Mass spectrometric characterization of stathmin isoforms separated by 2D PAGE

In proteome analysis, the determination of the phosphorylation status of pr oteins and protein isoforms, which have been separated by two-dimensional p olyacrylamide gel electrophoresis (2D PAGE), is of prime importance in addi tion to their identification. In this study, the extent to which such infor mation can be directly extracted from the mass spectrometric data used for identification was evaluated. By searching for metastable peaks which are c haracteristic 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. Furth ermore, by employing a double enzymatic strategy using trypsin and Glu-C in parallel, improved sequence coverage and additional separation of tbe pote ntial phosphorylation sites of the isoforms were achieved. The precise loca tion of the modified sites within an identified phosphopeptide was obtained by submitting the corresponding molecular ions directly to nano-electrospr ay. tandem mass spectrometric analysis. In this way the detailed phosphoryl ation status of six isomers of stathmin separated by 2D PAGE was determined . Two of these sin isomers were phosphorylated at all four known sites (ser ines 15, 24, 37 and 62) and Here probably derived from the previously repor ted alpha and beta forms, which differ by a yet unknown modification, In ad dition, isomers phosphorylated at serines 15, 24 and 37, serines 24, 37 and 62, serines 24 and 37 and serine 37 only were characterized. Copyright (C) 1999 John Wiley & Sons, Ltd.