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Fig. 2. Two 2D gels were run with unlabeled (
a
) and NHS-Cy5-labeled (
b
) proteins. Gels were Coomassie-stained, and
equivalent protein spots (1-10 from A, and 11-20 from B) were excised from the gels, digested with trypsin, and identifi ed
by MALDI MS.
The protein standard mixture run in one gel was previously labeled
with Cy5 minimal label, while the other was left unlabeled. After
running, both gels were stained with Coomassie blue, and twenty
equivalent protein spots were “picked” from both gels (see Fig.
2
),
digested with trypsin, and identifi ed following the procedures
described above. The results are shown in Table
2
. All equivalent
proteins, labeled and unlabeled, were identifi ed as identical pro-
teins; however, two pairs of protein spots (7 and 17, 10 and 20)
were identifi ed by MALDI MS as proteins not expected to be in
the protein standard mixture. However, all identifi cations were
consistent between the two gels. Despite the unexpected identifi -
cations, it is clear that the Cy5 label did not affect the protein
identifi cation process, as indicated by the MS and MS/MS scores
(column 7 in Table
2
) and the peptide sequenced ion scores (column
8 in Table
2
), even the peptide counts were very similar (column 6
in Table
2
).
4. Notes
1. When covering the gel with pure water, be careful not to add
too much water, but rather add just enough to cover the gel
surface, leaving the picking references visible. Otherwise, it will
be diffi cult for the camera to detect the picking references. In
some cases, e.g., after Coomassie blue staining, it is necessary to
remove the acrylamide that is covering the picking references
and replace the original picking tags with unstained tags.
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