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TRANSCRIPT
Supporting Information for: Effect of laser intensity on the apparent isotope patterns of heme and peptide ions in MALDI-
TOF MS
Taehee Kim, Jihyeon Lee, and Jeongkwon Kim*
Department of Chemistry, Chungnam National University, Daejeon, Republic of Korea
*Corresponding authors:
J. Kim
(Address) Department of Chemistry, Chungnam National University
99 Daehak-Ro, Yuseong-Gu, Daejeon, 305-764, Republic of Korea
(E-mail) [email protected]
Hemecm/z 617.18
[10-14]m/z 634.44 [81-87]
m/z 779.49
[29-39]m/z 1168.65
[29-40]m/z 1296.72
[89-101]m/z 1606.95 [10-23]
m/z 1633.58
[88-101]m/z 1735.02
500 900 1300 1700 2100 2500Mass (m/z)
2.8E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
1433.781478.82
2209.91
Hemecm/z 617.33
[10-14]m/z 634.59 [81-87]
m/z 779.69
[29-39]m/z 1168.97
[10-23]m/z 1634.11
500 900 1300 1700 2100 2500Mass (m/z)
2.5E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
801.66
1190.941434.20
1297.09 2232.35
(A) DHB
(B) CHCA
Supplementary Figure S1. MALDI mass spectra of tryptically digested peptides from 80 pmol of cytochrome c using the (A) DHB or (B) CHCA matrix. Laser intensities of 2019 and 1700 (arb. unit) were used with the DHB and CHCA matrix, respectively
(A) Myoglobin (60 pmol) - DHB
1400 1600 1800 2000 2200 2400 2600 2800 30000
10000
20000
30000
40000
Pea
kIn
tens
ity
Laser Intensity
m/z 616.17m/z 684.37m/z 748.44m/z 1086.56m/z 1271.66m/z 1502.67m/z 1606.85m/z 1661.85m/z 1853.96m/z 1885.02m/z 1982.06m/z 2110.15
(B) Myoglobin (60 pmol) - CHCA
1000 1200 1400 1600 1800 2000 22000
500
1000
1500
2000
2500
3000
3500
4000
4500
Pea
k In
tens
ity
Laser Intensity
m/z 616.17m/z 748.44m/z 1606.85
(C) Myoglobin (12 pmol) - DHB
1400 1600 1800 2000 2200 2400 2600 2800 30000
10000
20000
Pea
k In
tens
ity
Laser Intensity
m/z 616.17m/z 684.37m/z 748.44m/z 1086.56m/z 1271.66m/z 1502.67m/z 1606.85m/z 1661.85m/z 1853.96m/z 1885.02m/z 1982.06m/z 2110.15
(D) Myoglobin (12 pmol) - CHCA
1400 1600 1800 2000 2200 2400 2600 28000
1000
2000
3000
4000P
eak
Inte
nsity
Laser Intensity
m/z 616.17m/z 748.44m/z 1606.85
(E) Cytochrome c (80 pmol) - DHB
1400 1600 1800 2000 2200 2400 2600 2800 30000
10000
20000
30000
40000
50000
60000
Pea
k In
tens
ity
Laser Intensity
m/z 617.18m/z 634.39m/z 779.45m/z 1168.62m/z 1296.72m/z 1606.92m/z 1633.61m/z 1734.01
(G) Cytochrome c (16 pmol) - DHB
1400 1600 1800 2000 2200 2400 2600 2800 30000
10000
20000
30000
40000
Pea
k In
tens
ity
Laser Intensity
m/z 617.18m/z 634.39m/z 779.45m/z 1168.62m/z 1296.72m/z 1606.92m/z 1633.61m/z 1734.01
(F) Cytochrome c (80 pmol) - CHCA
1000 1200 1400 1600 1800 2000 22000
10000
20000
30000
40000
50000
Pea
k In
tens
ity
Laser Intensity
m/z 617.18m/z 634.39m/z 779.45m/z 1168.62m/z 1633.61
(H) Cytochrome c (16 pmol) - CHCA
1400 1600 1800 2000 2200 2400 26000
10000
20000
30000
Pea
k In
tens
ity
Laser Intensity
m/z 617.18m/z 779.45m/z 1168.62m/z 1633.61
Supplementary Figure S2. Change of the absolute peak intensities of tryptic peptides from myoglobin and cytochrome c upon the change of laser intensity in two different matrices of DHB and CHCA. Theoretical m/z values for peptides were provided in the inset.
