Reverse-phase capillary high performance liquid chromatography/high performance electrospray ionization mass spectrometry: an essential tool for the characterization of complex glycoprotein digests.

The B-domain of recombinant human Factor VIII comprises 909 amino acids and is extensively N- and O-glycosylated, in that at least 20 different sites are occupied by numerous carbohydrate structures. This domain was incubated with trypsin and subjected to liquid chromatography electrospray ionization mass spectrometry analysis, using an electrospray orthogonal acceleration time-of-flight mass spectrometer as the detector for a capillary reversed phase HPLC separation of the digest. The inherent high mass resolution afforded by this instrument provides both ion charge state determination and high accuracy mass measurement that are of significant advantage in defining such highly complex mixtures.