Biology Reference
In-Depth Information
protein candidates after the peptide mass fi ngerprint acquisition,
then isoelectric point and molecular weight range information
obtained from the 2D gel can help restrict the search parame-
ters. Should this approach also fail, one can compare the MS
spectra obtained for all the protein and blank gel spots in the
data acquisition set searching for small and unique MS peaks to
be used as precursors for MS/MS experiments (if available, AB
Sciex Peak Explorer software can be of assistance in this task).
Using a “Link Spectrum Traces” option allows resizing the
mass-to-charge axis range for all the displayed spectra facilitat-
ing the spectral comparison and analysis. Once a preliminary
protein identity emerges from the initial MS and MS/MS
experiments and from the analysis of the location of the spots
on the 2D gel, the remaining MALDI spots are used to increase
the protein coverage and to differentiate between different
protein forms. On the MALDI 4800 TOF/TOF instrument,
MS/MS data can be acquired either with or without the colli-
sion gas with similar results.
11. Both MS and MS/MS spectra are acquired during the initial
automated MALDI run. High-resolution MS spectrum of a
single protein digest can be used for protein identifi cation.
This is called peptide mass fi ngerprinting and is independent of
the protein identifi cation procedure that relies on matching
experimental MS/MS peptide spectra to the in silico theoreti-
cal MS/MS spectra generated for each protein digest in a data-
base. Proteomic “Aldente” software is frequently used in our
laboratory for this task (
20
). First, MALDI-MS peak lists are
background-subtracted in a Microsoft Excel spreadsheet to
remove MS contaminant peaks (see Notes 9 and 10). Then the
resulting MS peak lists are submitted to Aldente, initially for
further contaminant identifi cation (usually human keratins),
followed by the protein identifi cation to give an initial protein
and peptide report. This report is then compared with the pro-
tein identifi cation data obtained from the automated data
acquisition MS/MS experiments processed by the Protein
Pilot or Mascot software in search for additional MS peptide
peaks lacking their MS/MS counterparts. These MS peaks
proposed by Aldente are used to acquire more MS/MS data
using the remaining MALDI spot(s) in order to maximize pro-
tein coverage and thus protein identifi cation probability. The
fi nal cumulative data report in Excel shows a list of peptides
and proteins with their probability scores. The probability
scores depend on the proteomic software platform, for exam-
ple, Protein Pilot characterizes quality of every match between
a MS/MS spectrum of an identifi ed peptide and its theoretical
barcode-like spectrum with a peptide probability score 0-99.
The protein probability score corresponds to a product of the
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