Characterization of O-glycosylation sites in recombinant B-chain of platelet-derived growth factor expressed in yeast using liquid secondary ion mass spectrometry, tandem mass spectrometry and Edman sequence analysis.
High-performance tandem mass spectrometry has been employed to structurally characterize intact O-linked glycopeptides and establish the complexity and extent of glycosylation for recombinant human platelet-derived growth factor B chain (rhPDGF-B) expressed in yeast. In addition, liquid secondary ion mass spectrometry (LSIMS) and Edman degradation have been employed to verify the protein sequence. LSIMS of high-performance liquid chromatographically fractionated proteolytic digests confirmed the complete amino acid sequence predicted by the human PDGF-B gene structure. Potential glycopeptides (as indicated by a mass shift of 162 or 324 Da from the mass of a predicted cleavage product) were sequenced using tandem mass spectrometry and Edman degradation. Ultraviolet matrix laser desorption mass spectrometry of rhPDGF-B dimer was used to determine the molecular weight distribution for the intact recombinant glycoprotein. In addition to the presence of unmodified peptides, corresponding peptides bearing monomannosyl moieties were found on serine 26 and threonines 20, 63, 88, 90 and 101. Further, dimannosyl moieties were found on threonines 6 and 63. These data reveal the presence of O-linked glycosylation at sites which do not fortify the concept of a consensus sequence involving proline residues, but which strengthen the concept of secondary and tertiary structure requirements. The advantages of high-energy collisionally induced dissociation analysis of O-linked glycopeptides over conventional base elimination and borohydride reduction and other mass spectrometric techniques are presented for the first time.
Settineri
C A
CA
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
Medzihradszky
K F
KF
Masiarz
F R
FR
Burlingame
A L
AL
Chu
C
C
George-Nascimento
C
C
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000032:analysis* / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000235:genetics; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012697:Serine Endopeptidases; D014357:Trypsin
D002241:Carbohydrates; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin