Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests.
Identification of glycoproteins in complex mixtures derived from either human blood serum or a cancer cell line was achieved in a process involving the steps of (1) reduction and alkylation, (2) proteolysis of all proteins in the mixture with trypsin, (3) affinity chromatographic selection of the glycopeptides with an immobilized lectin, (4) direct transfer of the glycopeptide fraction to a reversed-phase liquid chromatography (RPLC) column and further fractionation by gradient elution, (5) matrix-assisted laser desorption ionization mass spectrometry of individual fractions collected from the RPLC column, and (6) peptide identification based on a database search. The types of glycoproteins analyzed were; (1) N-type glycoproteins of known primary structure, (2) N-type glycoproteins of unknown structure, and (3) O-type glycoproteins glycosylated with a single N-acetylglucosamine. Identification of peptides from complex mixtures was greatly facilitated by either C-terminal sequencing with a carboxypeptidase mixture or by comparing chromatographic behavior and mass to standards, as in the case of a known protein. In addition, deglycosylation of peptides with N glycosidase F was necessary 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 not discriminate between glycoforms.
Geng
M
M
Department of Chemistry, Purdue University, Lafayette, IN 47907-1393, USA.
Zhang
X
X
Bina
M
M
Regnier
F
F
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D019075:Electrophoresis, Capillary; D006020:Glycopeptides / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020543:Proteome / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry*
D006020:Glycopeptides; D006023:Glycoproteins; D020543:Proteome; D014357:Trypsin