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Utrecht University, Bruker Collaborating on TimsTOF Methods for Crosslinking Mass Spec

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NEW YORK – Researchers at Utrecht University are collaborating with Bruker to develop improved methods for protein crosslinking mass spectrometry experiments.

The two parties are using crosslinking technologies developed by the Utrecht researchers with Bruker's timsTOF Pro mass spec to enable more effective isolation and measurement of target proteins. Bruker plans to commercialize reagents and software tools developed through the collaboration.

Commonly used to study protein structure and protein-protein interactions, crosslinking mass spec involves the use of reagents to covalently link nearby amino acids to each other. These links are retained following digestion of proteins for mass spec workflows, and by analyzing the patterns of crosslinking, researchers are able to identify proteins that likely interact with each other or identify parts of a protein that are in close proximity to each other, providing structural information about the molecule.

One of the major challenges of the approach is the small proportion of proteins that bind to the crosslinking reagents. According to Albert Heck, head of the biomolecular mass spectrometry and proteomics group at Utrecht and one of the researchers in the Bruker collaboration, in a typical crosslinking experiment, crosslinked peptides make up only around 1 percent of the total sample.

"This has really hampered the whole field of crosslinking mass spectrometry," he said.

One way scientists have tried to address this issue is by developing crosslinking reagents that can be enriched, allowing them to pull crosslinked peptides out of the sample prior to mass spec analysis. One commonly used approach is to include a biotin handle, which can be included as part of the reagent itself or can be added via click-chemistry following the crosslinking reaction.

Heck noted, though, that eluting the crosslinked peptides from the biotin following the pull down reaction is a laborious process. Biotin is also a relatively large molecule, which can limit where biotin-linked reagents can penetrate and crosslink within protein structures.

To address these issues, Heck and colleagues including Richard Scheltema, an assistant professor at Utrecht who leads the crosslinking mass spec work in Heck's lab, developed a new crosslinking reagent. Called PhoX, the reagent uses a phosphonate-based affinity tag that allows researchers to pull out crosslinked peptides using IMAC or titanium dioxide-based approaches commonly used for phosphopeptide enrichment.

In addition to being easier to elute, the phosphonate molecule is much smaller than biotin, making for a crosslinker that is much smaller than biotin-linked crosslinkers, Heck said.

"We think that [PhoX] is more amenable to making crosslinks in places where the biotin crosslinker could probably not get into the protein structure," he said. "We think it is easier to use [than biotin], and for anyone who can do standard phosphoproteomic, for them it is just as easy to do enrichment of these PhoX-linked peptides as it is to do phosphoproteomics."

Bruker has licensed the PhoX reagent and plans to commercialize it, Heck said.

Crosslinking mass spec faces another challenge beyond the enrichment issue. Even when the crosslinking reaction proceeds efficiently and enrichment is effective, there will still be a proportion of crosslinkers bound to only one peptide.

The PhoX reagent is designed to bind and connect two lysines, Heck said, adding that "in many cases, what you see in practice is that only one of the [reagent] warheads gets attached to a lysine and the other gets hydrolyzed," leaving it unbound and generating so-called dead-end cross-linked peptides.

These dead-end formations are typically as abundant as the crosslinked peptides researchers are looking to analyze. And because they contain the crosslinking reagent, they are enriched along with the truly crosslinked peptides.

In a recent paper in Molecular and Cellular Proteomics, Heck, Scheltema (who was senior author on the study), and their colleagues demonstrated that the timsTOF Pro's ion mobility system could be used to separate a substantial portion of the dead-end peptides from the crosslinked peptides, improving the mass spec analysis.

Ion mobility separates molecules based on their collisional cross section (CCS), which is determined by their shape, charge, and size. The timsTOF Pro uses an ion mobility approach called trapped ion mobility, which allows ions to be trapped within the device and then released into the analyzer based on their collision cross section, with low mobility ions (meaning a high CCS scores) moving into the analyzer before high mobility ions (those with low CCS scores).

Given that dead-end peptides would have different CCS scores than crosslinked peptides, Scheltema thought the researchers could use ion mobility to separate the two, Heck said. It could also help further separate crosslinked peptides from unlinked peptides.

"We teamed up with Bruker and started to investigate how the ion mobility on the timsTOF could be useful for such an approach," he said, noting that this led to the work published in the MCP paper.

In the study, the researchers found that they were able to use the instrument's ion mobility to separate out between 50 percent and 70 percent of dead-end peptides, preventing the machine from spending time sequencing these peptides and allowing it to concentrate on the crosslinked peptides.

The researchers are now working with Bruker to further develop the approach.

Heck said that the combination of the trapped ion mobility approach and the timsTOF Pro's high speed provided opportunities for more targeted proteomic analyses.

"I almost see it like FACS sorting, but instead of sorting cells you are sorting ions based on their properties, on their mass, on their charge, and on their ion mobility characteristics," he said.

"I've been impressed by how well this instrument is able to use the ion mobility as an extra separations space to select only the ions you really want to fragment," he said. "We use the timsTOF not to get the most exclusive depth in the proteome, but really to look in an almost targeted way only at the peptides we want to analyze, and in this case those are the crosslinked peptides."

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