Proteomic analysis of human biopsy samples by single two-dimensional electrophoresis: Coomassie, silver, mass spectrometry, and Western blotting.
Proteomic analysis of myocardial tissue from patient populations is critical to our understanding of cardiac disease, but has been limited until now by the small size of biopsies (approximately 20-50 microg), and complicated by the difference in relative abundance of contractile proteins over other cellular components. Here we describe an approach to analysis of myocardial biopsies from patients undergoing coronary artery bypass surgery. First, individual biopsies are selectively extracted, producing subfractions that correspond to the contractile proteins and the cytosolic proteins. Two-dimensional electrophoresis separated proteins are detected by first staining with Coomassie blue then silver, to permit a wider range of accurate quantification. Western blotting of two-dimensional separated samples, to validate peptide mass fingerprinting data, previously required additional gel separations for transfer since staining protocols are not compatible with transfer to membranes or immunoblotting. An existing silver destaining protocol was adapted to allow removal of silver from a whole gel, followed by transfer and Western blotting. An existing Coomassie blue removal protocol was also adapted to permit Western blotting of gels stained with Coomassie blue and silver. Together, these techniques permit peptide mass fingerprinting concurrent with Western blotting of a single protein spot from a single biopsy, eliminating the need for repeated gel separations, and improving spot alignment between immunoblots and stained gels. In the end, this approach may allow a more complete characterization of protein changes in small human biopsies, and also reduce the number of repeated gel separations necessary for a standard proteomic analysis.
McDonough
Jason L
JL
Department of Physiology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Neverova
Irina
I
Van Eyk
Jennifer E
JE
D000818:Animals; D001706:Biopsy; D015153:Blotting, Western / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology; D020543:Proteome / Q000032:analysis*; D051381:Rats; D012394:Rosaniline Dyes / Q000737:chemistry; D016622:Silver Staining
D020543:Proteome; D012394:Rosaniline Dyes; C048139:Coomassie blue