Crosslinking Mass Spectrometry (XL-MS)
After finishing my PostDoc within the group of Prof. Matthias Mann (MPIB) at the beginning of 2015, I started the Scheltema laboratory within the wider context of the group of Prof. Albert Heck. The focus lies on mass spectrometry-based structural proteomics, for which we develop LC-MS/MS approaches in close collaboration with Prof. Alexander Makarov (Thermo Scientific, DE), data analysis software and novel chemistry. As part of this toolbox, crosslinking mass spectrometry (XL-MS) is a very attractive approach for the in-solution determination of protein structure, protein-protein interactions and protein dynamics, as it is not limited to protein size, is capable of dealing with highly complex protein mixtures and plays well with other structural techniques like Cryo-EM and crystallography. The technique utilizes small reagents capable of creating a covalent link between two residues in close proximity. The identity of these residues can be determined from the peptide identifications made by mass spectrometry. From these residue identities, both the proteins who are interacting as well as the position in the 3D structure of their interaction interface can be retrieved. Some of the major highlights of the recent developments we achieved include the development of quantitative XL-MS data analysis software (Nature Protocols) and the development of a novel crosslinking reagent (under review). Based on these developments we have created a well-rounded workflow, which we apply in collaboration with notable groups.
Figure 1 | Generic workflow for XL-MS experiments. (a) Cells or tissue are lysed gently, leaving protein complexes intact. (b) After optimized incubation with the cross-linking reagent, and proteolytic digestion 4 peptide products can be categorized. (c) Enrichment and pre-fractionation of XL-peptides using techniques like SCX. (d) Advanced data acquisition techniques utilizing multiple steps of fragmentation techniques (CID, HCD) are used to identify the peptides. (e) Advanced data analysis software (e.g. XlinkX into Proteome Discoverer v. 2.3) is used to identify the XL-peptides, (f) from which the data can be integrated well into structural modeling software (e.g. HADDOCK, I-TASSER, DisVis).
The whole workflow can be applied to any protein (-complex) of interest. Our laboratory has tackled for example CRIPR/Cas systems in the past (Benda et al, Mol Cell & Fagerlund et al; PNAS).
Figure 2 | Molecular architecture of Cas14–Cas2-32. (A) Schematic of intralinks (depicted as gray lines; thin lines are outliers) and interlinks (black) (Table S2). Cas1 is in green, the Cas2-3 domain organization is shown with Cas2 in yellow, the HD nuclease of Cas3 in red, and the remainder of Cas3 in blue (SF2 helicase, composed of RecA1 and RecA2, and a C-terminal domain). (B and C) Modeling of Cas1–Cas2-3 into EM density. Interprotein cross-links are shown (black lines) and EM data are shown in Fig. S1 B–F. (D) DNA-interacting peptides were mapped on Cas1 (positions 201 to 213 and 273 to 294; green) and the Cas2 domain of Cas2-3 (82 to 95; yellow). (E and F) The final Cas1–Cas2-3 model with features labeled and protospacer DNA shown in orange.
To simplify the whole process we actively develop the data analysis software XlinkX, which is incorporated in Proteome Discoverer and supports the transition from RAW data acquisition to reliable identification of crosslinked peptide pairs from which distance information can be derived.
For more information see the link to XlinkX below.
Key references:
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- B. Steigenberger, R.J. Pieters, A.J.R. Heck, R.A. Scheltema$; PhoX - an IMAC-enrichable Crosslinking Reagent; ACS Central Science; 2019
- P. Albanese, S. Tamara, G. Saracco, R.A. Scheltema$, C. Pagliano$; How paired PSII-LHCII supercomplexes mediate the stacking of plant thylakoid membranes unveiled by integrative structural mass-spectrometry; Nature Communications; 2020
- O. Klykov, C. van der Zwaan, A.J.R. Heck, A.B. Meijer, R.A. Scheltema$; Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling; PNAS; 2020
- O. Klykov*, B. Steigenberger, S. Pektas, D. Fasci, A.J.R. Heck, R.A. Scheltema$; Efficient and robust proteome wide approaches for crosslinking mass spectrometry; Nature Protocols; 2018