LCQ  - EXPERIMENTAL PROCEDURE
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	Saccharomyces cerevisiae DBY8724 cells11 (MATa GAL2 ura3 bar1::URA3) were grown with aeration at 30 °C until O.D. 0.6-0.8 in either rich YPD medium (2 % yeast extract, 1 % peptone, 2 % glucose) or synthetic YMD minimal medium (0.7 % yeast nitrogen base without amino acids and ammonium sulfate (DIFCO Bacto), 2 % glucose, 5 g/L ammonium sulfate, and 20 mg/L uracil), pelleted, washed, resuspended in buffer (20 mM Tris-HCl, 100 mM NaCl) containing 1% protease inhibitor cocktail (Calbiochem, CA) and lysed with glass beads. Soluble protein extracts were diluted to 4 mg/ml into digestion buffer (50 mM Tris HCL pH 8.0, 1.0 M Urea, 2.0 mM CaCl2), denatured at 95 °C for 10 min, and digested with sequencing grade trypsin (Sigma, MO) at 37 °C for ~20 hours.  

     Tryptic peptide mixtures were separated by automated two-dimensional high performance liquid chromatography. Chromatography was performed at 2 µL/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 minute 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 minute gradient from 5 % to 55 % ACN, and then purged at 95 % ACN.  Peptides were analyzed online with electrospray ionization ion trap mass spectrometry using a ThermoFinnigan Surveyor/DecaXP+ instrument. In each MS spectrum, the 5 tallest individual peaks 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: 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 by BioWorks (ThermoFinnegan). 

 In total, 246,820 MS/MS scans were collected for yeast rich medium (YPD) data, 241,288 scans for yeast minimal medium (YMD) data.  The probability of observing each protein and the total number of observed peptides were calculated using PeptideProphet and ProteinProphet, selecting proteins above a 5% false discovery rate (FDR) for protein identification threshold.