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Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry

A Corrigendum to this article was published on 27 April 2017

This article has been updated

Abstract

We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS)-cleavable cross-linkers, sequential collision-induced dissociation (CID)–tandem MS (MS/MS) and electron-transfer dissociation (ETD)-MS/MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,426 unique cross-links at a 5% FDR (2,013 intraprotein and 413 interprotein cross-links) or 1,822 cross-links at a 1% FDR (1,622 intraprotein and 200 interprotein cross-links), indicating the detection of 326 or 134 protein-protein interactions at 5% FDR or 1% FDR, respectively, in HeLa cell lysates. We validated the confidence of our cross-linking results by using a target-decoy strategy and mapping the observed cross-link distances onto existing high-resolution structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors.

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Figure 1: The proteome-wide XL-MS strategy.
Figure 2: Statistical analysis of FDR and comparison of CID-ETD and CID-only fragmentation strategies for our HeLa cell lysate cross-linking data set.
Figure 3: Mapping detected cross-links on the 80S ribosome.
Figure 4: Cross-link analysis of selected protein complexes.

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Change history

  • 17 February 2017

    In the version of this article initially published, the number of interprotein cross-links was overstated, as protein grouping during cross-link categorization was not properly considered. 314 intraprotein cross-links were mistakenly annotated as interprotein cross-links if shared peptides between different protein entries were involved. The cross-links were reannotated after protein grouping, and cross-links were removed from contaminants (e.g., keratin). The sentence in the abstract, "This approach allowed us to detect 2,179 unique cross-links (1,665 intraprotein cross-links at a 5% false discovery rate (FDR) and 514 interprotein cross-links at 1% FDR) in HeLa cell lysates," has been changed to "This approach allowed us to detect 2,426 unique cross-links at a 5% FDR (2,013 intraprotein and 413 interprotein cross-links) or 1,822 cross-links at a 1% FDR (1,622 intraprotein and 200 interprotein cross-links), indicating the detection of 326 or 134 protein-protein interactions at 5% FDR or 1% FDR, respectively, in HeLa cell lysates." The sentence, "We unambiguously identified 2,179 cross-links (1,665 intraprotein cross-links at 5% FDR and 514 interprotein cross-links at 1% FDR) in HeLa cell lysates using the whole human proteome database (~40,000 entries) as the search space," has been changed to, "We unambiguously identified 2,426 total cross-links at 5% FDR or 1,822 total cross-links at 1% FDR in HeLa cell lysates using the whole human proteome database (~40,000 entries) as the search space." The sentence in the Results, "At 5% FDR, 2,473 unique cross-links were identified via the combined CID-ETD strategy, whereas 1,113 unique cross-links were identified in CID-only analysis, which corresponds to a difference of more than twofold (Fig. 2a)," has been changed to, "At 5% FDR, 2,426 unique cross-links were identified via the combined CID-ETD strategy, whereas 1,089 unique cross-links were identified in CID-only analysis, which corresponds to a difference of more than twofold (Fig. 2a)." The sentence in the Results, "When we repeated the analysis with a more stringent FDR cutoff of 1%, the discrepancy between CID-ETD and CID-only data increased to threefold (1,867 cross-links identified with CID-ETD and 594 cross-links identified with CID only) (Fig. 2a)," has been changed to, "When we repeated the analysis with a more stringent FDR cutoff of 1%, the discrepancy between CID-ETD and CID-only data increased to threefold (1,822 cross-links identified with CID-ETD and 580 cross-links identified with CID only) (Fig. 2a)." The sentence in the Results, "On the basis of the statistical analysis of the distribution of target and decoy hits, we decided to use a more stringent FDR cutoff for interprotein cross-links than for intraprotein cross-links. Thus the reported 2,179 cross-links were filtered at 5% FDR for intraprotein cross-links (1,665 cross-links) and at 1% FDR for interprotein cross-links (514 cross-links) (Fig. 2a)," has been changed to, "On the basis of the statistical analysis of the distribution of target and decoy hits, we suggest using a more stringent FDR cutoff for interprotein cross-links compared than for intraprotein cross-links. All protein-protein interactions we detected based on our interprotein cross-link findings are reported in Supplementary Data 2." We added this sentence about our reanalysis in the Online Methods section: "Cross-links were categorized as 'interprotein' if the two linked peptides derived from two different protein groups (i.e., a set of proteins containing shared peptides); otherwise, the cross-link was annotated as 'intraprotein'. We note that if homo-oligomers or complexes wherein multiple copies of a single protein are present (co-)exist, cross-links comprising two peptides of the same protein group may either be 'intermolecular' (between homo-oligomeric subunits) or 'intramolecular' (within one subunit)." We also added the sentences, "Cross-links from contaminants (e.g., keratin) were removed" and "Protein-protein interactions detected in our cross-linking data set are reported in Supplementary Data 2. Annotated spectra for interprotein cross-links are available in Supplementary Data 3; annotated spectra for intraprotein cross-links are available in Supplementary Data 4." Figure 2 has been replaced to correctly reflect the results of our updated reanalysis described above. The Supplementary Data "all 2,179 crosslinks" table has been replaced with a new table, named Supplementary Data 1, which reports the crosslinks identified in our reanalysis. Another table, named Supplementary Data 2, has been added to report the protein-protein interactions detected in the cross-linking data set. The two folders containing annotated mass spectra in the "Supplementary Data" zip file have been renamed Supplementary Data 3 and 4. These errors have been corrected in the PDF and HTML versions of the article.

