Direct mass spectrometric characterization of disulfide linkages

MAbs
Xiaoyan GuanJette Wypych

Abstract

Disulfide linkage is critical to protein folding and structural stability. The location of disulfide linkages for antibodies is routinely discovered by comparing the chromatograms of the reduced and non-reduced peptide mapping with location identification confirmed by collision-induced dissociation (CID) mass spectrometry (MS)/MS. However, CID product spectra of disulfide-linked peptides can be difficult to interpret, and provide limited information on the backbone region within the disulfide loop. Here, we applied an electron-transfer dissociation (ETD)/CID combined fragmentation method that identifies the disulfide linkage without intensive LC comparison, and yet maps the disulfide location accurately. The native protein samples were digested using trypsin for proteolysis. The method uses RapiGest SF Surfactant and obviates the need for reduction/alkylation and extensive sample manipulation. An aliquot of the digest was loaded onto a C4 analytical column. Peptides were gradient-eluted and analyzed using a Thermo Scientific LTQ Orbitrap Elite mass spectrometer for the ETD-triggered CID MS 3 experiment. Survey MS scans were followed by data-dependent scans consisting of ETD MS2 scans on the most intense ion in the survey scan, ...Continue Reading

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Citations

Oct 20, 2018·MAbs·Lily Pei-Yao Liu-ShinGayathri Ratnaswamy
Dec 24, 2019·Accounts of Chemical Research·Yunlong Shi, Kate S Carroll

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Methods Mentioned

BETA
Edman sequencing
electron-capture dissociation

Software Mentioned

SEQUEST
MassAnalyzer Proteome Discoverer
Proteome Discoverer
SEQUEST®
RapiGest

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