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Chemical Cross-linking Mass Spectrometry (CX-MS) Service

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What Is Chemical Cross-Linking Mass Spectrometry (CXMS)

Chemical Cross-Linking Mass Spectrometry (CXMS) is a method that utilizes chemical cross-linkers to covalently connect two amino acids in a protein or protein complex when their spatial distance is close enough. After enzymatic digestion into peptide segments, mass spectrometry is used to identify the cross-linked sites, providing low-resolution structural information. This technique aids in inferring the folding state of proteins in three-dimensional space and approximating the regions of protein-protein interactions.

The principle of chemically cross-linking mass spectrometryThe principle of chemically cross-linking mass spectrometry (Simon Hauri et al,. Nature Communications 2019)

Our Chemical Cross-linking Mass Spectrometry (CX-MS) Service

Based on our excellent technology and expertise, we offer Cross-linked research services to a wide range of scientific and industrial clients. This method is dedicated to studying areas such as single protein analysis, complex protein complexes, and protein interactions research.

We utilizes a specialized XL-MS approach for in vitro and in vivo applications towards the determination of pathways and structures of protein complexes that have been challenging to define. These include complexes which contain transient interactions, (e.g. substrate-enzyme complexes), flexible subunits, heterogeneous composition or conformation, and subunits with poor solubility or stability.

Identification of cross-linked residues can provide a map of interacting protein surfaces that can be used for:
Building direct PPI networks (versus inferred); and
Mapping PPI interfaces for integrative structural determination of protein complexes.

Workflow of Chemical Cross-linking Mass Spectrometry (CX-MS)

Lolita Piersimoni et al,. Chem. Rev. 2022, 122, 8, 7500–7531 2021Lolita Piersimoni et al,. Chem. Rev. 2022, 122, 8, 7500–7531 2021

The Application of Chemical Cross-Linking Mass Spectrometry (CXMS)

CXMS is mainly applied to unravel protein structures and study protein-protein interactions. Deciphering protein structures is crucial for understanding their functions, as protein function regulation heavily relies on dynamic modulation of their conformations and interactions. Several techniques exist for characterizing protein-protein interface structures, with X-ray crystallography being the most common. While X-ray crystallography precisely determines antigen-antibody interaction sites, it has strict requirements regarding sample purity, concentration, and crystallinity, making it unsuitable for resolving the structures of large protein complexes and capturing protein-protein interaction dynamics. CXMS, when combined with protein tertiary structure prediction software, can determine protein spatial conformations, capturing weak/transient interactions and distinguishing direct versus indirect interactions. It helps define protein-protein interaction interfaces effectively.

Advantages of Chemical Cross-Linking Mass Spectrometry (CXMS)

Integration in Structural Biology: The increasing popularity of CXMS is largely due to its role as an integrated method, enabling the creation of more reliable target protein models.

Enhanced Accuracy: CXMS can be combined with various low-resolution techniques to generate more precise protein and protein complex structure models, especially for structures that are challenging to study using traditional methods.

Guided Molecular Modeling: When combined with cryo-electron microscopy and native mass spectrometry, CXMS provides spatial constraint data that guides molecular modeling and depicts the dynamic behavior of protein complexes.

Capturing Transient Interactions: Cross-linking reagents covalently connect non-covalently interacting proteins, preserving even short-lived and weak Protein-Protein Interactions (PPIs).

Mapping Interaction Sites: CXMS helps determine the locations of cross-linked amino acid side chains, restricting physical interaction sites to specific structural regions.

Wide Application: CXMS is extensively used in cell lysates, intact cells, or organelles to monitor PPIs and their spatial information across the entire proteome.

Studying Drug Resistance: Quantitative CXMS methods are used to study interactions in drug-resistant cancer cells.

Key for Visualizing Protein Structure and Interaction Dynamics: Quantitative CXMS will play an increasingly important role in visualizing protein structures and interaction dynamics.

Chavez et al. demonstrated the application of CX-MS technology in identifying protein structural features and interactions in tissue samples. This provided a systematic structural biology perspective on protein complexes present in mouse hearts. Each identified cross-linked peptide pair served as a valuable molecular probe, enabling quantification of protein conformational changes or protein-protein interaction variations in tissues. Additionally, it has the potential to assist in identifying molecular interactions, making it a promising target for mitochondrial-targeted therapies for cardiovascular or other diseases.

Chemical Cross-linking Mass Spectrometry (CX-MS) Service

Huang et al. utilized this method to investigate the three-dimensional structure of bovine serum albumin.

Chemical Cross-linking Mass Spectrometry (CX-MS) Service

Quantitative CX-MS methods have been used to study interactions in drug-resistant cancer cells, changes in protein conformation, and interactions induced by heat shock protein 90 (Hsp90) and mitotic inhibitors. It has also been applied to investigate the effects of pseudo-phosphorylation mutations and nucleotide binding on the interaction between Hsp90 and its co-chaperone Aha1.

Chemical Cross-linking Mass Spectrometry (CX-MS) Service

Courouble et al. combined HDX-MS with CX-MS to elucidate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex.

Chemical Cross-linking Mass Spectrometry (CX-MS) Service


  1. CHAVEZ J D, LEE C F, CAUDAL A, et al.. Chemical crosslinking mass spectrometry analysis of protein conformations and super complexes in heart tissue. Cell Syst., 2018, 6(1): 136-141.
  2. HUANG B X, KIM H Y, DASS C. Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry. J. Am. Soc. Mass Spectrom., 2004, 15(8):1237-1247.
  3. XU W, BEEBE K, CHAVEZ J D, et al.. Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1. Nat. Commun., 2019, 10(1): 2574[2021-08-26]. https://sci-hub. se/ 10.1038/s41467-019-10463-y.
  4. COUROUBLE V V, DEY S K, YADAV R, et al.. Revealing the structural plasticity of SARS-CoV-2 nsp7 and nsp8 using structural proteomics. J. Am. Soc. Mass Spectrom., 2021, 32(7): 1618-1630.
* For Research Use Only. Not for use in diagnostic procedures.
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