Increased proteome coverage by combining PAGE and peptide isoelectric focusing: comparative study of gel-based separation approaches

Abstract

The in-depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.

Keywords: IPGs; PAGE; Peptide IEF; Shotgun proteomics; Technology.

Figure 1

Proteome profiling by PAGE-pIEF-LC-MS/MS. (A) Schematic representation of the workflow. Proteins were first separated according to MW by 1D PAGE. Gels were fractionated, and proteins from gel slice #10 of the 1D PAGE were digested in gel and then analyzed by LC-MS/MS (PAGE#10-LC-MS/MS) or, after additional separation of the extracted peptides, by pIEF prior to LC-MS/MS (PAGE#10-pIEF-LC-MS/MS). (B) Overlap between proteins identified in PAGE#10-LC-MS/MS and PAGE#10-pIEF-LC-MS/MS and box plots showing the MS intensity distribution of “common” proteins, i.e. those identified by both PAGE#10-LC-MS/MS and PAGE#10-pIEF-LC-MS/MS (I) and of the “newly identified” proteins, i.e. those identified only when pIEF was also performed (II). (C) Overlap of identified peptides between PAGE#10-LC-MS/MS and PAGE#10-pIEF-LC-MS/MS.

Figure 2

(A) Schematic presentation of the three workflows employed in the study, showing the number of identified peptides (*) and proteins (**). (B) Overlap of the proteins identified by GeLC-MS/MS, pIEF-LC-MS/MS, or PAGE-pIEF-LC-MS/MS. (C) Intensity distributions of proteins identified in all three workflows (common proteins) and those identified only by PAGE-pIEF-LC-MS/MS.

Figure 3

Overlap of identified proteins across replicate pIEF analyses of tryptic peptides from the same PAGE subfraction (A–C), correlation of intensity of proteins identified in both pIEF replicate analyses (D–F) and distribution of identified peptides on the IPG strips (G–I).

Figure 4

Distribution of identified peptides across the IPG strip from pIEF analyses of tryptic peptides from the same PAGE subfraction of three replicate PAGE lanes.