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Phosphorylation Site Mapping
Identify sites of protein phosphorylation using chromatography columns that provide a high yield of
phosphopeptides plus Data Dependent Neutral Loss MS 3
scanning with the Finnigan LTQ to selectively
perform MS 3
on phosphopeptides.
Relative Abundance
100
50
100
90
80
70
60
50
40
30
20
10
20 .1 5
22 .6 4
27 .6 3
33 .1 3
28 .2 0
33 .5 9
35 .7 6
37 .3 6
39 .5 2
41 .4 0
MS
MS 2
MS 3
41 .7 1
19 .7 3
0
4. 55 9. 44
15 .4 6 42 .7 6 49 .0 4 57 .3 4
0 10 20 30 40 50 60
100
0
476.27 706.08
688.29
1031.78
1058.62
982.624
747.31 1013.78
503.37 632.39 876.39
1105.42
390.18
844.91
1375.65
1490.75
1361.58
1619 .71
1619.72
80
964.67
60
747.34
1688.81
632.38
40 328.26
503.42
20
603.30
345.07
827.52
1088.34
859.53
1105.56 1361 .71 1601.85
1332 .52
1233.69
1491.61
1070.57
1199.39
1574.61
1817.71
1800 .74
Time (min) m/z
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Time (min)
100
50
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
100
50
0
0 10 20 30 40 50
100
50
0
40 m M
150 160
0 10 20 30 40 50 60 70 80
Time (min)
90 100 110 120 130 140
100
50
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
100
50
80 m M
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
100
50
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
100
50
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
100
50
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
100
50
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
100
50
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
100
50
(Shows
neutral loss)
0 m M
10 m M
20 m M
60 m M
100 m M
120 m M
150 m M
200 m M
400 m M
800 m M
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (min)
Relative Abundance
40 0 60 0 80 0 1000 1200 1400 1600 1800 2000
Analysis of a beta casein digest, showing base peak LC/MS analysis, MS spectrum, MS/MS spectrum showing neutral loss
indicative of a phosphopeptide, MS 3 spectrum showing excellent fragmentation, and definitive identification by SEQUEST.
FQS*EEQQQTEDELQDK
Multidimensional Protein ID (MudPIT)
Analyze complicated protein digest mixtures with automated, on-line multidimensional LC/MS analysis. Peptides
are retained on a strong cation exchange column, desalted and concentrated on peptide traps, then resolved on a
high-performance C18 column and extensively mined for peptides by the Finnigan LTQ. Results from all salt steps
are compiled in a final Multiple File Consensus Report by BioWorks.
Neurofilament triple-m protein (125 ng/L in normal CSF)
present at < 1 fmol in this analysis
Partial list of
proteins found
ProteomeX
Methods
Accession Unique Coverage
Number Protein Peptides Percentage
P02763 ALPHA-1-ACID GLYCOPROTEIN 2 PRECURSOR 5 18
P19652 ALPHA-1-ACID GLYCOPROTEIN 2 PRECURSOR 8 28
P04217 ALPHA-1B-GLYCOPROTEIN 12 25
P01009 Alpha-1-antitrypsin precursor 10 26
P02765 ALPHA-2-HS-GLYCOPROTEIN PRECURSOR 10 31
P01023 ALPHA-2-MACROGLOBULIN PRECURSOR 35 48
P01011 ALPHA-1-ANTICHYMOTRYPSIN PRECURSOR 25 84
P43652 AFAMIN PRECURSOR (ALPHA-ALBUMIN) 3 25
P02768 SERUM ALBUMIN PRECURSOR 67 58
P01876 IG ALPHA-1 CHAIN C REGION 2 25
P01019 ANGIOTENSINOGEN PRECURSOR 11 25
P01008 ANTITHROMBIN-III PRECURSOR (ATIII) 3 24
P02647 APOLIPOPROTEIN A-I PRECURSOR (APO-AI) 6 16
P02652 APOLIPOPROTEIN A-II PRECURSOR (APO-AII) 5 20
P06727 APOLIPOPROTEIN A-IV PRECURSOR (APO-AIV) 4 15
P06727 APOLIPOPROTEIN A-IV PRECURSOR (APO-AIV) 7 25
P05090 APOLIPOPROTEIN D PRECURSOR 8 49
P02649 APOLIPOPROTEIN E PRECURSOR (APO-E) 11 32
P02749 Beta-2-glycoprotein I precursor (Apolipopro 5 12
P32247 BOMBESIN RECEPTOR SUBTYPE-3 (BRS-3) 5 24
P09871 COMPLEMENT C1S COMPONENT PRECURSOR 2 35
P49454 CENP-F KINETOCHORE PROTEIN 3 45
P00450 CERULOPLASMIN PRECURSOR (FERROXIDASE) 4 25
P00751 COMPLEMENT FACTOR B PRECURSOR 23 37
P08603 COMPLEMENT FACTOR H PRECURSOR 15 42
P10909 Clusterin precursor (Complement-associated 15 25
P09871 Complement C1s component precursor 2 24
P01024 COMPLEMENT C3 PRECURSOR 38 37
P01028 COMPLEMENT C4 PRECURSOR 32 23
Q12860 CONTACTIN PRECURSOR (GLYCOPROTEIN GP135) 6 16
P11532 DYSTROPHIN 5 25
P02671 FIBRINOGEN ALPHA/ALPHA-E CHAIN PRECURSOR 13 34
P02751 FIBRONECTIN PRECURSOR (FN) (COLD-INSOLUBLE 3 24
P01857 IG GAMMA-1 CHAIN C REGION 21 60
P01859 IG GAMMA-2 CHAIN C REGION 14 30
P01860 IG GAMMA-3 CHAIN C REGION (HEAVY CHAIN DISEA 13 34
P01861 CHAIN C REGION 3 15
P01777 HAIN V-III REGION TEI 3 14
P01834 HAIN C REGION 3 50
P80362 HAIN V-I REGION WAT 7 21
P18136 HAIN V-III REGION HIC PRECURSOR 6 16
P06396 GELSOLIN PRECURSOR, PLASMA 16 27
P02023 HEMOGLOBIN BETA CHAIN 32 23
P02790 HEMOPEXIN PRECURSOR (BETA-1B-GLYCOPROTEIN) 12 25
P00738 HAPTOGLOBIN-2 PRECURSOR 15 25
P00739 HAPTOGLOBIN-RELATED PROTEIN PRECURSOR 2 24
P01042 Kininogen Alpha-2-thiol proteinase inhibitor 38 37
P01597 KAPPA CHAIN V-I REGION DEE 32 23
P01842 IG LAMBDA CHAIN C REGIONS 3 50
Q92876 KALLIKREIN 6 PRECURSOR (PROTEASE M) (NEURO 2 23
P29622 Kallistatin precursor (Kallikrein inhibitor) 2 25
Q9UM88 Beta 2-microglobulin protein 9 70
P36955 MENT EPITHELIUM-DERIVED FACTOR PRECURSOR 6 23
P05155 PLASMA PROTEASE C1 INHIBITOR PRECURSOR (C1 I 13 34
P00747 PLASMINOGEN PRECURSOR 33 44
P02787 SEROTRANSFERRIN PRECURSOR 34 44
P05452 TETRANECTIN PRECURSOR (TN) (PLASMINOGEN-KRI 6 16
P07477 TRYPSINOGEN I PRECURSOR 15 33
P02766 Transthyretin precursor (Prealbumin) (TBPA) 6 16
P02774 VITAMIN D-BINDING PROTEIN PRECURSOR (DBP) 7 18
P04004 TRONECTIN PRECURSOR (SERUM SPREADING FACTOR) 2 35
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