Electroblotting through a tryptic membrane for LC-MS/MS analysis of proteins separated in electrophoretic gels

Abstract

Digestion of proteins separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) remains a popular method for protein identification using mass-spectrometry based proteomics. Although robust and routine, the in-gel digestion procedure is laborious and time-consuming. Electroblotting to a capture membrane prior to digestion reduces preparation steps but requires on-membrane digestion that yields fewer peptides than in-gel digestion. This paper develops direct electroblotting through a trypsin-containing membrane to a capture membrane to simplify extraction and digestion of proteins separated by SDS-PAGE. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifies the extracted peptides. Analysis of peptides from different capture membrane pieces shows that electrodigestion does not greatly disturb the spatial resolution of a standard protein mixture separated by SDS-PAGE. Electrodigestion of an Escherichia coli (E. coli) cell lysate requires four hours of total sample preparation and results in only 13% fewer protein identifications than in-gel digestion, which can take 24 h. Compared to simple electroblotting and protein digestion on a poly(vinylidene difluoride) (PVDF) capture membrane, adding a trypsin membrane to the electroblot increases the number of protein identifications by 22%. Additionally, electrodigestion experiments using capture membranes coated with polyelectrolyte layers identify a higher fraction of small proteolytic peptides than capture on PVDF or in-gel digestion.

Fig. 1

Four methods for analysis of proteins separated using gel electrophoresis. (A) Electroblotting onto PVDF before on-membrane digestion and extraction. (B) Digrstion during electroblotting, capture on polyelectrolyte-modified nylon, and peptide elution. (C) Digestion during electroblotting, capture on PVDF, and peptide elution. (D) In-gel digestion using a multistep procedure (not shown)

Fig. 2

Number of peptides and % sequence coverage in the analysis of a standard protein mixture using electrophoresis and the four different protocols in Fig. 1. The number of peptides includes all peptides found from any piece of capture membrane or gel in either of the two replicates without redundancy.

Fig. 3

Average LFQ intensities (arbitrary units ÷ 10⁷) of six standard proteins in sequential pieces of a nylon[PAA/PEI] capture membrane. The x-axis shows pieces arranged in decreasing order of expected molecular weights. Intensities are the average of two experimental replicates.

Fig. 4

Comparison of shared protein identifications in the analysis of an E. Coli cell lysate using the four protocols shown in Fig. 1.

Fig. 5

Average Protein LFQ intensity per piece of gel or capture membrane plotted by molecular weight for (A) On-Membrane Digestion (B) Electrodigestion with a nylon[PAA/PEI] capture membrane, (C) Electrodigestion with a PVDF capture membrane, and (D) In-Gel Digestion.

Fig. 6

Comparison of shared peptide identifications in the analysis of an E. Coli cell lysate using the four protocols shown in Fig. 1.

Fig. 7

Percentages of identified peptides with 0, 1, 2, and 3 missed cleavages after electrodigestion, in-gel digestion, and on-membrane digestion of E. Coli cell lysate.