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Acknowledgements

The authors thank R.A. Scheltema and P. Lössl for critical reading of the manuscript. This work was partly supported by Proteins@Work, a program of the Netherlands Proteomics Centre financed by the Netherlands Organisation for Scientific Research (NWO) as part of the National Roadmap Large-scale Research Facilities of the Netherlands (project number 184.032.201).

Author information

Authors and Affiliations

Authors

Contributions

F.L. and A.J.R.H designed the research; D.T.S.R synthesized the cross-linker; F.L. and H.P. performed the experiments; F.L. developed the XlinkX software and analyzed the data; F.L. and A.J.R.H. wrote the paper.

Corresponding author

Correspondence to Albert J R Heck.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The correlation of n-score cutoff and FDR in both CID-only and CID-ETD fragmentation strategies.

A bar diagram of different n-score thresholds applied in the search, plotted against the corresponding FDRs. The actual dataset (shown as grey bars) is fitted to a power function (shown as blue lines). The red triangles indicate the natural log scaled n-score cutoffs at FDR 5% and 1%. These n-score cutoff values are calculated based on the fitted curve.

Supplementary Figure 2 Illustrative CID-only and CID-ETD-MS/MS spectra of an interpeptide cross-link.

The MS2 product ion spectra are reconstructed based on the deconvoluted neutral monoisotopic masses. The four signature cross-linker-cleaved fragment ions are labeled as αS, αL, βS, and βL. Peptide fragment ions are labeled as b-/y-/c-/z- ion series.

Supplementary Figure 3 The long-distance interpeptide cross-link of eEF2.

The cross-link is shown in magenta and the amino acid sequence between the two linked lysines is colored in blue.

Supplementary Figure 4 The schematic positioning of SERBP1 onto the rabbit 80S ribosome in complex with a 34-nt mRNA fragment (PDB 4UJE).

Our cross-linking data on SERBP1 is highly in agreement with the cyro-EM structure of the 80S ribosome bound mRNA fragment. mRNA is shown in blue. mRNA contacting proteins that are previously reported are shown as cyan (from left to right, RPS14, RPS26 and RPS30). SERBP1, RPS28, RPS3, RPS12 and RPS27A are presented as red, yellow, yellow-orange, orange, and magenta, respectively.

Supplementary Figure 5 A zoomed-in view of the five intraprotein cross-links of SERBP1.

The five intra-protein cross-links of SERBP1 are SERBP1-K281-K299, SERBP1-K286 to K299, SERBP1-K299 to K303, K310-K321, K320-K327). All four observed intra-protein cross-links are located at the C-terminus of the protein indicates SERBP1 may possess a globular structure in this domain as opposed to the extended conformation at the remaining region. The color scheme is shown the same as Supplementary Figure 4.

Supplementary Figure 6 Illustrative CID and HCD MS2 spectra of an interpeptide cross-link.

The MS2 product ion spectra are reconstructed based on the deconvoluted neutral monoisotopic masses. The four signature ions generated by the cross-linker cleavage are indicated in purple under both HCD and CID conditions.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1 and 2, and Supplementary Notes 1 and 2 (PDF 2141 kb)

Supplementary Data 1

All identified cross-links (XLSX 928 kb)

Supplementary Data 2

Detected protein-protein interactions (XLSX 60 kb)

Supplementary Data 3

Annotated spectra for interprotein cross-links (ZIP 2944 kb)

Supplementary Data 4

Annotated spectra for intraprotein cross-links (ZIP 434 kb)

Supplementary Software

XlinkX software (ZIP 165 kb)

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Liu, F., Rijkers, D., Post, H. et al. Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. Nat Methods 12, 1179–1184 (2015). https://doi.org/10.1038/nmeth.3603

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