6 0 6.0 6 1 1 .2 6 1 6 .4 6 2 1. 6 6 2 6.8 6 3 2.0M a ss (m /z )
1 .4 E+ 4
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Intensity
Voyager Spec #1=>MC[BP = 1168.6, 28055]6 1 7 .1 7 9 6
6 1 8.1 7 1 0
6 1 9 .1 7 0 5
6 2 0.1 7 9 66 1 5 .1 9 1 3
6 0 7.6 1 4 9 6 2 1 .1 9 6 86 1 1 .3 2 7 0
6 2 7 6 3 1 6 3 5 6 3 9 6 4 3 6 4 7M a s s (m /z )
1 8 0 8 .3
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Intensity
Voyager Spec #1=>MC[BP = 1168.6, 28055]6 3 4 .4 4 5 7
6 3 5 .4 5 6 5
7 7 1 7 7 5 7 7 9 7 8 3 7 8 7 7 9 1M a ss (m /z )
2 6 9 9.6
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Intensity
Voyager Spec #1=>MC[BP = 1168.6, 28055]7 7 9 .4 9 8 0
7 8 0 .4 9 9 6
7 8 1 .5 0 3 1
1 1 5 1 .0 1 1 5 9 .6 1 1 6 8.2 1 1 7 6 .8 1 1 8 5 .4 1 1 9 4 .0M a ss (m /z )
2 .8 E+ 4
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Intensity
Voyager Spec #1=>MC[BP = 1168.6, 28055]1 1 6 8 .6 5 3 3
1 1 6 9.5 9 3 0
1 1 7 0 .5 2 4 3
1 1 7 1.5 0 5 0
1 1 7 2 .3 9 8 61 1 5 2 .1 9 6 2 1 1 9 0 .5 7 1 3
1 2 8 2 1 2 8 9 1 2 9 6 1 3 0 3 1 3 1 0 1 3 1 7M a s s (m /z )
7 8 8 5 .6
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Intensity
Voyager Spec #1=>MC[BP = 1168.6, 28055]1 2 9 6 .72 7 5
1 2 9 7 .6 9 8 0
1 2 9 8 .6 8 3 6
1 2 9 9 . 66 2 41 3 0 5 .55 7 0
1 5 8 3. 0 1 5 9 3.4 1 6 0 3.8 1 6 1 4. 2 1 6 2 4.6 1 6 3 5.0M a ss (m /z )
1 . 1 E+ 4
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Intensity
Voyager Spec #1=>MC[BP = 1168.6, 28055]1 6 0 6.9 5 8 7
1 6 0 7 .8 8 2 8
1 6 0 8. 8 2 5 4
1 6 0 9.7 3 7 9
1 5 9 8.8 1 4 2 1 6 1 0.8 1 3 1 1 6 3 1.7 3 7 81 6 2 3.8 2 1 5
1 6 1 5.0 1 6 2 3 .6 1 6 3 2.2 1 6 4 0.8 1 6 4 9 .4 1 6 5 8. 0M a ss (m /z )
7 2 6 3.1
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Intensity
Voyager Spec #1=>MC[BP = 1168.6, 28055]1 6 3 3.5 8 3 6
1 6 3 5.6 0 0 7
1 6 3 6 .4 9 8 9
1 6 3 7.5 8 3 41 6 3 2 .6 3 7 91 6 2 3.8 2 1 5
1 72 0 .0 1 72 6 .8 1 73 3 .6 1 74 0 .4 1 74 7 .2 1 75 4 .0M a s s (m / z )
4 2 4 6 . 7
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% Intensity
Voyager Spec #1=>MC[BP = 1168.6, 28055]1 73 5 .0 2 45
1 73 6 .0 7 17
1 7 3 6 . 9 3 8 8
1 73 7 .9 1 85
617.18
618.17
619.17
620.18615.19
634.45
635.46
779.50
780.50
781.50
1168.65
1170.52
1171.51
1169.59
1735.02
1736.94
1737.92
1736.071633.58
1635.60
1636.50
1634.59
1637.58
1606.96
1608.83
1609.74
1607.881296.73
1298.681299.66
1297.70
616.4 621.6 635.0 779.0 1168.2 1176.8
1296.0 1303.0 1603.8 1614.2 1733.6 1740.4
783.0
1632.2 1640.8
(A) (B) (C) (D)
(E) (F) (G) (H)
Supplementary Figure S3. Enlarged mass spectra from Supplementary Figure S1(A) show the identified peptides from cytochrome c using the DHB matrix
y = 0.950x + 0.045R² = 0.986
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120 140
Theo
retic
al a
bund
ance
of S
IP (%
)
Experimentally observed relative abundance of SIP (%)
(A) Bruker Daltonics IsotopePattern
y = 0.988x - 0.102R² = 0.987
