Mass spectrometric characterization of stathmin isoforms separated by 2D PAGE.
In proteome analysis, the determination of the phosphorylation status of proteins and protein isoforms, which have been separated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), is of prime importance in addition to their identification. In this study, the extent to which such information can be directly extracted from the mass spectrometric data used for identification was evaluated. By searching for metastable peaks which are characteristic for loss of phosphoric acid, the Ser-phosphorylated peptides were identified with a high success rate in reflector matrix-assisted laser desorption/ionization (MALDI) mass maps of in-gel digested proteins. Furthermore, by employing a double enzymatic strategy using trypsin and Glu-C in parallel, improved sequence coverage and additional separation of the potential phosphorylation sites of the isoforms were achieved. The precise location of the modified sites within an identified phosphopeptide was obtained by submitting the corresponding molecular ions directly to nano-electrospray tandem mass spectrometric analysis. In this way the detailed phosphorylation status of six isomers of stathmin separated by 2D PAGE was determined. Two of these six isomers were phosphorylated at all four known sites (serines 15, 24, 37 and 62) and were probably derived from the previously reported alpha and beta forms, which differ by a yet unknown modification. In addition, isomers phosphorylated at serines 15, 24 and 37, serines 24, 37 and 62, serines 24 and 37 and serine 37 only were characterized.
Müller
D R
DR
Novartis Pharma AG, Functional Genomics Area, Protein Sciences, Basle, Switzerland.
Schindler
P
P
Coulot
M
M
Voshol
H
H
van Oostrum
J
J
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry; D008868:Microtubule Proteins; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry; D050777:Stathmin
D008868:Microtubule Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; C493995:STMN1 protein, human; D050777:Stathmin