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

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 pro-teins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers im-proved protein coverage, especially for membrane pro-teins, 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 spec