Characterization of disulfide linkages in recombinant human granulocyte-colony stimulating factor
Article first published online: 10 APR 2013
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Volume 27, Issue 9, pages 940–946, 15 May 2013
How to Cite
Mo, J., Tymiak, A. A. and Chen, G. (2013), Characterization of disulfide linkages in recombinant human granulocyte-colony stimulating factor. Rapid Commun. Mass Spectrom., 27: 940–946. doi: 10.1002/rcm.6530
- Issue published online: 10 APR 2013
- Article first published online: 10 APR 2013
- Manuscript Accepted: 30 JAN 2013
- Manuscript Revised: 8 JAN 2013
- Manuscript Received: 6 SEP 2012
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.
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.
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.
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. Copyright © 2013 John Wiley & Sons, Ltd.