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120 140
Theo
retic
al a
bund
ance
of S
IP (%
)
Experimentally observed relative abundance of SIP (%)
(B) MS-isotope from Protein Prospector
Supplementary Figure S4. Plots comparing experimental and theoretical values of relative abundance where the theoretical values were obtained using (A) Bruker Daltonics IsotopePattern and (B) MS-isotope from Protein Prospector
Supplementary Table S1. Intensities of the monoisotopic peaks and second isotopic peaks from the identified tryptic peptides of myoglobin and cytochrome c at different laser intensities using DHB or CHCA matrix.
(A) Myoglobin (60 pmol) with DHB matrix
Position Heme b 44-48 135-140 49-57 33-43 120-134 18-32 33-46 81-97 104-119 80-97 79-97
Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio
Laser Intensity 616.17 617.17 684.37 685.37 748.44 749.44 1086.56 1087.56 1271.66 1272.66 1502.67 1503.67 1606.85 1607.86 1661.85 1662.85 1853.96 1854.96 1885.02 1886.02 1982.06 1983.06 2110.15 2111.15
1519 41 43 1.049 159 67 0.421 929 414 0.446 157 74 0.471 244 159 0.652 210 176 0.838 340 344 1.012 59 88 1.492 86 96 1.116 - 73 67 0.918 58 53 0.914
1619 216 185 0.856 1420 602 0.424 8444 3401 0.403 1222 677 0.554 1829 1274 0.697 1724 1412 0.819 2906 2461 0.847 391 547 1.399 637 609 0.956 219 194 0.886 463 575 1.242 353 401 1.136
1719 1374 960 0.699 1533 623 0.406 15365 6709 0.437 1021 618 0.605 2760 1976 0.716 2387 1898 0.795 7162 5930 0.828 689 1066 1.547 899 973 1.082 1030 1071 1.040 774 797 1.030 490 658 1.343
1819 1701 1326 0.780 1625 598 0.368 19917 9165 0.460 1060 598 0.564 3550 2354 0.663 3124 2314 0.741 11904 9841 0.827 1055 1570 1.488 1235 1189 0.963 2020 2025 1.002 970 1075 1.108 702 839 1.195
1919 2500 1934 0.774 2798 1170 0.418 32276 16301 0.505 1663 1085 0.652 5338 3674 0.688 4433 3503 0.790 17790 15104 0.849 1342 2090 1.557 1653 1793 1.085 2196 2322 1.057 1348 1544 1.145 1022 1219 1.193
2019 2409 1838 0.763 2474 1089 0.440 38657 20335 0.526 1603 996 0.621 6090 4382 0.720 4698 3793 0.807 22281 18513 0.831 1527 2227 1.458 1965 2043 1.040 2091 2355 1.126 1626 1809 1.113 1180 1477 1.252
2119 4334 3103 0.716 2938 1187 0.404 41260 22014 0.534 1882 1140 0.606 6888 4533 0.658 5587 4227 0.757 23305 20045 0.860 1485 2099 1.413 2347 2387 1.017 2031 2148 1.058 1904 2204 1.158 1481 1770 1.195
2219 4709 3417 0.726 2235 980 0.438 39647 20740 0.523 1646 981 0.596 5054 3516 0.696 3780 3157 0.835 19358 16523 0.854 1027 1614 1.572 1775 1878 1.058 1442 1669 1.157 1476 1671 1.132 1158 1425 1.231
2319 3598 2842 0.790 2068 834 0.403 37225 18574 0.499 1261 802 0.636 4280 2963 0.692 3111 2428 0.780 17613 15153 0.860 980 1414 1.443 1309 1537 1.174 1168 1300 1.113 1196 1408 1.177 909 1135 1.249
2419 1183 937 0.792 2476 1092 0.441 33132 16705 0.504 1533 865 0.564 4777 3354 0.702 3615 2823 0.781 14141 12047 0.852 986 1425 1.445 1572 1660 1.056 1313 1499 1.142 1302 1474 1.132 1040 1246 1.198
