-

41 114 402 500

Observed 2552.2717 2698.1821 1719.6868 1972.8424

Mr (expt) 2551.2644 2697.1748 1718.6795 1971.8351

Mr (calc) 2551.2603 2697.2023 1718.728 1971.907

Missed Cleavage 0 2 0 0

Score 90 105 55 44

Peptide K.ALVLIAFAQYLQQCPFEDHVK.L + Cysteinyl (C34) K.QEPERNECFLQHKDDNPNLPR.L + Cysteinyl (C101) K.QNCELFEQLGEYK.F + Cysteinyl (C392) R.RPCFSALEVDETYVPK.E + Cysteinyl (C487)

B Position 18 87 90 101 189 377

-

45 100 100 119 208 393

Observed 3397.6338 1799.6262 1544.5686 2410.1028 2256.0796 2148.0085

Mr (expt) 3396.6265 1798.6189 1543.5613 2409.0955 2255.0723 2147.0012

Mr (calc) 3396.6886 1798.6742 1543.5523 2409.1682 2255.1514 2147.0391

Missed Cleavage 2 2 1 2 0 1

Score 52 44 28 32 65 82

Peptide E.NFKALVLIAFAQYLQQCPFEDHVKLVNE.V + Cysteinyl (C34); Deamidated (Q29) E.MADCCAKQEPERNE.C + Carbamidomethyl (C90); Cysteinyl (C91) D.CCAKQEPERNE.C + 2 Cysteinyl (C90, C91) E.CFLQHKDDNPNLPRLVRPE.V + Cysteinyl (C101) E.GKASSAKQRLKCASLQKFGE.R + Cysteinyl (C200) E.FKPLVEEPQNLIKQNCE.L + Cysteinyl (C392)

Supplemental Table 4. Masses of S-homocysteinylated peptides detedcted with MALDI-TOF/TOF MS. Masses of Trypsin digested (A) and V8 protease digested (B) peptides detected with MALDI-TOF/TOF MS. A Position 94 485 -

114 500

Observed 2712.1492 1986.8774

Mr (expt) 2711.1419 1985.8701

Missed Cleavage 2711.218 2 1985.9227 0

Mr (calc)

Score

Peptide

48 K.QEPERNECFLQHKDDNPNLPR.L + HCysteinyl (C101) 47 R.RPCFSALEVDETYVPK.E + HCysteinyl (C487)

B Position 87 90 377 -

100 100 393

Observed 1813.637 1573.6201 2161.9646

Mr (expt) 1812.6297 1572.6128 2160.9573

Missed Cleavage 1812.6899 2 1572.5676 1 2161.0547 1

Mr (calc)

Score

Peptide

36 E.MADCCAKQEPERNE.C + Carbamidomethyl (C90); HCysteinyl (C91) 12 D.CCAKQEPERNE.C + Deamidated (Q94); 2 HCysteinyl (C90, C91) 65 E.FKPLVEEPQNLIKQNCE.L + HCysteinyl (C392)

6

Supplemental Table 5. Masses of cysteine-containing peptides detected with MALDITOF/TOF MS. Masses of Trypsin digested (A) and V8 protease digested (B) peptides detected with MALDI-TOF/TOF MS. A Position 74 94 198 263 314 390 485

– – – – – – –

81 114 205 274 323 410 500

Observed 933.4875 2636.1846 890.4734 1443.5825 1140.4976 2599.2068 1853.8645

Mr (expt) 932.4802 2635.1773 889.4661 1442.5752 1139.4903 2598.1995 1852.8572

Mr (calc) 932.5113 2635.2197 889.5055 1442.6347 1139.5281 2598.29 1852.9029

Missed Cleavage 0 2 1 0 1 1 0

Score 47 116 55 76 62 53 41

Peptide K.LCTVATLR.E + Carbamidomethyl (C75) K.QEPERNECFLQHKDDNPNLPR.L + Carbamidomethyl (C101) R.LKCASLQK.F K.YICENQDSISSK.L + Carbamidomethyl (C265) K.DVCKNYAEAK.D K.QNCELFEQLGEYKFQNALLVR.Y + Carbamidomethyl (C392) R.RPCFSALEVDETYVPK.E

B Position 49 87 101 189 312 312 359 369 369 377 377 426 480

– – – – – – – – – – – – –

60 100 119 208 321 321 368 376 376 393 393 442 495

Observed 1327.5275 1737.6477 2348.1121 2194.1082 1156.475 1213.491 1101.4072 974.3856 1031.4116 2028.95 2085.9751 1955.9292 1891.8563

