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A combined radiolabelling and silver staining technique for improved visualisation, localisation, and identification of proteins separated by two‐dimensional gel electrophoresis
Author(s) -
Westbrook Jules A.,
Yan Jun X.,
Wait Robin,
Dunn Michael J.
Publication year - 2001
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200103)1:3<370::aid-prot370>3.0.co;2-y
Subject(s) - proteome , peptide mass fingerprinting , proteomics , mass spectrometry , silver stain , chemistry , two dimensional gel electrophoresis , bottom up proteomics , chromatography , gel electrophoresis , matrix assisted laser desorption/ionization , peptide , tandem mass spectrometry , protein mass spectrometry , microbiology and biotechnology , biochemistry , biology , desorption , gene , organic chemistry , adsorption
Two‐dimensional gel electrophoresis (2‐DE) remains the method of choice for the Separation of protein mixtures whilst mass spectrometry (MS) is rapidly becoming the premier tool for protein identification. When combined, 2‐DE and MS form the current operating paradigm for classical proteomics. One of the key challenges of proteome research is that of detecting and identifying all of the elements (proteins) of a proteome. Silver staining and radiolabelling, e.g. with 35 S‐methionine ([ 35 S]‐met), represent two sensitive methods used to visualise many of the constitutive and synthesised elements of a proteome, respectively. The latter method allows a very low total protein loading on a two‐dimensional (2‐D) gel and challenges protein identification using current MS‐based technology. Therefore, it is necessary to refer to and locate a radiolabelled spot's cognate on a preparatively loaded stained gel, or Western blot, and use that protein spot for identification. Unfortunately, the images of autoradiographs and preparative gels or blots, even of the same sample, often do not correspond making it difficult to accurately locate and select spots of interest by visual comparison. We have established a technique that permits the unambiguous localisation of radiolabelled proteins on the same silver stained 2‐D gel. Protein identification of superimposed spots is described by peptide mass fingerprinting and database searching using matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry and by peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight MS (Q‐TOF).