Organic Solvents for Enhanced Proteolysis of Stable Proteins for Hydrogen-Deuterium Exchange Mass Spectrometry

Chunyang Guo, Lindsey K. Steinberg, Jeffrey P. Henderson, Michael L. Gross

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Protein digestion is a key challenge in mass spectrometry (MS)-based structural proteomics. Although using hydrogen-deuterium exchange kinetics with MS (HDX-MS) to interrogate the high-order structure of proteins is now established, it can be challenging for β-barrel proteins, which are important in cellular transport. These proteins contain a continuous chain of H-bonds that impart stability, causing difficulty in digestion for bottom-up measurements. To overcome this impediment, we tested organic solvents as denaturants during on-line pepsin digestion of soluble β-barrel proteins. We selected green fluorescent protein (GFP), siderocalin (Scn), and retinol-binding protein 4 (RBP4) as model proteins and screened six different polar-aprotic and polar-protic solvent combinations to disrupt the H-bonds and hydrophobic interactions holding together the β-sheets. The use of organic solvents improves digestion, generating more peptides from the rigid β-barrel regions, without compromising the ability to predict the retinol binding site on RBP4 when adopting this proteolysis with HDX.

Original languageEnglish
Pages (from-to)11553-11557
Number of pages5
JournalAnalytical Chemistry
Volume92
Issue number17
DOIs
StatePublished - Sep 1 2020

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