Proteome experiments

Two-dimensional gel-based proteome experiments

Quantitative protein profiling by 2-DE followed by gel image analyses and mass spectrometry was performed essentially as described (Kimura et al., 2006. PMID:16767787) with a few modifications(Kimura et al., manuscript in preparation). Briefly, whole-cell protein samples (250 µg) were separated by isoelectric focusing (IEF) in the first dimension and by SDS-PAGE in the second dimension, and subsequently the proteins in the gel were stained with SYPRO Ruby (Molecular Probes). The 2-DE gel images were scanned using ProXPRESS fluorescent imager (PerkinElmer, Inc.) and analyzed using Progenesis Workstation (Nonlinear Dynamics Ltd.). The protein levels for each spot on a given gel were normalized by median centering.
  In order to identify the proteins in each spot on the gels, the mass spectra of digest fragments originating from the proteins excised from each gel were obtained using MALDI/TOF mass spectrometry (AXIMA-CFR, Shimazu Co.) and/or nanoLC-ESI/IT mass spectrometry (Esquire 3000plus, Bruker Daltonics Inc.). The obtained mass spectra data were searched against IPI database using MASCOT program (Matrix Science) with Peptide Mass Fingerprint (PMF) or MS/MS Ion Search (MIS).
  To define a spot on a gel that corresponded to one on another gel, all gel images were superimposed. When the position of a spot on a gel matched that of one on another, we considered them as identical protein spots.
  The reliability of protein identification was categorized into the following five classes, primarily according to their degree of confidence of the database search results of MS analysis data (dispersion of peptide mass tolerance, the extent of sequence coverage, the number of matched peptides, and the protein mass accuracy).

Reliability ClassDescription
A Conclusive; based solely on the MS analysis data
B Most likely; based solely on the MS analysis data
C Likely; supported by comigration of the previously identified proteins on 2-DE and consistent with the predicted MS data for the identified proteins, although the MS data alone could not identify the protein with sufficient confidence.
D Tentative; supported only by their electrophoretic behaviors on 2-DE
E Unidentified; could not identified even after combination analysis of 2-DE data and MS data