Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry.
The success attributed to identification and characterization of gel separated proteins by mass spectrometry (MS) is highly dependent on the percentage of an entire sequence covered by matching peptides derived from enzymatic digestion. Desalting and concentration of peptide mixtures on reversed-phase (RP) microcolumns prior to mass spectrometric analysis have resulted in increased signal-to-noise ratio and sensitivity, and consequently higher sequence coverage. A large proportion of peptides, however, remains undetected by MS presumably because they are lost during sample preparation on microcolumns, or are suppressed in the ionization process. We report here the use of graphite powder packed in constricted GELoader tips as an alternative to RP microcolumns for desalting and concentration of peptide mixtures prior to MS. Such columns are able to retain small and/or hydrophilic peptides that can be lost when using RP microcolumns. In addition, we show that samples contaminated with small biological polymers can readily be analyzed using graphite powder rather than RP microcolumns, since the polymer molecules bind strongly to graphite and are not eluted with the peptides.
D015180:Electrophoresis, Gel, Two-Dimensional; D006108:Graphite / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010455:Peptides; D006108:Graphite