Reversed-phase high-performance liquid chromatography is the standard method for separating peptides generated from proteolytic digests of proteins. Typically, the small hydrophilic peptides from a proteolytic digest are recovered in the flowthrough fraction along with nonvolatile buffers and salts. Unfortunately, the presence of these salts can interfere with subsequent mass spectrometric analysis or Edman sequencing. To overcome this limitation, and thus enable these small peptides to be identified and characterized, we have investigated a porous graphitic carbon (PGC) column for desalting the peptides found in the unretained fraction. Using a volatile mobile phase combined with a PGC column (Hypercarb), we demonstrate that small hydrophilic peptides at the picomole level can be desalted and characterized by matrix-assisted laser desorption and ionization-time-of-flight-mass spectrometry. Even after desalting, we show that the choice of matrix still plays a significant role in distinguishing the small peptides from the matrix background. The utility of this approach is demonstrated with the flowthrough fraction of an endoproteinase Lys-C digest of a recombinant immunoglobulin. In addition, we demonstrate that a PGC column offers an alternative approach for the separation of hydrophilic, phosphorylated peptides from their unphosphorylated counterparts.
Copyright 1999 Academic Press.