2519 1958 1421 0.726 1487 627 0.422 18765 8796 0.469 824 515 0.625 2333 1503 0.644 1653 1258 0.761 6927 5884 0.849 454 621 1.368 732 769 1.051 511 574 1.123 630 698 1.108 468 595 1.271
2619 1292 967 0.748 883 434 0.492 14386 6614 0.460 539 346 0.642 1489 1080 0.725 1211 948 0.783 5231 4474 0.855 333 442 1.327 548 590 1.077 460 498 1.083 467 507 1.086 335 434 1.296
2719 893 780 0.873 472 216 0.458 12312 5569 0.452 250 152 0.608 1150 800 0.696 838 673 0.803 4795 4255 0.887 259 373 1.440 319 376 1.179 378 458 1.212 - -
2819 671 582 0.867 682 241 0.353 9589 3967 0.414 335 248 0.740 984 699 0.710 703 601 0.855 3512 2985 0.850 237 349 1.473 303 311 1.026 399 479 1.201 244 282 1.156 -
Average 0.797 0.421 0.474 0.606 0.690 0.796 0.862 1.459 1.063 1.092 1.116 1.206
Standard deviation 0.091 0.035 0.042 0.060 0.026 0.033 0.046 0.070 0.065 0.087 0.077 0.107
(B) Myoglobin (60 pmol) with CHCA matrix
Position Heme b 135-140 18-32
Theoretical m/z value Ratio Theoretical m/z value Ratio Theoretical m/z value Ratio
Laser Intensity 616.17 617.17 748.44 749.44 1606.85 1607.86
1084 166 87 0.524 220 80 0.364
1184 573 369 0.644 482 208 0.432 181 162 0.895
1284 784 393 0.501 832 416 0.500 417 345 0.827
1384 1498 1050 0.701 1432 695 0.485 753 636 0.845
1484 3641 2021 0.555 2972 1301 0.438 1514 1423 0.940
1583 3984 2150 0.540 3434 1571 0.457 2597 2199 0.847
1682 3844 2040 0.531 2672 1266 0.474 2303 1888 0.820
1782 3025 1655 0.547 2242 1016 0.453 2050 1644 0.802
1882 2189 1276 0.583 2154 1013 0.470 1666 1410 0.846
1982 2138 1210 0.566 1540 770 0.500 1074 1006 0.937
2082 1781 971 0.545 1151 568 0.493 467 372 0.797
Average 0.567 0.461 0.856
Standard deviation 0.058 0.040 0.052
(C) Cytochrome c (80 pmol) with DHB matrix
Position Heme c 10-14 81-87 29-39 29-40 89-101 10-23 88-101
Theoretical m/z value Ratio Theoretical m/z
value Ratio Theoretical m/z value Ratio Theoretical m/z
value Ratio Theoretical m/z value Ratio Theoretical m/z
value Ratio Theoretical m/z value Ratio Theoretical m/z
value Ratio
Laser Intensity 617.18 618.19 634.39 635.39 779.45 780.45 1168.62 1169.62 1296.72 1297.72 1606.92 1607.92 1633.61 1634.62 1735.01 1736.01
1519 40 13 0.325 81 31 0.383 89 40 0.449 287 204 0.711 51 45 0.882 83 83 1.000 38 29 0.763 44 40 0.909
1619 66 47 0.712 202 69 0.342 360 169 0.469 1901 1128 0.593 247 192 0.777 413 357 0.864 348 343 0.986 156 134 0.859
1719 1105 591 0.535 622 270 0.434 1164 496 0.426 9207 6248 0.679 1556 1105 0.710 2451 1947 0.794 1638 1685 1.029 906 861 0.950
1819 3482 2051 0.589 1310 483 0.369 1986 918 0.462 20288 16435 0.810 5104 3587 0.703 7285 6316 0.867 4645 4642 0.999 2878 2700 0.938
1919 8253 5583 0.676 1552 548 0.353 2823 1260 0.446 27388 22720 0.830 6983 5207 0.746 10680 9768 0.915 6458 5486 0.849 4698 4286 0.912
