Characterization of the Mouse Brain Proteome Using Global Proteomic Analysis Complemented with Cysteinyl-Peptide Enrichment

We report a global proteomic approach for analyzing brain tissue and for the first time a comprehensive
characterization of the whole mouse brain proteome. Preparation of the whole brain sample incorporated
a highly efficient cysteinyl-peptide enrichment (CPE) technique to complement a global enzymatic
digestion method. Both the global and the cysteinyl-enriched peptide samples were analyzed by SCX
fractionation coupled with reversed phase LC−MS/MS analysis. A total of 48 328 different peptides
were confidently identified (>98% confidence level), covering 7792 nonredundant proteins (∼34% of
the predicted mouse proteome). A total of 1564 and 1859 proteins were identified exclusively from the
cysteinyl-peptide and the global peptide samples, respectively, corresponding to 25% and 31%
improvements in proteome coverage compared to analysis of only the global peptide or cysteinyl-peptide samples. The identified proteins provide a broad representation of the mouse proteome with
little bias evident due to protein pI, molecular weight, and/or cellular localization. Approximately 26%
of the identified proteins with gene ontology (GO) annotations were membrane proteins, with 1447
proteins predicted to have transmembrane domains, and many of the membrane proteins were found
to be involved in transport and cell signaling. The MS/MS spectrum count information for the identified
proteins was used to provide a measure of relative protein abundances. The mouse brain peptide/protein database generated from this study represents the most comprehensive proteome coverage
for the mammalian brain to date, and the basis for future quantitative brain proteomic studies using
mouse models. The proteomic approach presented here may have broad applications for rapid
proteomic analyses of various mouse models of human brain diseases.
Keywords: cysteinyl-peptide enrichment • mass spectrometry • mouse brain proteome • protein categorization •
proteome coverage