A fast and convenient MALDI-MS based proteomic approach: identification ofcomponents scaffolded by the actin cytoskeleton of activated human thrombocytes

A recently developed concentration and purification method (Gevaert, K., De mol, H., Puype, M., Broekaert, D., De Boeck, S., Houthaeve, T., Vandekerckh ove, J., 1997. Electrophoresis 18, 2950-2960) for the analysis of diluted p eptide samples by matrix-assisted laser desorption ionization-time-of-fligh t-mass spectrometry (MALDI-TOF-MS) is compared with conventional MALDI samp le preparation methods. In the procedure developed, reverse-phase chromatog raphic beads are added to diluted peptide solutions and act as a peptide-tr apping device. Peptides concentrated on the added beads are subsequently ha rvested, transferred to the MALDI-target disc and efficiently on target des orbed from the beads in a very small volume of an organic-aqueous mixture c ontaining the aromatic MALDI-matrix components. Using this procedure, we sh ow that it is possible to use the totality of in gel protein digests withou t negative interference of buffers and chaotropes that may be present in th e digestion mixture. This method links MALDI-MS peptide analysis more effic iently to 2-D gel electrophoresis in the concept of proteome analysis. The procedure is illustrated by the identification of a class of proteins, whic h translocate to the actin cytoskeleton of human platelets upon thrombin st imulation. (C) 2000 Elsevier Science B.V. All rights reserved.