2019 14359 11072 0.771 1783 791 0.444 2700 1299 0.481 28055 23696 0.845 7862 5451 0.693 10559 9505 0.900 7256 6956 0.959 4247 3856 0.908
2119 26113 20203 0.774 2541 1143 0.450 4759 2113 0.444 43655 37160 0.851 12390 8673 0.700 16775 14439 0.861 10831 10269 0.948 6196 6220 1.004
2219 48139 41993 0.872 3120 1451 0.465 4718 2195 0.465 55809 46538 0.834 13818 9746 0.705 18502 15436 0.834 10918 10671 0.977 6951 6704 0.964
2319 52585 46567 0.886 3109 1229 0.395 3779 1919 0.508 46854 36293 0.775 10183 7161 0.703 13691 11972 0.874 7848 7681 0.979 5389 5374 0.997
2419 42563 34920 0.820 2659 1182 0.445 3201 1505 0.470 30765 20586 0.669 5763 3957 0.687 7377 6301 0.854 4584 4355 0.950 2984 2828 0.948
2519 32818 26819 0.817 1566 835 0.533 18637 12571 0.675 4050 2683 0.662 4724 4368 0.925 3034 2921 0.963 1759 1808 1.028
2619 24778 19119 0.772 1024 423 0.413 10328 6129 0.593 1452 1073 0.739 2052 1776 0.865 1131 1077 0.952 836 871 1.042
2719 19421 15551 0.801 1608 715 0.445 1575 857 0.544 13960 9524 0.682 2569 1715 0.668 3549 3155 0.889 1592 1652 1.038 1513 1491 0.985
2819 16290 13171 0.809 8377 5014 0.599 1438 916 0.637 1561 1489 0.954 1288 1223 0.950 569 578 1.016
2919 10507 7475 0.711 0.383 386 207 0.536 4649 2890 0.622 725 411 0.608 907 814 0.897 693 650 0.938 386 391 1.013
Average 0.726 0.411 0.470 0.725 0.715 0.885 0.953 0.961Standard deviation 0.152 0.044 0.039 0.097 0.060 0.051 0.070 0.053
(D) Cytochrome c (80 pmol) with CHCA matrix
Position Heme c 10-14 81-87 29-39 10-23
Theoretical m/z value Ratio Theoretical m/z value Ratio Theoretical m/z value Ratio Theoretical m/z value Ratio Theoretical m/z value Ratio
Laser Intensity 617.18 618.19 634.39 635.39 779.45 780.45 1168.62 1169.62 1633.61 1634.62
1300 72 46 0.639 654 237 0.362 552 224 0.406 452 281 0.622
1400 1582 873 0.552 1263 386 0.306 685 273 0.399 1271 808 0.636 963 982 1.020
1500 3959 2592 0.655 3940 1320 0.335 2202 889 0.404 3673 2313 0.630 1983 2080 1.049
1600 17018 12270 0.721 6287 2215 0.352 3406 1394 0.409 8732 5501 0.630 6145 5974 0.972
1700 25273 21250 0.841 8036 2879 0.358 6121 2558 0.418 14775 9148 0.619 7687 7397 0.962
1800 37412 31385 0.839 9995 3466 0.347 7455 3252 0.436 14455 9190 0.636 8588 8604 1.002
1900 45548 41019 0.901 5937 2216 0.373 5266 2310 0.439 8713 5311 0.610 7223 6889 0.954
2000 48173 41820 0.868 5804 2321 0.400 5919 2601 0.439 9888 6209 0.628 6109 5998 0.982
2100 51489 46566 0.904 4397 1740 0.396 - 7340 4675 0.637 6069 5809 0.957
Average 0.769 0.359 0.419 0.628 0.987 Standard deviation 0.130 0.029 0.017 0.009 0.034
(E) Myoglobin (12 pmol) with DHB matrix
Position Heme b 44-48 135-140 49-57 33-43 120-134 18-32 33-46 81-97 104-119 80-97 79-97
Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio Theoretical m/z value Ratio Theoretical
m/z value Ratio
Laser Intensity 616.17 617.17 684.37 685.37 748.44 749.44 1086.56 1087.56 1271.66 1272.66 1502.67 1503.67 1606.85 1607.86 1661.85 1662.85 1853.96 1854.96 1885.02 1886.02 1982.06 1983.06 2110.15 2111.15
