<p>Top-Down Analysis of highly postranslationally modified peptides by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry</p><p>§* § †,‡ §,* Andres Guerrero , Larry Lerno , Daniela Barile , Carlito B. Lebrilla</p><p>§Department of Chemistry, University of California at Davis, One Shields Avenue, Davis, CA, 95616, USA</p><p>†Department of Food Science, University of California at Davis, One Shields Avenue, Davis, CA, 95616, USA</p><p>‡Foods for Health Institute, University of California at Davis, One Shields Avenue, Davis, CA, 95616, USA</p><p>Supplementary material</p><p> a b c d Table S1. ECD product ion assignments of GMPa-P and GMPb-P. Dalton. Mass accuracy in ppm. Phosphorylated serine in minuscule Identified charge states.</p><p>For m Exact Massa Mass Accuracyb Type Sequencec m/z (Monoisotopic)d MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA A 6784.3425 -0.1 Unfrag. V 970.0547 (7+), 1131.5636 (6+) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA</p><p>A 6766.3320 -0.2 Unfrag. -H2O V 967.4826 (7+), 1128.5601 (6+) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA</p><p>A 6748.3214 -0.2 Unfrag. -2H2O V 964.9096 (7+), 1125.5592 (6+) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA</p><p>A 6731.2948 -3.1 Unfrag. -2H2O, NH3 V 1122.7254 (6+) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA</p><p>A 6730.3108 2.8 Unfrag. -3 H2O V 962.3337 (7+) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA</p><p>A 6713.2843 -2.5 Unfrag. -3H2O, NH3 V 1119.7229 (6+) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA</p><p>A 6712.3003 1.3 Unfrag. -4H2O V 959.7622 (7+)</p><p>A 6667.2635 1.3 c63 -NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA 1112.0499 (6+), 1334.2553 (5+)</p><p>A 6649.2530 0.2 c63 - H2O, NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA 1109.0481 (6+), 1330.6559 (5+)</p><p>A 6632.2264 -1.2 c63 - H2O, 2NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA 1327.2527 (5+)</p><p>A 6631.2424 2.0 c63 -2H2O, NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA 948.1836 (7+), 1106.0439 (6+) A 6613.2530 4.1 c62 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTST 945.6081 (7+), 1103.0450 (6+)</p><p>A 6613.2319 0.9 c63 -3H2O, NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTSTA 945.6081 (7+), 1103.0450 (6+) A 6595.2424 3.9 c62 - H2O MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTST 1100.0422 (6+)</p><p>A 6559.2213 1.6 c62 -3H2O MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTVQVTST 1094.0412 (6+)</p><p>A 6061.9615 1.4 c57 - H2O, NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTV 1213.1964 (5+)</p><p>A 6043.9509 0.8 c57 -2H2O, NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIESPPEINTV 1209.5951 (5+)</p><p>A 5329.5808 -0.4 c50 -NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIES 1066.7224 (5+)</p><p>A 5242.5488 0.9 c49 -NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIE 1049.3146 (5+)</p><p>A 5224.5382 1.0 c49 - H2O, NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVIE 1045.7124 (5+)</p><p>A 5113.5062 -3.6 c48 -NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEVI 1023.5107 (5+)</p><p>A 4999.4381 3.7 c47 - H2O MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEV 1250.6104 (4+)</p><p>A 4982.4116 0.5 c47 - H2O, NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPEV 997.2876 (5+) A 4918.3803 -1.0 c46 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPE 1230.3518 (4+)</p><p>A 4900.3697 2.4 c46 - H2O MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDsPE 1225.8450 (4+) A 4410.2596 