Characterization of protein variants and post-translational modifications: ESI-MSn analyses of intact proteins eluted from polyacrylamide gels.
We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and post-translational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MSn analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the modification. In the case of labile post-translational modifications, like phosphorylations and glycosylations, we conducted electrospray ionization MSn analyses of the intact proteins to confirm their presence. Finally, after digestion of the proteins in solution, we performed tandem MS analyses of the modified peptides to locate the modifications. Using this strategy, we have determined the molecular mass of 5-10 pmol of a protein up to circa 50 kDa loaded on a gel with a 0.01% mass accuracy. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of a mixture of kappa-casein isoforms, for which we were able to identify the two major variants and their phosphorylation site and glycosylation motif. We believe that this strategy, which combines two-dimensional gel electrophoresis and mass spectrometric analyses of gel-eluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics.
Claverol
Stéphane
S
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, 205 route de Narbonne, 31077 Toulouse cedex, France.
Burlet-Schiltz
Odile
O
Gairin
Jean Edouard
JE
Monsarrat
Bernard
B
D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002364:Caseins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional; D014644:Genetic Variation; D006736:Horses; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D002364:Caseins; D009211:Myoglobin; D020033:Protein Isoforms; D011506:Proteins; D013205:Staphylococcal Protein A; D012710:Serum Albumin, Bovine; D002256:Carbonic Anhydrases