Peptide separations by on-line MudPIT compared to isoelectric focusing in an off-gel format: application to a membrane-enriched fraction from C2C12 mouse skeletal muscle cells

Abstract

High-resolution peptide separation is pivotal for successful shotgun proteomics. The need for capable techniques propels invention and improvement of ever more sophisticated approaches. Recently, Agilent Technologies has introduced the OFFGEL fractionator, which conducts peptide separation by isoelectric focusing in an off-gel setup. This platform has been shown to accomplish high resolution of peptides for diverse sample types, yielding valuable advantages over comparable separation techniques. In this study, we deliver the first comparison of the newly emerging OFFGEL approach to the well-established on-line MudPIT platform. Samples from a membrane-enriched fraction isolated from murine C2C12 cells were subjected to replicate analysis by OFFGEL (12 fractions, pH 3-10) followed by RP-LC-MS/MS or 12-step on-line MudPIT. OFFGEL analyses yielded 1398 proteins (identified by 10,269 peptides), while 1428 proteins (11,078 peptides) were detected with the MudPIT approach. Thus, our data shows that both platforms produce highly comparable results in terms of protein/peptide identifications and reproducibility for the sample type analyzed. We achieve more accurate peptide focusing after OFFGEL fractionation with 88% of all peptides binned to a single fraction, as compared to 61% of peptides detected in only one step in MudPIT analyses. Our study suggests that both platforms are equally capable of high quality peptide separation of a sample with medium complexity, rendering them comparably valuable for comprehensive proteomic analyses.

Figure 1

A) Flow-chart of experimental design: After isolation via silica-bead coating from murine C2C12 cells, membrane-enriched samples were digested and subjected to either OFFGEL followed by RP-LC-MS/MS or MudPIT analysis B) Western Blotting shows high enrichment of typical proteins for the respective fractions homogenate (H) and membrane (M)

Figure 2

A) Protein identifications are comparable between methods and across samples B) The majority of proteins identified with the two approaches are identical C/D) 4-way Venn-diagrams for OFFGEL and MudPIT analyses comparing protein identifications in all sample runs show comparable reproducibility

Figure 3

A) Comparison of transmembrane proteins detected with both techniques shows the same percentage of overlap as the comparison of all protein identifications depicted in Fig. 2B B) The average number of peptide detections per sample for both platforms is comparable C) Visualization of peptide distribution into steps/fractions shows a trimodal repartition after OFFGEL separation whereas the majority of peptides are detected in the first steps of MudPIT analyses

Figure 4

Pie charts and heat maps depict the superior focusing quality of OFFGEL: 88 % of all peptides are confined to one single fraction, whereas in MudPIT analyses only 61 % of all peptides are detected in a single step. More common detection of peptides in multiple steps is also mirrored in the heat maps, depicting summed spectral counts for all samples across steps/fractions