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Development of a Cross-linking Workflow with a Team of Collaborators (without Getting Cross-linked)

Team TFS
Team TFS

tyhrthbJust last year, at the 20th Annual Lorne Proteomics Symposium in Australia, a number of scientists interested in structural proteomics approached me about Thermo Fisher Scientific’s solution for analysis of crosslinked peptides. Their interest was sparked by an excellent presentation by Professor Albert Heck from Utrecht University, titled "Exploring alternative proteases and fragmentation methods for proteomics." Listening to Professor Heck, I realized that the last time I studied protein structure and used crosslinkers was more than 15 years ago when I worked at UC Davis and studied brine shrimp (Artemia franciscana) p26, a small heat shock/alpha crystalin protein by cryo microscopy. After the Lorne conference, I visited number of different laboratories in Australia and almost everyone brought up the question whether Thermo Fisher Scientific have a cross-linking mass spectrometry (XL-MS) workflow.

Since my time at the UC Davis Bodega Bay marine lab, a lot of things have happened in the area of protein structural analysis. New generation mass spectrometers such as the Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer have been developed, making experiments presented by professor Heck possible. To perform wide proteome interactome mapping, not only do mass spectrometerss need to be able to perform the task, but this work requires novel crosslinkers that are mass spec cleavable. During fragmentation in the mass spectrometer, these MS cleavable crosslinkers produce special signature ions that can be used for data analysis. This has enabled the development of novel type of search engines, solving the “the n-square problem” (the quadratic expansion of the computational search space), and can search full proteomes relatively quickly. Of course the central piece of this 21st century crosslink-MS (a.k.a. XL-MS) workflow is a mass spectrometer which can perform multiple types of fragmentations at high speed, high resolution and high mass accuracy.

Working at Thermo Fisher Scientific, I have the unique opportunity to interact and collaborate with many key opinion leaders in many areas of proteomics research. One such collaboration is with Albert Heck’s group at Utrecht University, which enabled us to develop our new XL-MS workflow (see figure 1). Albert Heck’s graduate student and now post-doc Fan Liu came up with XL-MS novel software XlinkX which solved the N2 search problem using DSSO, a mass spec cleavable crosslinker. This crosslinker was developed by another Thermo Fisher collaborator, Lan Huang from UC Irvine. In collaboration with our Thermo Scientific™ Proteome Discoverer™ software team and Thermo Fisher Pierce colleagues, we are in process to make this workflow accessible for everyone who wants to perform such studies, and the next time I’m in Australia, I can happily answer, “Yes, we do have our XL-MS workflow.”


    1. Poster: Optimization of Crosslinked Peptide Analysis on an Orbitrap Fusion Lumos Mass Spectrometer


    1. Brochure: Orbitrap Fusion Lumos MS: Test new limits of detection, characterization and quantitation with the latest Tribrid™ mass spectrometer


    1. Crosslinking Reagents Technical Handbook: the basics of crosslinking explained, including chemical reactivity, molecular properties and applications.


    1. Bioconjugate Techniques, 3rd Edition: order your copy today


    1. Crosslinker Selection Tool: this tool provides quick access to customized lists crosslinkers that meet specific criteria, including target functional group, solubility, and cell membrane permeability.


    • Kao A, Chiu CL, Vellucci D, Yang Y, Patel VR, Guan S, Randall A, Baldi P, Rychnovsky SD, Huang L. Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes. 2011. Mol Cell Proteomics, 10(1): M110.002212.

    • Liu F, Rijkers D, Post H, Heck AJ. Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. 2015. Nat Methods, 12(12):1179-84.


Fig 1: Crosslinking mass spectrometry workflow