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FIGURE 2 Map of predicted peptides and their sequence coverage for all proteins in the S. cerevisiae ribosome. Peptides in
red are within the 500 to 5,000 Da mass range. Peptides in teal are heavier than 5,000 Da. The proteins are indicated along the
X axis. The top graph illustrates Asp-selective acid cleavage and the bottom graph illustrates tryptic digestion. (Reprinted with
permission from reference #18.)
microwave device for 30 minutes at 140 C.
The resulting peptides were extracted by
vortexing after addition of a 1:2 solution of 5%
formic acid: acetonitrile, concentrated, and
injected for LC-MS/MS analysis. A total of
1,241 distinct peptides were identi
nitrogen cavitation, the proteins were precipi-
tated and redissolved and four aliquots of
approximately 170
g each were loaded in
four lanes onto an 8% to 16% polyacrylamide
gel for SDS-PAGE fractionation. Each lane was
cut into eight sections and related sections
were pooled. Bands were prepared for digestion
by dehydrating and then rehydrating in acidic
aqueous solvent as proposed by Shevchenko
and colleagues for in-gel tryptic digestion. 26
Once rehydrated, the macerated gel pieces
were digested in 12.5% acetic acid in the
m
ed, repre-
senting 642 proteins from the lysate. Average
sequence coverage per protein was 8.25%.
Despite the larger protein sample required for
in-gel acid digestion, the traditional advantages
of electrophoresis d sample concentration,
cleanup, and visualization d are fully realized.
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