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Characterization of disulfide linkages in recombinant human granulocyte‐colony stimulating factor
Author(s) -
Mo Jingjie,
Tymiak Adrienne A.,
Chen Guodong
Publication year - 2013
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6530
Subject(s) - chemistry , peptide , recombinant dna , cysteine , scrambling , electron transfer dissociation , mass spectrometry , biochemistry , disulfide bond , peptide sequence , chromatography , tandem mass spectrometry , enzyme , gene , linguistics , philosophy
RATIONALE 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 C 36 –C 42 and C 64 –C 74 , leaving C 17 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 C 18 was involved in S‐S linkages and C 37 , C 65 or C 75 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. Copyright © 2013 John Wiley & Sons, Ltd.