1600 37 21 0.568 - - 35 24 0.686 - - - - - - - - - - - - - - - - - -
1700 53 33 0.623 - - 131 47 0.359 - - 28 24 0.857 25 17 0.680 28 28 1.000 - - - - - - - - - -
1800 228 144 0.632 107 37 0.346 362 133 0.367 - - 74 52 0.703 35 26 0.743 90 73 0.811 - - - - - - - - - -
1900 860 559 0.650 780 272 0.349 2906 1268 0.436 263 166 0.631 651 511 0.785 345 296 0.858 711 561 0.789 123 208 1.691 130 140 1.077 83 103 1.241 103 98 0.951 65 82 1.262
2000 3273 2099 0.641 2808 1124 0.400 10447 4333 0.415 752 489 0.650 2201 1571 0.714 1056 802 0.759 2530 2223 0.879 419 613 1.463 398 378 0.950 318 397 1.248 273 321 1.176 195 257 1.318
2100 4886 3266 0.668 5424 2235 0.412 20181 8184 0.406 1698 895 0.527 4436 2954 0.666 2057 1714 0.833 5069 4181 0.825 799 1226 1.534 806 835 1.036 495 560 1.131 587 647 1.102 398 475 1.193
2200 6166 4058 0.658 1750 707 0.404 7795 3254 0.417 525 328 0.625 1565 1035 0.661 957 725 0.758 2689 2308 0.858 366 555 1.516 344 389 1.131 628 715 1.139 292 314 1.075 - -
2300 6180 4194 0.679 2715 1214 0.447 13979 5757 0.412 852 579 0.680 2402 1790 0.745 1355 1006 0.742 4128 3679 0.891 495 761 1.537 523 591 1.130 515 590 1.146 - - - -
2400 8983 5345 0.595 1053 545 0.518 5731 2373 0.414 289 192 0.664 750 560 0.747 406 326 0.803 1497 1399 0.935 184 248 1.348 - - 201 230 1.144 - - - -
2500 6159 3482 0.565 698 347 0.497 3905 1697 0.435 187 157 0.840 485 347 0.715 289 240 0.830 1096 933 0.851 - - 119 138 1.160 155 196 1.265 - - - -
2600 7935 4520 0.570 408 228 0.559 3058 1306 0.427 - - 388 323 0.832 264 189 0.716 945 785 0.831 - - - - - - - - - -
2700 6769 3773 0.557 324 162 0.500 2145 956 0.446 - - 262 197 0.752 166 128 0.771 505 475 0.941 - - - - - - - - - -
2800 5588 3177 0.569 283 160 0.565 1821 805 0.442 - - 261 200 0.766 161 143 0.888 599 564 0.942 - - - - - - - - - -
2900 3025 1575 0.521 248 145 0.585 1439 571 0.397 - - 186 121 0.651 106 113 1.066 386 323 0.837 - - - - - - - - - -
Average 0.607 0.465 0.433 0.660 0.738 0.804 0.876 1.515 1.081 1.188 1.0761.258
Standard deviation 0.049 0.084 0.077 0.094 0.063 0.098 0.062 0.112 0.078 0.060 0.093 0.062
(F) Myoglobin (12 pmol) with CHCA matrixPosition Heme b 135-140 18-32
Theoretical m/z value Ratio Theoretical m/z value Ratio Theoretical m/z value Ratio
Laser Intensity 616.17 617.17 748.44 749.44 1606.85 1607.86
1400 499 289 0.579 - - - -
1500 726 365 0.503 191 93 0.487 120 105 0.875
1600 1448 794 0.548 264 156 0.591 - -
1700 1723 878 0.510 - - - -
1800 2024 1200 0.593 546 356 0.652 - -
1900 1947 1113 0.572 525 244 0.465 331 293 0.885
2000 2070 1165 0.563 572 351 0.614 424 383 0.903
2100 3917 2024 0.517 871 485 0.557 967 791 0.818
2200 3106 1627 0.524 658 404 0.614 601 592 0.985
2300 2217 1249 0.563 454 282 0.621 303 281 0.927
2400 1622 944 0.582 331 238 0.719 277 223 0.805
