PH Dependence of Succinimide-Ester-Based Protein Cross-Linking for Structural Mass Spectrometry Applications.

ABSTRACT: Within the research field of cross-linking mass spectrometry (XL-MS), the most commonly used cross-linking reagents are succinimide-ester-based (e.g., disuccinimidyl suberate (DSS)). These reagents primarily cross-link lysine side chains. So far, they have predominantly been used to investigate protein structures at neutral to slightly basic pH (7.0-8.5) to ensure the reactivity of the primary amine of the lysine side chain. However, disease-related molecular processes are not limited to such pH ranges; e.g., some important biological pathways are active in acidic intracellular compartments. The applicability of lysine-reactive cross-linking reagents to low-pH conditions remains unclear. Here, we cross-linked a mixture of eight model proteins at eight different pH conditions (pH 4.0-7.5) to investigate the pH dependency of DSS. DSS was able to cross-link proteins even at pH 4.0, but a clear decrease in the cross-linking efficiency was observed when the pH was lowered. Nevertheless, at pH 5.0, approximately half of the number of cross-links observed at pH 7.5 could still be identified. These findings highlight the ability of succinimide-based cross-linking reagents to be useful in probing the structure of proteins in a slightly acidic environment.

SUBMITTER: Trabjerg E 

PROVIDER: S-EPMC9838815 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature