Proteomic analysis of human biopsy samples by single two-dimensional electrophoresis: Coomassie, silver, mass spectrometry, and Western blotting

Abstract

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.

Myosin and actin expression and oxidation in aging muscle

“…Muscles were trimmed of visible fat and connective tissue and were frozen in liquid nitrogen and stored at −80°C until analysis. Fractionation of muscle samples into extracts 1 and 2 (enriched for cytosolic and myofibrillar proteins, respectively) was performed on ice according to McDonough et al (27). Briefly, muscle samples (60 mg) were diced with a razor and exposed to 500 µl of subfractionation buffer 1 (20 mM imidazole, 0.25 mM phenylmethylsulfonyl fluoride, pH 7.4) and immediately homogenized with a glass homogenizer (Kontes Duall).…”

Effects of Left Ventricular Assist Device (LVAD) Placement on Myocardial Oxidative Stress Markers

“…Images were captured using a GS-800 calibrated densitometer (BioRad). In order to determine the remaining carbonylated cytosolic protein band, the gel bands were manually excised from the silver stained SDS-PAGE gel with a clean razor blade and in-gel digestion was performed essentially as described [22][23][24]. Raw spectral data were extracted with ReAdW (Institute for Systems Biology) and converted to mzXML files.…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…In TFA extraction, cells are lysed with low ionic buffer, and myofilament proteins are extracted from the resulting pellet with 1% TFA v/v [23]. This method has been applied to extract myofilament proteins from minute amounts (20,50 mg) of biopsy samples [24]. Recently, this method has been modified to produce two additional sub-proteomes, cytoplasmic-enriched and membrane-enriched fractions [25].…”