2500 1604 917 0.572 314 194 0.618 298 245 0.822
Average 0.552 0.594 0.878Standard deviation
0.031 0.075 0.062
(G) Cytochrome c (16 pmol) with DHB matrix
Position Heme c 10-14 81-87 29-39 29-40 89-101 10-23 88-101
Theoretical m/z value Ratio Theoretical m/z
value Ratio Theoretical m/z value Ratio Theoretical m/z
value Ratio Theoretical m/z value Ratio Theoretical m/z
value Ratio Theoretical m/z value Ratio Theoretical m/z
value Ratio
Laser Intensity 617.18 618.19 634.39 635.39 779.45 780.45 1168.62 1169.62 1296.72 1297.72 1606.92 1607.92 1633.61 1634.62 1735.01 1736.01
1500 105 42 0.400 111 36 0.324 86 53 0.616 39 31 0.795 61 46 0.754 - -
1600 484 179 0.370 352 174 0.494 245 168 0.686 104 80 0.769 143 118 0.825 - -
1700 1240 470 0.379 925 409 0.442 850 508 0.598 331 226 0.683 453 368 0.812 221 222 1.005
1800 3282 1151 0.351 2926 1279 0.437 2210 1382 0.625 752 595 0.791 996 965 0.969 218 259 1.188 516 555 1.076
1900 11563 4434 0.383 10137 4780 0.472 7732 5171 0.669 2801 2017 0.720 3512 3130 0.891 1203 1386 1.152 1852 1854 1.001
2000 18953 7173 0.378 14397 6477 0.450 12394 7960 0.642 4777 3502 0.733 6721 5857 0.871 1727 2009 1.163 3466 3307 0.954
2100 1763 1205 0.683 32880 12850 0.391 29011 13812 0.476 27660 18620 0.673 11256 7656 0.680 14825 13036 0.879 4111 4610 1.121 7081 6931 0.979
2200 4360 2791 0.640 25418 9583 0.377 20137 9057 0.450 21152 13585 0.642 7956 5695 0.716 11844 10452 0.882 2506 2755 1.099 6153 6055 0.984
2300 10066 6432 0.639 18566 7107 0.383 18758 8212 0.438 25077 15752 0.628 9626 6641 0.690 12680 11358 0.896 3531 3752 1.063 6338 6403 1.010
2400 8716 5845 0.671 11043 4150 0.376 8780 4050 0.461 11934 7276 0.610 4398 3212 0.730 6416 5674 0.884 1447 1517 1.048 3333 3451 1.035
2500 6458 4272 0.662 10277 3897 0.379 8186 3552 0.434 9990 6328 0.633 3698 2643 0.715 5122 4619 0.902 1089 1181 1.084 2784 2908 1.045
2600 8306 5672 0.683 3976 1518 0.382 4795 2137 0.446 7623 5000 0.656 2851 2057 0.722 3573 3261 0.913 1087 1181 1.086 1787 1875 1.049
2700 4066 2870 0.706 3165 1202 0.380 2364 1086 0.459 3560 2228 0.626 1269 986 0.777 1724 1554 0.901 398 403 1.013 - -
2800 6941 4573 0.659 4028 1567 0.389 4230 1841 0.435 5283 3341 0.632 1906 1410 0.740 2613 2351 0.900 736 790 1.073 1312 1310 0.998
2900 3306 2303 0.697 1779 719 0.404 1634 760 0.465 2279 1422 0.624 786 585 0.744 1078 1012 0.939 309 328 1.061 - -
average 0.671 0.381 0.446 0.637 0.734 0.881 1.096 1.012Standard deviation 0.023 0.012 0.038 0.024 0.037 0.052 0.051 0.036
(H) Cytochrome c (16 pmol) with CHCA matrix
Position Heme c 81-87 29-39 10-23
Theoretical m/z value Ratio Theoretical m/z value Ratio Theoretical m/z value Ratio Theoretical m/z value Ratio
Laser Intensity 617.18 618.19 779.45 780.45 1168.62 1169.62 1633.61 1634.62
1500 2535 1470 0.580 1029 442 0.430 9519 5831 0.613 1420 1363 0.960
1600 4802 2784 0.580 1203 539 0.448 11098 6800 0.613 1922 1833 0.954
