Characterization of protein variants and post-translational modifications:: ESI-MSn analyses of intact proteins eluted from polyacrylamide gels

被引:50
作者
Claverol, S
Burlet-Schiltz, O
Gairin, JE
Monsarrat, B [1 ]
机构
[1] CNRS, Inst Pharmacol & Biol Struct, 205 Route Narbonne, F-31077 Toulouse, France
[2] CNRS Pierre Fabre, Ctr Rech Pharmacol Sante, F-31077 Toulouse, France
关键词
D O I
10.1074/mcp.T300003-MCP200
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and posttranslational 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 geleluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics.
引用
收藏
页码:483 / 493
页数:11
相关论文
共 38 条
[1]   NOMENCLATURE FOR PEPTIDE FRAGMENT IONS (POSITIVE-IONS) [J].
BIEMANN, K .
METHODS IN ENZYMOLOGY, 1990, 193 :886-887
[2]  
Buzás Z, 2001, PROTEOMICS, V1, P691
[3]   Identification of bacteriophage MS2 coat protein from E-coli lysates via ion trap collisional activation of intact protein ions [J].
Cargile, BJ ;
McLuckey, SA ;
Stephenson, JL .
ANALYTICAL CHEMISTRY, 2001, 73 (06) :1277-1285
[4]   A procedure for protein elution from reverse-stained polyacrylamide gels applicable at the low picomole level: An alternative route to the preparation of low abundance proteins for microanalysis [J].
CastellanosSerra, LR ;
FernandezPatron, C ;
Hardy, E ;
Huerta, V .
ELECTROPHORESIS, 1996, 17 (10) :1564-1572
[5]   One step microelectroelution concentration method for efficient coupling of sodium dodecylsulfate gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry for protein analysis [J].
Clarke, NJ ;
Li, F ;
Tomlinson, AJ ;
Naylor, S .
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 1998, 9 (01) :88-91
[6]   Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels [J].
Cohen, SL ;
Chait, BT .
ANALYTICAL BIOCHEMISTRY, 1997, 247 (02) :257-267
[7]   Coupling 2D SDS-PAGE with CNBr cleavage and MALDI-TOFMS:: A strategy applied to the identification of proteins induced by a hypochlorous acid stress in Escherichia coli [J].
Dukan, S ;
Turlin, E ;
Biville, F ;
Bolbach, G ;
Touati, D ;
Tabet, JC ;
Blais, JC .
ANALYTICAL CHEMISTRY, 1998, 70 (20) :4433-4440
[8]  
Ehring H, 1997, RAPID COMMUN MASS SP, V11, P1867
[9]  
Galvani M, 2000, RAPID COMMUN MASS SP, V14, P18, DOI 10.1002/(SICI)1097-0231(20000115)14:1&lt
[10]  
18::AID-RCM826&gt