2.0 c42 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLE 1470.7551 (3+) A 4281.2170 -1.2 c41 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATL 1071.0610 (4+) A 4168.1330 3.8 c40 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVAT 1042.7857 (4+), 1390.0415 (3+)</p><p>A 4150.1224 1.5 c40 - H2O MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVAT 1038.2845 (4+)</p><p>A 4132.1118 0.6 c40 -2H2O MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVAT 1033.7828 (4+) A 3409.7519 3.4 c32 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTE 1137.2517 (3+)</p><p>A 3262.6988 -0.5 c31 - H2O MAIPPKKNQDKTEIPTINTIASGEPTSTPTT 1088.2383 (3+) A 2880.5135 2.7 c27 MAIPPKKNQDKTEIPTINTIASGEPTS 960.8410 (3+), 1440.7544 (2+)</p><p>A 2578.3545 1.8 c24 -NH3 MAIPPKKNQDKTEIPTINTIASGE 645.3449 (4+), 860.1222 (3+), 1289.6728 (2+) A 2466.3385 1.9 c23 MAIPPKKNQDKTEIPTINTIASG 822.7839 (3+), 1233.6694 (2+) A 2409.3170 2.5 c22 MAIPPKKNQDKTEIPTINTIAS 1205.1592 (2+) A 2322.2850 2.4 c21 MAIPPKKNQDKTEIPTINTIA 1161.6434 (2+) A 2251.2479 2.7 c20 MAIPPKKNQDKTEIPTINTI 1126.1246 (2+)</p><p>A 2234.2213 -1.8 c20 -NH3 MAIPPKKNQDKTEIPTINTI 745.4133 (3+) A 2037.1161 2.9 c18 MAIPPKKNQDKTEIPTIN 1019.0588 (2+) A 1923.0732 1.9 c17 MAIPPKKNQDKTEIPTI 962.0385 (2+) A 1809.9891 1.5 c16 MAIPPKKNQDKTEIPT 905.4969 (2+) A 1708.9415 1.5 c15 MAIPPKKNQDKTEIP 854.9731 (2+)</p><p>A 1509.7557 -8.5 z15 -NH3 SPPEINTVQVTSTAV 1509.7686 (1+) A 1498.8046 -0.2 c13 MAIPPKKNQDKTE 749.9061 (2+)</p><p>A 1428.7304 -7.1 c13 -2H2O, 2NH3 MAIPPKKNQDKTE 1428.7405 (1+) A 1369.7620 -1.1 c12 MAIPPKKNQDKT 685.3854 (2+)</p><p>A 1351.6655 0.1 z14 -3H2O, 2NH3 PPEINTVQVTSTAV 1351.6653 (1+) A 1268.7144 -2.1 c11 MAIPPKKNQDK 634.8621 (2+) A 1140.6194 -0.1 c10 MAIPPKKNQD 570.8147 (2+), 1140.6158 (1+)</p><p>A 1099.5756 6.3 z11 -NH3 INTVQVTSTAV 1099.5686 (1+) A 1025.5925 0.7 c9 MAIPPKKNQ 513.3013 (2+), 1025.5892 (1+) A 897.5339 2.6 c8 MAIPPKKN 897.5316 (1+) A 783.4910 1.0 c7 MAIPPKK 783.4902 (1+) A 655.3960 -1.6 c6 MAIPPK 655.3970 (1+) B 6752.3891 -1.1 Unfrag. MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEAsPEVIESPPEINTVQVTSTAV 965.4914 (7+)</p><p>B 6735.3625 -3.0 Unfrag. -NH3 MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEAsPEVIESPPEINTVQVTSTAV 1123.4032 (6+) B 4180.1694 0.5 c40 MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVAT 1045.7973 (4+) B 2880.5135 1.0 c27 MAIPPKKNQDKTEIPTINTIASGEPTS 960.8411 (3+), 1440.7597 (2+)</p><p>B 2578.3545 1.1 c24 -NH3 MAIPPKKNQDKTEIPTINTIASGE 860.1221 (3+) B 2466.3385 0.0 c23 MAIPPKKNQDKTEIPTINTIASG 1233.6729 (2+) B 2322.2850 0.6 c21 MAIPPKKNQDKTEIPTINTIA 1161.64549 (2+) B 2037.1161 1.3 c18 MAIPPKKNQDKTEIPTIN 1019.0604 (2+) B 1923.0732 1.2 c17 MAIPPKKNQDKTEIPTI 962.0391 (2+) B 1708.9415 0.5 c15 MAIPPKKNQDKTEIP 854.9740 (2+) B 1498.8046 1.2 c13 MAIPPKKNQDKTE 749.9051 (2+) B 1369.7620 1.8 c12 MAIPPKKNQDKT 685.3834 (2+) B 1268.7144 1.6 c11 MAIPPKKNQDK 634.8598 (2+) B 1140.6194 0.9 c10 MAIPPKKNQD 1140.6183 (1+)</p><p>B 1139.5494 -0.2 z12 -4H2O, 2NH3 EINTVQVTSTAV 1139.5496 (1+) B 1025.5925 1.7 c9 MAIPPKKNQ 513.2993 (2+), 1025.5905 (1+) B 897.5339 1.7 c8 MAIPPKKN 897.5324 (1+) B 783.4910 3.0 c7 MAIPPKK 783.4886 (1+) B 655.3960 2.0 c6 MAIPPK 655.39470 (1+) Table S2. Theoretical and experimental isotopic distribution of GMPa-P</p><p>Relative ion intensity Isotope Experimental Theoretical M 15.15 12.58 M+1 48.34 43.86 M+2 79.23 79.65 M+3 93.61 100 M+4 100 97.28 M+5 80.46 77.98 M+6 57.25 53.53 M+7 36.13 32.28 M+8 19.62 17.43 M+9 12.6 8.54 Figure S1. ECD Tandem-MS results of GMPa-P (a) and GMPb-P (b).</p>