In-Source Reduction of Disulfide-Bonded Peptides Monitored by Ion Mobility Mass Spectrometry

• Published in: Journal of the American Society for Mass Spectrometry 2018-04, Vol.29 (4), p.742-751
• Main Authors: Stocks, Bradley B., Melanson, Jeremy E.
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Animals; Bioinformatics; Biotechnology; Cattle; Chemical bonds; Chemistry; Chemistry and Materials Science; Disulfides - chemistry; Hepcidins - chemistry; Humans; Insulin; Insulin - chemistry; Ion Mobility Spectrometry - methods; Ionic mobility; Ionization; Ions; Mass spectrometry; Mathematical models; Organic Chemistry; Oxytocin - chemistry; Peptides; Peptides - chemistry; Proteomics; Reduction; Research Article; Scientific imaging; Spectrometry, Mass, Electrospray Ionization - methods; Spectroscopy
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-018-1894-1

Many peptides with antimicrobial activity and/or therapeutic potential contain disulfide bonds as a means to enhance stability, and their quantitation is often performed using electrospray ionization mass spectrometry (ESI-MS). Disulfides can be reduced during ESI under commonly used instrument conditions, which has the potential to hinder accurate peptide quantitation. We demonstrate that this in-source reduction (ISR) is predominantly observed for peptides infused from acidic solutions and subjected to elevated ESI voltages (3–4 kV). ISR is readily apparent in the mass spectrum of oxytocin—a small, single disulfide-containing peptide. However, subtle m / z shifts due to partial ISR of highly charged ( z  ≥ 3) peptides with multiple disulfide linkages may proceed unnoticed. Ion mobility (IM)-MS separates ions on the basis of charge and shape in the gas phase, and using insulin as a model system, we show that IM-MS arrival time distributions (ATDs) are particularly sensitive to partial ISR of large peptides. Isotope modeling allows for the relative quantitation of disulfide-intact and partially reduced states of the mobility-separated peptide conformers. Interestingly, hepcidin peptides ionized from acidic solutions at elevated ESI voltages undergo gas-phase compaction, ostensibly due to partial disulfide ISR. Our IM-MS results lead us to propose that residual acid is the likely cause of disparate ATDs recently measured for hepcidin from different suppliers [Anal. Bioanal. Chem. 409 , 2559–2567 (2017)]. Overall, our results demonstrate the utility of IM-MS to detect partial ISR of disulfide-bonded peptides and reinforce the notion that peptide/protein measurements should be carried out using minimally activating instrument conditions. Graphical Abstract ᅟ