Protein identification using tryptic peptides for protein array pixelation was performed using an LCQ DecaXP + ITmass spectrometer (Thermo Electron) interfaced with an Eldex MicroPro pump and an autosampler. Tryptic peptides were separated by
RP-HPLC on a C18 nanocapillary column (0.75 mm id x 100 mm packed with 5 mm Magic particles, Michrom BioResources). Solvent A was 0.58% acetic acid in Milli-Q water, and solvent B was 0.58% acetic acid in ACN. Peptides were eluted into the mass spectrometer at 200 nL/min using an ACN gradient. In some experiments with complex samples, gradient lengths were increased to enhance detection of additional proteins. The mass spectrometer was set to repetitively scan m/z from 375 to 1600 followed by data-dependent MS/MS scans on the three most abundant ions with dynamic exclusion enabled. The MS/MS spectra were searched against the International Protein Index (IPI, version 2.21) for protein identifications using SEQUEST. Consistent with the HUPO recommendations, peptide identifications were filtered using the following criteria: Xcorr > 1.9 for charge state 11, Xcorr > 2.2 for charge state 2+,or Xcorr > 3.75 for charge state 31; DCn > 0.1; Rsp < 4.
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