Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography

Abstract

Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe.

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

“…An overview of the chromatographic separation and fractions is shown in Fig. S3 A and B. Extracted tandem mass spectra (MS/MS) of the SEC fractions analyzed with xQuest/xProphet (25) demonstrate good enrichment of cross-linked species in the early fractions, in agreement with published observations (26). Based on these initial tests, we focused our subsequent analyses on fractions A2 to A5.…”

Integrative structure and functional anatomy of a nuclear pore complex

“…First, peptide size chromatography 68 was used for size-based separation into 2 – 4 fractions (~2–10 kDa). Then, a secondary fractionation, using a self-packed basic (pH10) C18 resins (Dr. Masch GmbH), resulted in 10 – 12 peptide fractions which were subsequently analyzed by LC/MS.…”

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

“…For the identification of the crosslinks listed in Table 1, we had to analyze a large number of datasets. Low yields of identified crosslinks with mass spectrometry can mainly be attributed to technical reasons, and the development of ways to tackle this problem is an intensive area of research in the field (Fritzsche et al 2012;Leitner et al 2012). Particularly, cation exchange chromatography (SCX) and affinity tags incorporated in the crosslinking reagent seem promising strategies to enrich crosslinks and will hopefully improve the yields and the intensity in the MS spectra also for crosslinks representing the sHsp-substrate interaction.…”