Mass Spectrometric Determination of Disulfide Linkages in Recombinant Therapeutic Proteins Using Online LC−MS with Electron-Transfer Dissociation

Abstract

In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an over-expressed CHO cell line (disulfide scrambling), is often a great concern as such incorrectly folded forms may not be completely removed in the final product. Thus, significant efforts have been devoted to map disulfide bonds to assure drug quality. Similar to ECD, disulfide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an on-line LC-MS strategy combining collision induced dissociation (CID-MS 2 ), electron transfer dissociation (ETD-MS 2 ), and CID of an isolated product ion derived from ETD (MS 3 ) has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS 3 step. The on-line LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of recombinant human growth hormone (Nutropin), a monoclonal antibody (Herceptin) and tissue plasminogen activator (Activase). The characterization of disulfide-dissociated or partially dissociated peptide ions in the MS 3 step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the unexpected disulfide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disulfide-linked and disulfide-dissociated peptide ions with high abundance provided not only facile interpretation with high confidence but also simplified the conventional approach for determination of disulfide linkages, which often requires two separate experiments (with and without chemical reduction). The on-line LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins.

Defining the Disulfide Bonds of Insulin-like Growth Factor-binding Protein-5 by Tandem Mass Spectrometry with Electron Transfer Dissociation and Collision-induced Dissociation

“…1C). Purified IGFBP-5 was digested with trypsin under non-reducing conditions and subjected to a MS3 protocol with ETD (ETD-MS2) followed by CID (CID-MS3) (40). CID-MS2 also was employed to help identify disulfide-linked peptides (Fig.…”

“…Mass spectrometric (MS) methods for identifying disulfide bonds have improved over the last few years, and several proteins recently have been mapped using a tandem MS approach in which peptides, including disulfide-linked species, are selected in the MS1 scan and then subjected to ETD (ETD-MS2) followed by CID (CID-MS3) (39,40). ETD fragments peptides via electron transfer from a radical anion to a protonated peptide, causing cleavage between C␣-N bonds which results in c and z ions (41).…”

“…Traditional MS disulfide mapping methodologies have employed CID, but only to compare protease-digested peptides in proteins treated with or without reducing agents (44). However, because ETD preferentially cleaves disulfide bonds, this approach may be applied to protein samples without prior chemical reduction (40), as subsequent CID fragmentation of the peptides liberated by ETD will then identify the cysteines involved in disulfide bonds (40).…”

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

“…15,17,19,27–30 Several mass spectrometry (MS)/MS approaches for characterizing disulfide linkages have been described using collision-induced dissociation (CID), electron-transfer dissociation (ETD), or electron-capture dissociation (ECD). 11–13,29,31–35 These approaches often require multiple analysis, comparison of reduced and non-reduced peptide maps, interpretation of complex data sets, or large sample consumption. Electrospray ionization MS/MS characterization of IgG2 hinge disulfides can also be difficult due to the large size of hinge peptides (5 – 10 kDa, depending on disulfide configuration and number of missed cleavages) that generate low-intensity, multiply charged peak clusters that complicate the deconvolution of the MS/MS spectra.…”

High-Efficiency Recognition and Identification of Disulfide Bonded Peptides in Rat Neuropeptidome Using Targeted Electron Transfer Dissociation Tandem Mass Spectrometry

“…17 By preserving the post translational modifications (PTMs) to the greatest extent, it is considered to be an ideal tool for elucidation of sequences and identification of PTMs including disulfide bonding sites. 18 Despite these advantages, ETD still suffers from a low fragmentation rate of precursor ions with weak intensity just like other fragmentation techniques.…”