1. B. Neutral loss triggered MS3 scan of 983.1, 2+
A MS/MS of , 2+
Probe4 #
3759
RT:
35.77
NL:
3.98E4 F:
ITMS + c NSI d Full ms2 [ ]
100
983.1 95 90 85 80 75
FQSpEEQQQTEDELQDK
Xcorr: 5.20 70 65 60 55
Relative Abundance 50 45 40 35
y13+2
810.5
b13+2
836.9 30
b10+1
1316.5
b7+1
957.3
b8+1
1086.5 25
b6+1
829.2
b13+1
1673.4 20
b5+1
701.2
y6+1
748.3
b14+1
1801.4 15
y5+1
633.3
y7+1
877.4
y9+1
1106.4
y10+1
1234.5
y11+1
1362.5
y12+1
1491.6 10
y14+1
1787.6
y4+1
504.3
y13+1
1620.6 5
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 [ ]
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
Relative Abundance 50
y13+2
810.9
b5+1
603.1 45
y11+2
681.9
y7+1
877.4
y14+1
1689.6 40
b9+1
1089.5
b11+1
1333.4
b4+1
474.4 35
b3+1
345.2
y12+1
1491.8
y5+1
632.6
y6+1
748.3
y10+1
1234.5 30
y9+1
1106.5
y11+1
1362.5
b13+1
1575.5
y3+1
390.1
b14+1
1703.3 25 20
y4+1
504.3 15
b15+1
1818.9 10 5
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 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 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. Neutral loss triggered MS3 scan of 976.45, 3+
RELEELNVPGEIVESpLSp(Dha)(Dha)EESITR
Xcorr: 5.60
y4+1
476.4
y8+1
872.5
b7+1
884.4
y7+1
804.5
b13+2
740.3
y5+1
605.5
y12+2
725.2
y14+2
830.8
y15+2
895.7
y9+1
1039.3
y10+1
1153.4
y11+1
1319.6
b13+1
1479.7
b10+1
1138.4
y14+1
1661.4
y16+1
1847.8
Probe4 #
4343
RT:
41.46
NL:
5.53E3 F:
ITMS + c NSI d Full ms3 [ ]
400
600
800
1000
1200
1400
1600
1800
2000
m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
Relative Abundance
943.8
y16+2
923.6
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 Dha (Dehydroalanine) indicates the sites of the neutral loss of phosphoric acid from phosphoSer. Only prominent y- and b-fragment ions have been labeled.