title: Peptide, disulfide, and glycosylation mapping of recombinant human thrombopoietin from ser1 to Arg246.
abstract: Thrombopoietin (TPO) is a hematopoietic factor involved in the regulation of megakaryocytopoiesis. Full length recombinant human TPO (332 residues) has been expressed in BHK cells and purified to homogeneity using conventional means. Peptide, disulfide, and glycosylation mapping of human TPO from residues 1 to 246 has been carried out using liquid chromatography-electrospray mass spectrometry (LC-ESMS). A modification of the ramped orifice method of Carr and co-workers [Carr et al. (1993) Protein Sci. 2, 183-196] is employed, providing additional information for assignment of the LC-ESMS chromatograms. With the modification, b- and y-series peptide ions are produced via front-end CID which confirms the mass-based assignments. The results of our analysis of TPO indicate that the amino acid sequence of TPO 1-246 is as expected from the transfected cDNA with complete cleavage of the signal peptide. Two unique disulfides are formed between the four cysteines in the cytokine domain of TPO: Cys7-Cys151 and Cys29-Cys85. The glycosylation map indicates the position, occupancy, and structures of the N- and O-glycans in TPO 1-246. In addition, site specific structural characterization of the PNGase F-liberated N-glycans has been performed following purification by high-pH anionic exchange chromatography with pulsed amperometric detection (HPAEC-PAD); the results corroborate the LC-ESMS data. The N-glycans are of the complex type with the core-fucosylated disialylated biantennary and trisialylated triantennary structures predominating. The O-glycans are of the mucin type with the monosialylated and disialylated GalGalNAc-S/T structures predominating. Furthermore, we propose that the C-terminal domain of TPO be further divided into two domains on the basis of sequence homology among the cloned sequences and glycosylation/structural features: an N-glycan domain (154-246) and an O-glycan domain (247-332).
authors.affiliation: Department of Biological Structure, ZymoGenetics, Inc., Seattle, Washington 98102, USA.
authors.forename: R C
authors.initials: RC
authors.lastname: Hoffman
authors.forename: H
authors.initials: H
authors.lastname: Andersen
authors.forename: K
authors.initials: K
authors.lastname: Walker
authors.forename: J D
authors.initials: JD
authors.lastname: Krakover
authors.forename: S
authors.initials: S
authors.lastname: Patel
authors.forename: M R
authors.initials: MR
authors.lastname: Stamm
authors.forename: S G
authors.initials: SG
authors.lastname: Osborn
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002845:Chromatography / Q000379:methods; D004220:Disulfides / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011134:Polysaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods; D013926:Thrombopoietin / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D011134:Polysaccharides; D011994:Recombinant Proteins; D012694:Serine; D013926:Thrombopoietin; D001120:Arginine