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Platform [ 1 , 2 ]. In this platform, up-front fractionation of pro-
teomic samples is performed by one-dimensional SDS-PAGE fol-
lowed by shotgun liquid chromatography- electrospray tandem
mass spectrometry (LC-MS/MS) analysis of proteins from indi-
vidual gel bands. The resulting sequence and gel-migration infor-
mation are then assembled into two- dimensional “peptographs,”
which, when combined with spectral counting data obtained
through LC-MS/MS analysis, provide a semiquantitative topo-
graphical map for all detectable proteins in a sample.
In a typical PROTOMAP experiment (Fig. 1 ), control and
experimental samples, one in which the protease or proteolytic
pathway of interest is activated or inactivated, are run on SDS-
PAGE. Each distinct gel lane is cut into bands of defi ned length
intervals. The proteins in each band are then separately digested
into peptides, analyzed, and sequenced using LC-MS/MS.
Through the use of custom software detailed later in the protocol,
the data are reassembled into a two-dimensional “peptograph” for
each protein identifi ed in the sample. The peptograph displays the
sequence coverage for that protein in each band as well as the rela-
tive migration rate on the gel. Proteolyzed proteins are identifi ed
as proteins that experience a shift to a lower molecular weight in
the protease-active sample. By comparing the spectral counts
obtained for the full-length parental form of a substrate in the
protease- active sample to the control sample, one can estimate the
magnitude of cleavage event. This combined with near-complete
topographical descriptions of cleavage patterns (often yieldingFig. 1 Typical workfl ow for PROTOMAP experiment. Proteomes from control and experimental systems are
separated by one-dimensional SDS-PAGE and the gel lanes cut into bands at defi ned intervals. Proteins are
digested in-gel with trypsin, and the resulting peptides are analyzed by one-dimensional reverse-phase
LC-MS/MS. The proteomic data are integrated into peptographs, which plot sequence coverage for a given
protein versus SDS-PAGE migration ( left panel ). In the right panel, the peptograph also displays average spec-
tral counts for each protein in each gel band. Proteins that undergo a proteolytic cleavage event are identifi ed
by a shift in migration from higher ( parental, red ) to lower ( fragments, blue ) MW species in control versus
experimental samples. The sequence coverage shown in the left panel provides a topographical map of the
protein fragments that persist following proteolysis. The magnitude of proteolysis is estimated by comparing
spectral counts of the parental protein in the control versus experimental samples
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