Characterization of the mouse brain proteome using global proteomic analysis complemented with cysteinyl-peptide enrichment

Abstract

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 ( approximately 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.

Figure 1

A flowchart showing the experimental strategy: preparation of the whole mouse brain using a combination of global tryptic digestion and cysteinyl-peptide enrichment (CPE) methodology, followed by SCX fractionation and LC-MS/MS analysis of each fraction, and the peptide/protein identification results from both preparation methods.

Figure 2

(A) Comparisons of peptide and protein identifications resulting from the global tryptic peptide and cysteinyl-enriched peptide samples. The comparisons were plotted as Venn diagrams at both the peptide level (left) and the protein level (right). (B) Distribution of total sequence coverage in identified proteins. The number of identified proteins is plotted against the percent sequence coverage.

Figure 3

Comparisons of protein distributions, based on their physiochemical characteristics: (A) pI, and (B) molecular weight (MW), between the identified proteins in the dataset and the entire mouse proteome. Predicted pI values were obtained from the IPI mouse protein database and the pI distributions were plotted with 0.2 pH unit increment. MW values were also obtained from the IPI mouse protein database and the MW distributions were plotted with 2 kDa increments.

Figure 4

Protein categorization using GO identification numbers based on their cellular location, comprising 44% of the total 7792 identified proteins in the dataset.

Figure 5

(A) Histogram illustrating the distributions of the identified proteins that have transmembrane domains (TMD), as predicted by TMHMM. The two axes represent the number of TMDs (1-19) and the corresponding total number of proteins having the same amount of TMDs. (B) Comparison of the distributions of functional categories between membrane proteins and all proteins according to GO biological processes. A total of 4428 proteins were annotated by GO biological processes from the entire dataset, among which 1332 annotated proteins were membrane proteins.

Figure 6

The distribution of spectrum counts for identified proteins. The number of identified proteins was plotted against the spectrum count for each protein.