Peptide, disulfide, and glycosylation mapping of recombinant human thrombopoietin from ser1 to Arg246

Biochemistry
R C HoffmanS G Osborn

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 performe...Continue Reading

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Citations

Oct 10, 1998·Stem Cells·T KatoH Miyazaki
Sep 30, 2000·Stem Cells·T KatoH Miyazaki
Jul 4, 2013·International Journal of Hematology·David J Kuter
Apr 16, 2003·Analytical Biochemistry·Jeffrey S Rohrer, Harvey I Miller
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Nov 10, 2013·International Journal of Cell Biology·Maurizio RomanoDiana Baralle
May 25, 2010·Blood Reviews·Roberto StasiEdward C Gordon-Smith
Mar 31, 1999·Baillière's Clinical Haematology·A E von dem BorneM de Haas
Apr 25, 2008·Biochimica Et Biophysica Acta·Roberto Marcucci, Maurizio Romano
Jul 9, 2002·The Journal of Biological Chemistry·Hannah M Linden, Kenneth Kaushansky

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