Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting

Abstract

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.

Fig. 1

Steps required for in-gel, and on-membrane digestion with (OM) or without (OM/NC-Free) removal of the nitrocellulose.

Fig. 2

Comparison of MALDI MS spectra obtained from (a–c) 10 pmol of UPII and from (d–i) 3 pmol of UPIII after (a, d, g) in-gel digestion, (b, e, h) on-membrane digestion followed by direct dissolution in the MALDI matrix and (c, f, i) on-membrane digestion followed by removal of the NC before MS analysis. Spectra a–f were collected in reflectron mode and spectra g–i in linear mode. Asterisks denote peaks corresponding to peptides found only after on-membrane but not in-gel digestion. "Coverage" refers to percentage of UPII or UPIII amino acid sequence accounted for by observed peptides.

Fig. 3

Comparison of LC-Q-TOF-MS/MS spectra obtained from 1 pmol BSA after (a, c) in-gel digestion and (b, d) on-membrane digestion (NC-free method).

Fig. 4

Western blot of UPII and UPIII using a commercial protein-free blocking agent. Left lane (UPs): Coomassie blue stain of uroplakin preparation blotted to the membrane.

Fig. 5

Chromatograms, MS and MS/MS spectra obtained by LC-MS/MS (Q-TOF) of peptides from UPIII identified after Western blotting followed by on-membrane digestion (NC-free method) but not by conventional in-gel digestion. (a, e) extracted ion chromatogram (m/z 747 and 1014, respectively) after in-gel digestion, (b, f) extracted ion chromatogram (m/z 747 and 1014, respectively) after on-membrane digestion, (c, g) MS spectra after on-membrane digestion and (d, h) MS/MS spectra after on-membrane digestion.