title: Characterization of post-translational modifications in full-length human BMP-1 confirms the presence of a rare vicinal disulfide linkage in the catalytic domain and highlights novel features of the EGF domain.
abstract: Bone morphogenetic protein 1 (BMP-1) is an essential metalloproteinase to trigger extracellular matrix assembly and organogenesis. Previous structural studies on the refolded catalytic domain of BMP-1 produced in E. coli have suggested the existence of a rare vicinal disulfide linkage near the active site. To confirm that this was not an artifact of the refolding procedure, the full-length human BMP-1 produced in mammalian cells was investigated via sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution LC-MS/MS analysis to interrogate the post-translational modifications. Ten disulfide linkages of BMP-1, including the vicinal disulfide linkage C185-C186 could be unambiguously identified. Further, around 50% of this vicinal disulfide bond was found to be modified by N-ethylmaleimide (NEM), a cysteine protease inhibitor supplied when the BMP-1-containing medium was collected, suggesting that this bond was highly unstable. In the absence of NEM, BMP-1 has a higher tendency to form aggregates, but after aggregate removal, C185 and C186 are almost quantitatively engaged in the vicinal disulfide bond and BMP-1 activity remains unchanged. In addition, three consensus N-glycosylation sites at N142, N363, and N599 could be identified together with a previously unknown O-glycosylation site and an Asn-hydroxylation.
  
  
  CONCLUSIONS
  An in-depth characterization of post-translational modifications of the full-length human BMP-1 produced in mammalian cells by MS was performed. A rare vicinal disulfide bond in the catalytic domain could be confirmed for the first time by mass spectrometry along with nine other proposed disulfide linkages of mature BMP-1. This vicinal disulfide bond can transiently open to form covalent adducts with the cysteine protease inhibitor (NEM) supplied in cell medium during protein harvesting. Further, we report a previously unknown O-glycosylation site and Asn-hydroxylation site, indicating a novel feature of BMP-1 in the EGF domain. The study clearly outlines the benefit of in-depth characterization of overexpressed proteins to deduce important protein modifications.
authors.affiliation: Systematic Proteomics & Bioanalytics, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel, Germany.
authors.forename: Chien-Wen
authors.initials: CW
authors.lastname: Hung
authors.affiliation: Systematic Proteomics & Bioanalytics, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel, Germany.
authors.forename: Tomas
authors.initials: T
authors.lastname: Koudelka
authors.affiliation: UMR 5305, Tissue Biology and Therapeutic Engineering, CNRS/University of Lyon, 69367 Lyon, France.
authors.forename: Cyril
authors.initials: C
authors.lastname: Anastasi
authors.affiliation: Systematic Proteomics & Bioanalytics, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Becker
authors.affiliation: UMR 5305, Tissue Biology and Therapeutic Engineering, CNRS/University of Lyon, 69367 Lyon, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Moali
authors.affiliation: Systematic Proteomics & Bioanalytics, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel, Germany. Electronic address: a.tholey@iem.uni-kiel.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
mesh_terms: D055395:Bone Morphogenetic Protein 1 / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D006900:Hydroxylation; D013058:Mass Spectrometry; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational / Q000502:physiology*
chemical_list: D004220:Disulfides; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1
keywords: Disulfide bond; LC-MS; Posttranslational modification; Protein characterization; Protein mass spectrometry