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Peptides adsorbed on reverse‐phase chromatographic beads as targets for femtomole sequencing by post‐source decay matrix assisted laser desorption ionization‐reflectron time of flight mass spectrometry (MALDI‐RETOF‐MS)
Author(s) -
Gevaert Kris,
Demol Hans,
Puype Magda,
Broekaert Daniël,
De Boeck Stefaan,
Houthaeve Tony,
Vandekerckhove Joël
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181537
Subject(s) - reflectron , chemistry , mass spectrometry , chromatography , matrix assisted laser desorption/ionization , desorption , protein mass spectrometry , matrix (chemical analysis) , surface enhanced laser desorption/ionization , maldi imaging , ionization , analytical chemistry (journal) , yield (engineering) , adsorption , time of flight mass spectrometry , electrospray ionization , materials science , ion , organic chemistry , metallurgy
We here describe a procedure for concentrating peptides from solutions by adsorbing them onto reverse‐phase beads that were added to these solutions. The beads are then transferred to the target disc of the matrix assisted laser desorption ionization‐reflectron time of flight (MALDI‐RETOF) mass spectrometer. Because of their hydrophobic nature, these beads cluster in a very small area on the target disc assuring an important concentration step. After drying, peptides are desorbed from the beads by adding a small volume of 50% acetonitrile in 0.1% trifluroacetic acid in water containing the matrix components. Hereby we focus the original amount of peptide material on the target disc on a very small surface, producing highly concentrated peptide‐matrix mixtures. This permits high yield identification and sequence tagging by post‐source‐decay analysis on peptides derived from proteins only available in the femtomole range from one‐dimensional (1‐D) or two‐dimensional (2‐D) gels. The procedure is illustrated by the identification of 38 proteins from human thrombocyte membrane skeletons.