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 (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.
Luque-Garcia
Jose L
JL
Kimmel Center for Biology and Medicine, Skirball Institute and Department of Pharmacology.
Zhou
Ge
G
Spellman
Daniel S
DS
Sun
Tung-Tien
TT
Neubert
Thomas A
TA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002417:Cattle; D002853:Chromatography, Liquid; D003101:Collodion / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000379:methods*; D008562:Membrane Glycoproteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012710:Serum Albumin, Bovine; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D060170:Uroplakin II; D060171:Uroplakin III
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008567:Membranes, Artificial; D010455:Peptides; D011506:Proteins; D060170:Uroplakin II; D060171:Uroplakin III; D012710:Serum Albumin, Bovine; D003101:Collodion