Mr (expt) 1326.5202 1736.6404 2347.1048 2193.1009 1155.4677 1212.4837 1100.3999 973.3783 1030.4043 2027.9427 2084.9678 1954.9219 1890.849

Mr (calc) 1326.5762 1736.6916 2347.1855 2193.1688 1155.523 1212.5445 1100.4379 973.4215 1030.443 2028.035 2085.0564 1954.9829 1890.9258

Missed Cleavage 2 2 2 0 1 1 0 1 1 1 1 0 2

Score 78 82 64 106 74 65 36 47 51 67 106 73 29

7

Peptide E.FAKTCVADESAE.N + Carbamidomethyl (C53) E.MADCCAKQEPERNE.C + 2 Carbamidomethyl (C90, C91) E.CFLQHKDDNPNLPRLVRPE.V + Carbamidomethyl (C101) E.GKASSAKQRLKCASLQKFGE.R + Carbamidomethyl (C200) E.SKDVCKNYAE.A E.SKDVCKNYAE.A + Carbamidomethyl (C316) E.KCCAAADPHE.C + Carbamidomethyl (C361) E.CYAKVFDE.F E.CYAKVFDE.F + Carbamidomethyl (C369) E.FKPLVEEPQNLIKQNCE.L E.FKPLVEEPQNLIKQNCE.L + Carbamidomethyl (C392) E.VSRNLGKVGSKCCKHPE.A + 2 Carbamidomethyl (C438, C448) E.SLVNRRPCFSALEVDE.T + Carbamidomethyl (C487)

Supplemental Figures (Figure S1-S9)

Supplemental Figure 1. Analysis of serum protein from hyperlipidemia patients. Anion-exchange liquid chromatography of human serum. Serum from healthy subjects (No.1-5) and hyperlipidemia patients (No. 6-20) were submitted to HPLC using an anionexchange column.

8

1

DAHKSEVAHR

FKDLGEENFK

ALVLIAFAQY

LQQCPFEDHV

KLVNEVTEFA

51

KTCVADESAE

NCDKSLHTLF

GDKLCTVATL

RETYGEMADC

CAKQEPERNE

101

CFLQHKDDNP

NLPRLVRPEV

DVMCTAFHDN

EETFLKKYLY

EIARRHPYFY

151

APELLFFAKR

YKAAFTECCQ

AADKAACLLP

KLDELRDEGK

ASSAKQRLKC

201

ASLQKFGERA

FKAWAVARLS

QRFPKAEFAE

VSKLVTDLTK

VHTECCHGDL

251

LECADDRADL

AKYICENQDS

ISSKLKECCE

KPLLEKSHCI

AEVENDEMPA

301

DLPSLAADFV

ESKDVCKNYA

EAKDVFLGMF

LYEYARRHPD

YSVVLLLRLA

351

KTYETTLEKC

CAAADPHECY

AKVFDEFKPL

VEEPQNLIKQ

NCELFEQLGE

401

YKFQNALLVR

YTKKVPQVST

PTLVEVSRNL

GKVGSKCCKH

PEAKRMPCAE

451

DYLSVVLNQL

CVLHEKTPVS

DRVTKCCTES

LVNRRPCFSA

LEVDETYVPK

501

EFNAETFTFH

ADICTLSEKE

RQIKKQTALV

ELVKHKPKAT

KEQLKAVMDD

551

FAAFVEKCCK

ADDKETCFAE

EGKKLVAASQ

AALGL

Supplemental Figure 2. Identification of peak 1 protein by MALDI-TOF/TOF MS analysis. Protein sequence of HSA. Sequence in red is the peptide fragment that was identified in the peak 1.