1700 9477 5802 0.612 909 446 0.491 8810 5642 0.640 1409 1360 0.965
1800 14235 9641 0.677 916 529 0.578 11060 6644 0.601 2324 2195 0.944
1900 20862 14124 0.677 1320 734 0.556 12322 7689 0.624 2084 1970 0.945
2000 16583 11050 0.666 1211 647 0.534 12283 7648 0.623 2337 2150 0.920
2100 22209 14454 0.651 1638 827 0.505 16328 9671 0.592 2458 2260 0.919
2200 14701 10149 0.690 569 361 0.634 4541 2967 0.653 959 930 0.970
2300 11004 7373 0.670 1448 749 0.517 14481 8388 0.579 2107 2007 0.953
2400 6298 4377 0.695 963 567 0.589 9049 5896 0.652 1897 1898 1.001
2500 11130 7372 0.662 763 430 0.564 4753 2994 0.630 936 863 0.922
average 0.651 0.531 0.620 0.950
Standard deviation
0.041 0.061 0.024 0.024
Supplementary Table S2. Isotope masses and abundances for the elements used in the measurementIsotopePatterna) MS-isotopeb) Isotopic compositions of the elements 2009[1]
ElementMass number of isotope
mass abundance mass abundance Observed range of natural variations
Best measurement from a single terrestrial source
Representative isotopic composition
H 1 1.007825 0.99989 1.007825032 0.99985 0.999 816–0.999 974 0.999 844 26(5) 0.999 885(70)2 2.014102 0.00012 2.014101778 0.00015 0.000 026–0.000 184 0.000 155 74(5) 0.000 115(70)
C 12 12.000000 0.98930 12.0000 0.98893 0.988 53–0.990 37 0.988 922(28) 0.9893(8)13 13.003355 0.01070 13.003354838 0.01107 0.009 63–0.011 47 0.011 078(28) 0.0107(8)
N 14 14.003074 0.99636 14.003074005 0.996337 0.995 79–0.996 54 0.996 337(4) 0.996 36(20)15 15.000109 0.00364 15.000108898 0.003663 0.003 46–0.004 21 0.003 663(4) 0.003 64(20)
O16 15.994915 0.99757 15.994914622 0.99759 0.997 38–0.997 76 0.997 6206(5) 0.997 57(16)17 16.999132 0.00038 16.999131501 0.000374 0.000 37–0.000 40 0.000 3790(9) 0.000 38(1)18 17.999161 0.00205 17.999160419 0.002036 0.001 88–0.002 22 0.002 0004(5) 0.002 05(14)
S
32 31.972071 0.94990 31.972070690 0.9502 0.944 54–0.952 81 0.950 4074(88) 0.9499(26)33 32.971459 0.00750 32.971458497 0.0075 0.007 30–0.007 93 0.007 4869(60) 0.0075(2)34 33.967867 0.04250 33.967866831 0.0421 0.039 76–0.047 34 0.041 9599(66) 0.0425(24)36 35.967081 0.00010 35.967080880 0.0002 0.000 13–0.000 19 0.000 145 79(89) 0.0001(1)
Fe
54 53.939611 0.05845 53.939614836 0.0590 0.058 37–0.058 61 0.058 45(23) 0.058 45(35)56 55.934937 0.91754 55.934942133 0.9172 0.917 42–0.917 60 0.917 54(24) 0.917 54(36)57 56.935394 0.02119 56.935398707 0.0210 0.021 16–0.021 21 0.021 191(65) 0.021 19(10)58 57.933276 0.00282 57.933280458 0.0028 0.002 81–0.002 82 0.002 819(27) 0.002 82(4)
Electron 0.000549 0.0005458a) software from Bruker Daltonicsb) http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msisotope; The abundance values were obtained by personal communication with Peter Baker at UCSF.
Reference
1. Berglund, M., Wieser, M. E.: Isotopic compositions of the elements 2009 (IUPAC Technical Report). Pure and Applied Chemistry 83, 397-410 (2011).