Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography - PubMed (original) (raw)

Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography

Alexander Leitner et al. Mol Cell Proteomics. 2012 Mar.

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.

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Figures

Fig. 1.

Fig. 1.

Peptide separations by size exclusion chromatography. UV traces at 215 nm are shown. A, Separation of a model peptide mixture (1 μg per peptide injected) consisting of insulin (1; 5.7 kDa), oxidized insulin A chain (2; 2.5 kDa), and angiotensin II (3; 1.0 kDa). B, Separation of the eight-protein mix cross-linked with DSS and digested with trypsin as the protease (100 μg total protein digest injected). The fractions collected for LC-MS analysis are highlighted. Elution profiles using other proteases are shown in the

supplemental Material Fig. S1

.

Fig. 2.

Fig. 2.

Relative distributions of three classes of peptides (unmodified peptides, green; mono-links, orange; and cross-links, blue) among the SEC fractions from a trypsin digest of the eight-protein mix. Data points are normalized so that for each peptide class, the sum of identifications in all five fractions is set to 100%. Distributions for other proteases are shown in

supplemental material Fig. S2

.

Fig. 3.

Fig. 3.

Distribution of cross-link identifications in different SEC fractions. Shown are nonredundant cross-linked peptides in five individual SEC fractions per enzyme, with the two main fractions highlighted in red. (A) Trypsin, (B) Asp-N, (C) Glu-C, (D) Lys-C, (E) Lys-N.

Fig. 4.

Fig. 4.

Comparison of cross-link identifications with five different proteases. Shown are nonredundant cross-linked peptides combined over all SEC fractions (in blue) and found in the two main fractions (in red), respectively.

Fig. 5.

Fig. 5.

A, Lys-Lys contacts identified by the multiprotease approach mapped onto a bovine serum albumin homology structure obtained from ModBase. Distances (Cα-Cα) of less than 30 Å, between 30 and 35 Å and above 35 Å are colored in black, orange and red, respectively. Visualization was performed using PyMOL 1.3 (Schrödinger LLC). B, Histogram showing the distribution of the BSA distance restraints shown in (A).

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