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.