With cross-linking mass spectrometry (XL-MS) distances between amino acids within the folded protein structure and between two interacting proteins can be measured. It can be applied to almost any protein(-complex) regardless of size, solubility and strength of interaction. Even though XL-MS provides lower resolution compared to classical structural biology approach like Cryo-EM, it is highly complementary to these techniques.

It works by introducing small and agile reagents typically equipped with two amino acid reactive groups separated by a spacer to the proteins. Upon capturing two amino acids in close proximity the two positions have been connected with a covalent bond.

After proteolytic digestion, two peptides connected by a covalent bond have been created. The identities of the these peptides can de determined by mass spectrometry, after which the amino acids in close proximity in the protein structure are known.

Core technologies


Cross-linking proteins is not trivial due to the substoichiometric reaction efficiencies achieved, which render routine detection of cross-linked peptides problematic. We developed PhoX, which is a trifunctional cross-linking reagent decorated with a stable phosphonic acid handle. This makes the cross-linked peptides amenable to the well-established immobilized metal affinity chromatography (IMAC) enrichment. The handle allows for 300× enrichment efficiency and 97% specificity.

Relevant literature:

* Steigenberger et al; PhoX – an IMAC enrichable cross-linking reagent, ACS Central Science, 2019

The workflow consists of the following steps:

After cross-linking the proteins in their native state, the proteins are denatured, reduced, alkylated, and digested to peptides (I). The mixture of peptides contains 3 different kinds of products: unmodified peptides (gray), monolinks (orange), and cross-links (green) (II). Cross-linked and monolinked peptides are enriched using Fe-IMAC on a liquid sample handling platform providing high sample throughput (III). Direct measurement of the cross-linked peptides produces low counts of cross-link identifications due to their extremely low abundances (left panel), while measurement following Fe-IMAC enrichment results in no cross-link identifications in the flow-through with similar abundance levels for the linear peptides as detected in the No enrichment (middle panel) and many in the eluate due to their enhanced abundance levels (right panel).

Sample processing can be parallelized in a 96-well plate format and either reduces the measurement time required to a single ‘shot’ or allows for a much deeper coverage of the protein sample.

XlinkX – Proteome Discoverer

The highly complex fragmentation spectra in the recorded mass spectrometry data require sophisticated algorithms to be interpreted correctly. We developed XlinkX, a powerful XL-MS search engine embedded in the Proteome Discoverer data analysis suite.

Relevant literature:

* Klykov et al; Efficient and robust proteome-wide approaches for cross-linking mass spectrometry, Nature Protocols, 2018

* Steigenberger et al; Finding and using diagnostic ions in collision induced crosslinked peptide fragmentation spectra, IJMS, 2019

The engine produces correct results in clear and well defined table format within Proteome Discoverer, which can easily be navigated to extract all the information efficiently as depicted below.

The software is highly configurable and e.g. supports the definition of any cross-linking reagent and many different data acquisition approaches.