A combined radiolabelling and silver staining technique for improved visualisation, localisation, and identification of proteins separated by two-dimensional gel electrophoresis.
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 35S-methionine ([35S]-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).
Westbrook
J A
JA
National Heart & Lung Institute, Imperial College School of Medicine, Heart Science Centre, Harefield Hospital, Harefield, Middlesex UB9 6JH, UK. jules.westbrook@harefield.nthames.nhs.uk
Yan
J X
JX
Wait
R
R
Dunn
M J
MJ
D000595:Amino Acid Sequence; D001345:Autoradiography / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D020543:Proteome; D012834:Silver; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods*
D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome; D012834:Silver