Mass Spec Parameters:

The LC/LC/MS/MS analysis was performed using a ThermoFinnigan Surveyor/DecaXP+
instrument. Tryptic peptide mixtures were separated by automated two
dimensional-high performance liquid chromatography. Chromatography was
performed at 2µL/min with all buffers acidified with 0.1% formic acid.
Chromatography salt step fractions were eluted from a strong cation exchange
column (SCX) with a continuous 5% acetonitrile (ACN) background and 10 minutes
salt bumps of 0, 20, 60 and 900 mM ammonium chloride. Each salt bump was
eluted directly onto a reverse phase C18 column and washed free of salt.
Reverse phase chromatography was run in a 125 minutes gradient from 5% to 55%
ACN, and then purged at 95% ACN.  

Peptides were analyzed online with electrospray ionization (ESI) ion trap mass
spectrometry (MS) . In each MS spectrum, the 6 tallest individual peaks,
corresponding to peptides, were fragmented by collision-induced dissociation
(CID) with helium gas to produce MS/MS spectra. Gas phase fractionation (GFP)
was used to achieve maximum proteome coverage . In order to increase coverage
of lower abundance proteins, each tryptic peptide mixture was analyzed by
three sequential LC/LC/MS/MS analyses, in each case examining a different
mass/charge (m/z) range (300-650, 650-900, and 900-1500 m/z) for
data-dependent precursor ion selection for CID; fragmentation data from the
three runs were then combined for analysis.