Analysis of Electroblotted Proteins by Mass Spectrometry: Protein Identification after Western Blotting

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, or 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 mass spectrometry analysis. This method
offers improved protein coverage, especially for membrane proteins such as uroplakins, since 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.
http://www.mcponline.org/content/early/2007/10/15/mcp.M700415-MCP200.full.pdf