A fast and convenient MALDI-MS based proteomic approach: identification of components scaffolded by the actin cytoskeleton of activated human thrombocytes.
A recently developed concentration and purification method (Gevaert, K., Demol, H., Puype, M., Broekaert, D., De Boeck, S., Houthaeve, T., Vandekerckhove, J., 1997. Electrophoresis 18, 2950-2960) for the analysis of diluted peptide samples by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is compared with conventional MALDI sample preparation methods. In the procedure developed, reverse-phase chromatographic beads are added to diluted peptide solutions and act as a peptide-trapping device. Peptides concentrated on the added beads are subsequently harvested, transferred to the MALDI-target disc and efficiently on target desorbed from the beads in a very small volume of an organic-aqueous mixture containing the aromatic MALDI-matrix components. Using this procedure, we show that it is possible to use the totality of in gel protein digests without negative interference of buffers and chaotropes that may be present in the digestion mixture. This method links MALDI-MS peptide analysis more efficiently to 2-D gel electrophoresis in the concept of proteome analysis. The procedure is illustrated by the identification of a class of proteins, which translocate to the actin cytoskeleton of human platelets upon thrombin stimulation.
Gevaert
K
K
Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research, Faculty of Medicine, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000, Gent, Belgium. krgev@medusa.rug.ac.be
Eggermont
L
L
Demol
H
H
Vandekerckhove
J
J
D000199:Actins / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001709:Biotechnology; D001792:Blood Platelets / Q000737:chemistry* / Q000187:drug effects; D003599:Cytoskeleton / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D020543:Proteome / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013917:Thrombin / Q000494:pharmacology
D000199:Actins; D020543:Proteome; D013917:Thrombin