Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests

Identification of glycoproteins in complex mixtures derived from either hum an blood serum or a cancer cell line was achieved in a process involving th e steps of (1) reduction and alkylation, (2) proteolysis of all proteins in the mixture with trypsin, (3) affinity chromatographic selection of the gl ycopeptides with an immobilized lectin, (4) direct transfer of the glycopep tide fraction to a reversed-phase liquid chromatography (RPLC) column and f urther fractionation by gradient elution, (5) matrix-assisted laser desorpt ion ionization mass spectrometry of individual fractions collected from the RPLC column, and (6) peptide identification based on a database search. Th e types of glycoproteins analyzed were; (1) N-type glycoproteins of known p rimary structure, (2) N-type glycoproteins of unknown structure, and (3) O- type glycoproteins glycosylated with a single N-acetylglucosamine. Identifi cation of peptides from complex mixtures was greatly facilitated by either C-terminal sequencing with a carboxypeptidase mixture or by comparing chrom atographic behavior and mass to standards, as in the case of a known protei n. In addition, deglycosylation of peptides with N glycosidase F was necess ary to identify N-type glycoproteins of unknown structure. The strength of this approach is that it is fast and targets specific molecular species or classes of glycoproteins for identification. The weakness is that it does n ot discriminate between glycoforms. (C) 2001 Elsevier Science B.V. All righ ts reserved.