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

Leitner A, Reischl R, Walzthoeni T, Herzog F, Bohn S, Förster F, Aebersold R - Mol. Cell Proteomics (2012)

Bottom Line: Here, we propose two complementary experimental strategies to expand cross-linking data sets.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland.

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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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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Figure 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).

Mentions: To illustrate the extensive cross-link coverage obtained for BSA, Fig. 5A visualizes the BSA restraints on a homology model obtained from ModBase (28). As shown in Fig. 5B, the majority of the observed distances conform to the expected span of the cross-linking reagent (Cα-Cα distance of 23 Å excluding any structural flexibility), and with the exception of two contacts, the bridged distances lie within 28 Å. The calculated span between residues 117 and 489 is 34.8 Å and could result from a deviation of the homology model to the actual structure, whereas the second outlier corresponds to a theoretical distance of 57.1 Å. Even if both cases are classified as false positives, the error rate would still be at an acceptable level of 3.6% (2 of 55).


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

Leitner A, Reischl R, Walzthoeni T, Herzog F, Bohn S, Förster F, Aebersold R - Mol. Cell Proteomics (2012)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316732&req=5

Figure 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).
Mentions: To illustrate the extensive cross-link coverage obtained for BSA, Fig. 5A visualizes the BSA restraints on a homology model obtained from ModBase (28). As shown in Fig. 5B, the majority of the observed distances conform to the expected span of the cross-linking reagent (Cα-Cα distance of 23 Å excluding any structural flexibility), and with the exception of two contacts, the bridged distances lie within 28 Å. The calculated span between residues 117 and 489 is 34.8 Å and could result from a deviation of the homology model to the actual structure, whereas the second outlier corresponds to a theoretical distance of 57.1 Å. Even if both cases are classified as false positives, the error rate would still be at an acceptable level of 3.6% (2 of 55).

Bottom Line: Here, we propose two complementary experimental strategies to expand cross-linking data sets.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland.

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.

Show MeSH
Related in: MedlinePlus