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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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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.
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Figure 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.

Mentions: We used a polymeric FPLC size-exclusion column suitable for a separation range of 1000 to 7000 Da, according to the manufacturer's specifications. We first evaluated the efficiency of the SEC column by analyzing a peptide mixture consisting of insulin (5.7 kDa), oxidized insulin A chain (2.5 kDa), and angiotensin II (1.0 kDa). Careful optimization of the mobile phase was required as symmetric peaks were only observed in the presence of an acidic aqueous/organic mobile phase. The use of 30% acetonitrile and 0.1% trifluoroacetic acid resulted in acceptable separation of the three analytes, particularly in the range of 1–3 kDa that is most relevant for cross-linked peptides, as shown in Fig. 1A. This volatile mobile phase composition also ensured direct compatibility with downstream LC-MS analysis, requiring only an evaporation step and, in contrast to SCX fractionation, no further sample clean-up.


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)

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

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

Figure 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.
Mentions: We used a polymeric FPLC size-exclusion column suitable for a separation range of 1000 to 7000 Da, according to the manufacturer's specifications. We first evaluated the efficiency of the SEC column by analyzing a peptide mixture consisting of insulin (5.7 kDa), oxidized insulin A chain (2.5 kDa), and angiotensin II (1.0 kDa). Careful optimization of the mobile phase was required as symmetric peaks were only observed in the presence of an acidic aqueous/organic mobile phase. The use of 30% acetonitrile and 0.1% trifluoroacetic acid resulted in acceptable separation of the three analytes, particularly in the range of 1–3 kDa that is most relevant for cross-linked peptides, as shown in Fig. 1A. This volatile mobile phase composition also ensured direct compatibility with downstream LC-MS analysis, requiring only an evaporation step and, in contrast to SCX fractionation, no further sample clean-up.

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