The PMF data was acquired on 4800 Plus MALDI TOF/TOF (ABI, USA) system working in
a reflector mode with an accelerating voltage of 25 KV.  The machine was calibrated
to below 10ppm accuracy by using the standard peptide mix spanning a mass range of
800-4000 m/z.  The PMF data was screened for preliminary protein Ids using GPS software
v3.5 (Global Proteomic Solution, ABI, USA) by searching the databases NCBI (v2008)
and SWISS-PROT (v20090313) using MASCOT search engine.  Search parameters included
carbamidomethylation of cysteine as fixed modification, methionine oxidation as variable
modification, 1 missed cleavage site with an error tolerance of less than 50 ppm.
The protein Ids were further validated based on MASCOT score, sequence coverage,
number of peptides matched vs number submitted and matching of theoretical and experimental
pI and Mr of the protein spot obtained from gel analysis.  The screened Ids were
further confirmed by MS/MS of chosen peptides from the PMF list through fragmentation
by high laser in a CID tube.  The resulting MS/MS ion spectra were also analyzed
in a similar manner by a database search using MASCOT.   Protein Ids with high MS/MS
ion score in conjunction with PMF data were chosen as candidate Ids.