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Identification of Beta-Casein Phosphopeptides by Nano LC-ESI-MS2 and MS3 Techniques

This study focuses on the identification of phosphopeptides derived from beta-casein using advanced mass spectrometry techniques. Specifically, we employed nano LC-ESI-MS2 and neutral loss triggered MS3 to accurately characterize the phosphorylation sites and fragmentation patterns of relevant peptides. The results identified key peptides such as FQSpEEQQQTEDELQDK and RELEELNVPGEIVESpLSp(Dha)(Dha)EESITR, confirming their structural features based on the unique fragmentation profiles. Our findings contribute to understanding the functional roles of phosphorylated proteins in biological processes.

catherine-dudley
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Identification of Beta-Casein Phosphopeptides by Nano LC-ESI-MS2 and MS3 Techniques

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  1. A. MS/MS of 1032.17, 2+ Probe4 # 3759 RT: 35.77 NL: 3.98E4 F: ITMS + c NSI d Full ms2 1032.17@30.00 [ 270.00-2000.00] 100 983.1 95 90 85 80 FQSpEEQQQTEDELQDK Xcorr: 5.20 75 70 65 60 55 50 Relative Abundance 45 40 y13+2 810.5 b13+2 836.9 35 b10+1 1316.5 30 b7+1 957.3 b8+1 1086.5 b6+1 829.2 b13+1 1673.4 25 b14+1 1801.4 b5+1 701.2 y6+1 748.3 20 y7+1 877.4 y10+1 1234.5 y12+1 1491.6 15 y5+1 633.3 y9+1 1106.4 y11+1 1362.5 y14+1 1787.6 10 y13+1 1620.6 y4+1 504.3 5 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z B. Neutral loss triggered MS3 scan of 983.1, 2+ Probe4 # 3760 RT: 35.78 NL: 8.81E2 F: ITMS + c NSI d Full ms3 1032.17@30.00 983.10@35.00 [ 260.00-2000.00] y14+2 845.3 100 95 90 FQ(Dha)EEQQQTEDELQDK Xcorr: 5.44 85 80 75 70 65 60 y13+1 1620.6 55 b12+2 731.4 b10+1 1218.4 y13+2 810.9 50 Relative Abundance b5+1 603.1 y14+1 1689.6 45 y11+2 681.9 y7+1 877.4 b9+1 1089.5 b11+1 1333.4 40 b4+1 474.4 b3+1 345.2 y12+1 1491.8 35 y10+1 1234.5 y5+1 632.6 y6+1 748.3 b13+1 1575.5 y11+1 1362.5 y9+1 1106.5 30 y3+1 390.1 b14+1 1703.3 25 20 y4+1 504.3 15 b15+1 1818.9 10 5 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z FIG. S1. Identification of the monophosphopeptide from beta-casein by nano LC-ESI-MS2 and by neutral loss triggered MS3. (A) Identification of the phosphopeptide FQSpEEQQQTEDELQDK by the MS2 fragmentation pattern of the peptide ion 1032.17 from the full MS scan indicates the phosphorylation site on Ser. (B) Identification of the peptide FQ(Dha)EEQQQTEDELQDK by the MS3 fragmentation pattern of the detected neutral loss fragment 983.1. Dha (Dehydroalanine) indicates the site of the neutral loss of phosphoric acid from the phospoSer. Only prominent y- and b-fragment ions have been labeled.

  2. Probe4 # 4343 RT: 41.46 NL: 5.53E3 F: ITMS + c NSI d Full ms3 1009.25@30.00 976.45@35.00 [ 255.00-2000.00] 943.8 100 RELEELNVPGEIVESpLSp(Dha)(Dha)EESITR Xcorr: 5.60 95 90 85 80 75 70 65 y16+2 923.6 60 55 Relative Abundance 50 y15+2 895.7 45 40 b13+2 740.3 y14+2 830.8 b7+1 884.4 35 y7+1 804.5 30 y9+1 1039.3 y12+2 725.2 y10+1 1153.4 y8+1 872.5 25 y14+1 1661.4 20 b10+1 1138.4 y5+1 605.5 y11+1 1319.6 y16+1 1847.8 15 y4+1 476.4 b13+1 1479.7 10 5 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z Neutral loss triggered MS3 scan of 976.45, 3+ FIG. S2. Identification of the tetraphosphopeptide from beta-casein by neutral loss triggered nano LC-ESI-MS3. The MS2 fragmentation spectrum of the peptide ion revealed no sufficient fragment information for peptide identification. Identification of the peptide RELEELNVPGEIVESpLSp(Dha)(Dha)EESITR) by neutral loss triggered MS3 of peptide ion 976.45. Dha (Dehydroalanine) indicates the sites of the neutral loss of phosphoric acid from phosphoSer. Only prominent y- and b-fragment ions have been labeled.

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