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).
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.
Gevaert
K
K
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Belgium. krgev@gengenp.rug.ac.be
Demol
H
H
Puype
M
M
Broekaert
D
D
De Boeck
S
S
Houthaeve
T
T
Vandekerckhove
J
J
D000097:Acetonitriles; D000327:Adsorption; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000648:ultrastructure*; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008565:Membrane Proteins / Q000097:blood*; D008832:Microchemistry; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D000097:Acetonitriles; D008565:Membrane Proteins; C032159:acetonitrile