title: Characterization of disulfide linkages in recombinant human granulocyte-colony stimulating factor.
abstract: BACKGROUND
  Recombinant human G granulocyte-colony stimulating factor (rhG-CSF) produced in Escherichia coli is a non-glycosylated polypeptide containing five cysteine residues. The reported major disulfide (S-S) linkages in mature human G-CSF are C36 -C42 and C64 -C74 , leaving C17 as a free cysteine, which could potentially result in S-S scrambling. The purpose of this work is to illustrate different mass spectrometry (MS) approaches for characterization of S-S linkages in therapeutic proteins including S-S scrambling using rhG-CSF as a model protein.
  
  
  METHODS
  Peptide mapping analysis of both non-reduced and reduced digests of rhG-CSF was performed to demonstrate the presence of S-S linked peptides and their corresponding reduced peptides. High mass accuracy measurements of these peptides provided the initial identifications of S-S linkages. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) were used to fragment these peptides in order to obtain further sequence information and identify S-S linkages.
  
  
  RESULTS
  S-S linked peptides and their corresponding reduced peptides correlating with major S-S linkages were observed. Peptides that correlated with other S-S linkages as a result of S-S scrambling were also observed.
  
  
  CONCLUSIONS
  Presence of the reported major S-S linkages in rhG-CSF was confirmed. S-S scrambling was also observed in which C18 was involved in S-S linkages and C37 , C65 or C75 were present as free cysteines. This study demonstrates the practical utility of combining different MS methods for characterization of S-S linkages in therapeutic proteins.
authors.affiliation: Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA. Jingjie.mo@bms.com
authors.forename: Jingjie
authors.initials: J
authors.lastname: Mo
authors.forename: Adrienne A
authors.initials: AA
authors.lastname: Tymiak
authors.forename: Guodong
authors.initials: G
authors.lastname: Chen
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D004220:Disulfides / Q000032:analysis*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor