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.
Highlights
Cross-linking mass spectrometry (XL-MS) is a technique to delineate the structure of large proteins and protein complexes.[1−5] In recent years, the method has been successfully applied to study large and challenging protein systems and has been shown to be highly powerful in combination with complementary structural biology methods, e.g., X-ray crystallography, cryogenic electron microscopy, and small-angle X-ray scattering.[1−4,6−10]In the most common application of XL-MS, a protein or a protein complex of interest is treated with a chemical reagent that cross-links amino acid side chains under native conditions
After cross-linking and proteolytic cleavage, cross-linked peptides can be enriched by size exclusion, strong cation exchange chromatography, or affinity tags and separated by reverse-phase liquid chromatography before final mass analysis by high-resolution tandem MS (LC-MS/MS).[11−13] Subsequently, the identity of the cross-linked residues and the length of the cross-linker can be used to provide “molecular rulers” that translate into atomic distance restraints for integrative molecular modeling of protein structures or large multiprotein complexes.[6,7,14−17]
Experimental Design To assess the cross-linking efficiency of NHS-based reagents at slightly acidic conditions, we cross-linked a mixture of eight model proteins at eight different pH
Summary
Cross-linking mass spectrometry (XL-MS) is a technique to delineate the structure of large proteins and protein complexes.[1−5] In recent years, the method has been successfully applied to study large and challenging protein systems and has been shown to be highly powerful in combination with complementary structural biology methods, e.g., X-ray crystallography, cryogenic electron microscopy, and small-angle X-ray scattering.[1−4,6−10]. In the most common application of XL-MS, a protein or a protein complex of interest is treated with a chemical reagent that cross-links amino acid side chains under native conditions. The most widely used cross-linking reagents are homobifunctional, N-hydroxysuccinimide (NHS)-based esters (e.g., disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl sulfoxide (DSSO), and disuccinimidyl dibutyric urea (DSBU)) (Figure 1).[18] These reagents primarily connect primary amino groups (Lys residues and the N termini of proteins),[11] but reactivity toward hydroxyl groups (Ser, Thr, and Tyr residues) has been reported in the literature.[19−21] Many NHS-based reagents are commercially available, including those that facilitate the reliable identification of cross-linked peptides through enhanced features, e.g., stable isotope labeling or gas-phase cleavable bonds.[22−25].
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