10
D. R. GOODLETT et al.
Fig. 1.4. Two Dimensional Phosphopeptide
Map of CD3-~ peptides. After in vitro labeling CD3-z was digested with trypsin.
Recovery of 32P-peptides from the plate
ranged from 55-95 %. As assessed by radioactive decay from the date of labeling, spots
2 and 3 contained 8.0 and 1.5 picomoles of
peptide respectively
2 3 ,...---Fi gure 5
Rgure 6 4
10
6
14
5
12
+
9 13 15
7 • 11
origin
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1994). The protein was digested with trypsin, peptides were separated by 2DPP
mapping, extracted and quantitated by radioactive decay. Spot 3 (Fig. 1.4) was
analyzed by I!LC/MS/MS. Injecting 10 % of the extract from spot 1 (150 femtomoles of peptide) produced the spectrum shown in Fig. 1.5. Injecting more than
10 % of a spot often resulted in increased chemical noise originating from the cellulose slurry and failure to observe any peptide signal. The quality of the data was
good enough to enable the phosphorylation site to be unambiguously identified
within the known amino acid sequence of the protein. Spot 2 was analyzed twice
so that a comparison could be made between in-source cm (Fig. 1.6.A) and tandem cm (Fig. 1.6.B). These two different types of fragmentation produce spectra
that, when compared, can aid in locating phosphorylated residues. As can be
seen in Fig. 1.6A in-source CID produces a cleaner looking spectrum from dissociation of high charge states rather than from a single selected charge state. If sufFig. 1.5. Tandem MS of
[M + 3Hj3+ CD3-z peptide
(DTp YDALHMQTLAPR)
taken from spot 3 in Fig.
104. Data acquired using an
auto-MS/MS routine that
initiated tandem MS based
on preset signal intensity.
To ensure acquisition of
interpretable data in automated mode the instrument control language
routine changes V COFF =
[M + 2Hj2+1-25.5 by
about-1.5V per tandem
MS scan. Ten % of spot 3
or 150 fern tomales analyzed by IlLC-MS/MS
Q)
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y4
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200
400
600 BOO
1000
1200
1400
m/z