1

DAHKSEVAHR

FKDLGEENFK

ALVLIAFAQY

LQQCPFEDHV

KLVNEVTEFA

51

KTCVADESAE

NCDKSLHTLF

GDKLCTVATL

RETYGEMADC

CAKQEPERNE

101

CFLQHKDDNP

NLPRLVRPEV

DVMCTAFHDN

EETFLKKYLY

EIARRHPYFY

151

APELLFFAKR

YKAAFTECCQ

AADKAACLLP

KLDELRDEGK

ASSAKQRLKC

201

ASLQKFGERA

FKAWAVARLS

QRFPKAEFAE

VSKLVTDLTK

VHTECCHGDL

251

LECADDRADL

AKYICENQDS

ISSKLKECCE

KPLLEKSHCI

AEVENDEMPA

301

DLPSLAADFV

ESKDVCKNYA

EAKDVFLGMF

LYEYARRHPD

YSVVLLLRLA

351

KTYETTLEKC

CAAADPHECY

AKVFDEFKPL

VEEPQNLIKQ

NCELFEQLGE

401

YKFQNALLVR

YTKKVPQVST

PTLVEVSRNL

GKVGSKCCKH

PEAKRMPCAE

451

DYLSVVLNQL

CVLHEKTPVS

DRVTKCCTES

LVNRRPCFSA

LEVDETYVPK

501

EFNAETFTFH

ADICTLSEKE

RQIKKQTALV

ELVKHKPKAT

KEQLKAVMDD

551

FAAFVEKCCK

ADDKETCFAE

EGKKLVAASQ

AALGL

Supplemental Figure 3. Identification of peak 2 protein by MALDI-TOF/TOF MS analysis. Protein sequence of HSA. Sequence in red is the peptide fragment that was identified in the peak 2.

9

DAABD-GSH (mmol/mol Albumin)

4

***

2

0 red ox HSA HSA

Supplemental Figure 4. Quantification of HSA-bound glutathione. Protein bound glutathione in redHSA and oxHSA from human sera were quantified using LC-MS/MS with the MRM mode. Statistical significance was determined by unpaired Student’s t-tests comparing redHSA to oxHSA samples, ***p < 0.005.

10

Supplemental Figure 5. Mass spectra of S-cysteinylated peptides detedcted with MALDI-TOF/TOF MS. Mass spectra of Trypsin digested (A) and V8 protease digested (B) peptides detected with MALDI-TOF/TOF MS. Data represent CA; carbamidomethylation, CY; cysteinylation, and DE; deamidation.

11

Supplemental Figure 6. Mass spectra of S-homocysteinylated peptides detedcted with MALDI-TOF/TOF MS. Mass spectra of Trypsin digested (A) and V8 protease digested (B) peptides detected with MALDI-TOF/TOF MS. Data represent CA; carbamidomethylation, HC; homocysteinylation, and DE; deamidation.

12

A

B

100 50 0

Serum total GSH (μM)

6

Serum total homocysteine (μM)

Serum total cysteine (μM)

150

C

4 2 0

Normal Hypersubjects lipidemia

Normal Hypersubjects lipidemia

1.5

1.0 0.5 0

Normal Hypersubjects lipidemia

Supplemental Figure 7. Quantification of serum total thiols from normal subject and hyperlipidemia patients. Quantification of (A) cysteine, (B) homocysteine, and (C) GSH in sera from normal subjects and hyperlipidemia patients were quantified using LC-MS/MS with the MRM mode.

13

250

HSA-bound cysteine (mol/mol HSA)

HSA-bound cysteine (mol/mol HSA)

A

200 150

100 r = 0.24 P = 0.692

50 0

250 200 150

100 r = 0.20 P = 0.468

50 0

0

20

100

150

0

Serum total cysteine (μM)

B

100

150

Serum total cysteine (μM)

5.0

5.0 r = 0.24 P = 0.692

4.0

HSA-bound GSH (mol/mol HSA)

HSA-bound GSH (mol/mol HSA)

20

3.0 2.0

1.0 0

r = 0.20 P = 0.468

4.0 3.0 2.0

1.0 0

0

0.5

1.0

1.5

0

Serum total GSH (μM)

0.5

1.0

1.5

Serum total GSH (μM)

Supplemental Figure 8. Relationship between serum total and HSA-bound thiol. Graph illustrating the relationship between (A) serum total cysteine and HSA-bound cysteine in normal subjects (n=5) (left panel) and hyperlipidemia patients (n=15) (right panel), and (B) serum total GSH and HSA-bound GSH in normal subjects (n=5) (left panel) and hyperlipidemia patients (n=15) (right panel).

14

Supplemental Figure 9. HPLC chromatograph of modified rHSAs. rHSA (150 μM) and homocysteine (600 μM) or homocystine (300 μM) were incubated at 37℃ for 24 h in 0.1 M phosphate buffer (pH 6.7) containing 0.3 M NaCl for S-thiolation of HSA. rHSA (top) and homocysteine (middle) or homocystine treated rHSA (lower) was analyzed by anion-exchange chromatography by monitoring the excitation at 280 nm and emission at 340 nm.

15