BILLERICA, Mass.—In August, Bruker Corp. announced a collaboration with Utrecht University to advance the study of the 3D structures and interactions of proteins by mass spectrometry.
The Heck Lab at Utrecht University has been a leader in proteomics and the study of protein structure and interactions by mass spectrometry for over two decades. Dr. Richard Scheltema has recently joined the Heck Lab as group leader to focus on crosslinking mass spectrometry (XL-MS) for structural and interaction proteomics.
The collaborative work will focus on the development of trapped ion mobility spectrometry (TIMS) and parallel accumulation serial fragmentation (PASEF) methods, along with crosslinkers and XL-MS software for the timsTOF Pro 4D-Proteomics mass spectrometer.
“We are delighted to work with Bruker on the further development of workflows for XL-MS that take advantage of the speed of PASEF and the unique large-scale, accurate CCS [collision cross sections] data to enhance the detection of crosslinks in XL-MS,” said Dr. Albert Heck of Utrecht University. “We are excited by the initial results published in Molecular and Cellular Proteomics and look forward to advancing XL-MS even further. We are also interested in other applications of ion mobility separation and CCS on the timsTOF Pro to glycoproteomics and top-down proteomics.”
The CCS workflows have been described in a paper entitled “Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry.”
“Having personally been involved in some of the conceptual work in XL-MS in 2001 at Sandia National Laboratory, I believe the advances made by Heck’s group will make this technique more routinely available for structural biology studies using the timsTOF Pro,” noted Dr. Gary Kruppa, vice president of proteomics at Bruker. “Our collaboration with Utrecht University will accelerate adoption of XL-MS within the broader structural and interaction proteomics community.”
“XL-MS represents a powerful approach to uncover structural details of proteins and protein-complexes, even in highly complex samples. Despite its power, the technique has however suffered from limited analytical depth due to the low reaction efficiency of the used reagents,” according to the paper. “With the introduction of enrichable cross-linking reagents like PhoX, this can partly be resolved.”
“With these reagents the sample complexity can be reduced, focusing only on peptides modified by the cross-linking reagent, forming mono-linked and cross-linked peptide products. Further improvements are however still required to fully unlock the potential of XL-MS, as the mono-linked peptides do not provide the sought-after structural information and typically make up more than half of the sample load after enrichment,” the study states.
“[W]e described the development of a novel acquisition approach utilizing ion mobility to physically separate the mono-linked from the cross-linked peptides, providing better signal-to-noise to the latter class of ions,” the authors noted. “Additionally, we present a novel acquisition technique capable of preventing sequencing of a large majority of mono-linked peptides, while still sequencing the desired cross-linked peptides.”
“The approach is exemplified on the Bruker timsTOF Pro, which incorporates a trapped ion mobility device in a mass spectrometry platform geared towards shotgun proteomics. From the acquired data we have demonstrated that the data acquisition software can make the required a-priori distinction between mono-linked and cross-linked peptides,” they continue. “This focuses the acquisition, a feature largely beneficial for complex mixtures.”
Bruker plans to commercialize the results of the collaboration as integrated solutions for the study of protein structures and interactions using XL-MS. Combining the novel, enrichable PhoX crosslinker developed by Heck and Scheltema with the extreme speed and sensitivity of PASEF methods on the timsTOF Pro platform enables the discovery of more crosslinked products, which yield more information about protein structures and interactions.
Advanced analysis software is key, as XL-MS data is more complex and even more information-rich than typical shotgun proteomics experiments. Scheltema is working on enabling the innovative XlinkX software to process TIMS/PASEF data, and making it available to the community of timsTOF Pro users.
“My group intends to push the boundaries of PASEF to enhance XL-MS workflows by making them CCS-aware,” added Scheltema. “We have a significant ongoing effort in bioinformatics applied to analyzing XL-MS data using our XlinkX software. We are excited to work with the open data-format architecture of the timsTOF Pro in XlinkX to develop code that can use large-scale, accurate CCS values for the identification of crosslinks and to further improve false discovery rate calculations.”