Cells: wild-type yeast

Experiment:  
	- grown in YPD to log-phase
	
CYS modification: 
NO

Mass spectrometry:
LC/LC-MS/MS on LTQ-Orbitrap

 For mass spectrometry analysis, eight runs were performed on an LTQ-Orbitrap varying a range of parameters for optimization. SCX salt steps were performed by injecting 10 ul of Ammonium Chloride solutions of varying molarity, namely (0, 15, 60, 900) mM or (0, 20, 100, 900) mM in a 5% ACN, 0.1% Formic Acid background onto a strong cation exchange column (Thermo BioBasic-SCX 100mm X 0.180 mm ID) with a flow rate of 800 nl/min. Reverse phase chromatography was performed on a Thermo BioBasic-18 column 100mm X 0.10 mm ID running 38 nl/min for 90 or 120 minutes with varying ACN concentrations on a background of 0.1% Formic Acid. The column eluent was nano-electro-sprayed at 1.95kV from a 10 um tip (New Objective). FTMS resolution was set at 60,000 or 100,000. Between 6 and 10 MS/MS spectra (using 1 or 3 microscans) were acquired per MS scan. Many other individual parameters were varied including exclusion list settings, charge state rejection, mono-isotopic precursor selection, minimum signal required for MS2 scanning, MS2 isolation width, and use of a mass lock (445.120025).

Analysis:

SEQUEST (Bioworks)
Peptide/ProteinProphet v4.0