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Bruker's TimsTOF Pro Could Boost Shotgun Mass Spec Sensitivity

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NEW YORK (GenomeWeb) – Bruker's launch of its timsTOF Pro mass spectrometer at last week's annual meeting of the Human Proteome Organization features a new workflow that the company and collaborating researchers said could significantly up the depth and sensitivity of shotgun proteomic experiments.

The timsTOF Pro, and specifically its trapped ion mobility spectrometry (TIMS) technology, enables the PASEF (Parallel Accumulation - Serial Fragmentation) mass spec method developed by Bruker and the lab of Max Planck Institute of Biochemistry researcher Matthias Mann.

The method combines collection of ions via TIMS with rapid quadrupole switching on a QTOF instrument to enable the fragmentation of multiple simultaneously eluting precursor ions and, according to Mann, offers speed and sensitivity gains of up to 10-fold compared to other mass spec approaches.

Mann and his colleagues published a 2015 study in the Journal of Proteome Research detailing the method, but that was essentially a proof-of-principle analyzing a four-protein digest. The release of the timsTOF Pro makes proteome-scale PASEF analyses possible.

In these analyses, precursor ions are accumulated in the TIMS device and then released in discrete packets based on their collisional cross sections. Then, instead of looking at only one set of precursors per scan, the instrument's quadrupole is set to rapidly switch from one precursor to another, which lets the mass spec analyze multiple precursors per scan and increases the speed of the MS/MS analysis by the number of precursors multiplexed.

While the initial PASEF experiments used just one TIMS device, the timsTOF Pro features two TIMS devices in parallel. This, Mann said, allows researchers to accumulate ions in the first device and then pass them to the next device where they can be released over a time interval typically around 100 milliseconds. The dual TIMS device architecture "enables up to 100 percent utilization of incoming ions," he said.

In addition to the hardware advances, significant work was needed on the software side, as well, Mann said.

"There has been a lot of development in the acquisition engine that [is used] to pick the precursor ions to be sequenced as a function of [their] mass-to-charge, signal intensity, elution time, and ion mobility," he said. "There have been a lot of improvements there."

Additionally, Mann said, the group of his Max Planck colleague Juegen Cox refined the post-acquisition data processing tools to incorporate the new data from the TIMS system for identifying peaks amidst what he noted are "much larger data volumes" than in conventional experiments.

"This is a massively synchronized experiment," said Rohan Thakur, executive vice president at Bruker. "We're trying to synchronize precursor masses in the ion mobility domain and then cycle the quadrupoles to match the ions that co-locate in the ion mobility space, and then piece the MS/MS data together all the way back to the precursor ions that you select in the ion mobility domain. All that timing has to work precisely."

In a presentation at HUPO, Mann showed data from his group's experiments running the method on the timsTOF Pro. Using 5 μg of HeLa cell digest split into 24 fractions with each fraction run on a 45-minute gradient they identified 9,348 proteins. In a single-shot experiment using a 30-minute LC gradient they identified roughly 4,000 proteins and 800 unique proteins per minute. And in an experiment starting with 3.125 ng of HeLa digest, the equivalent of roughly 15 cells, they identified 1,803 proteins.

It is the high sensitivity exemplified by the latter experiment that Mann said has proved most powerful thus far.

"Based on the fact that we trap the ions in parallel and use 10 or more of the trapped ions, we should have a factor of 10 in speed and/or sensitivity compared to other instruments where this is done serially," he said. "This is indeed the case, and we now load about five to 10 times less [sample] than we do on other systems, while [achieving] comparable depths [of coverage]."

Mann added that there was room to start with sample sizes even smaller than the 15-cell HeLa digest experiment he presented at HUPO.

He said that he sees the instrument and PASEF method as well suited to clinical research where sample material is often limited. He noted work that he and his colleagues have done combining the Bruker instrument with the Evosep One LC system, which is aimed at the clinical proteomics space.

Also introduced at last week's HUPO meeting, the Evosep One instrument is, according to the company, optimized for rapidly running large-cohort experiments and can run around 200 samples per day at speeds of 1μL/min while operating around the clock.

The LC offers "very fast HPLC separation without losing robustness or sensitivity," said Mann, who noted that he has a business relationship with Evosep, though not with Bruker. "Given the fact that the Evosep in our hands allows thousands of injections of clinical material like plasma, and the fact that we have not had to clean the Bruker PASEF timsTOF for a year, this combination could be extremely attractive to bring proteomics to the clinic," he added.

Thakur likewise noted that Bruker envisioned the timsTOF as being well-suited to clinical research analyzing small samples like needle biopsies in cancer.

"That's exactly why we need to go to lower cell counts and hence more sensitive instruments," he said. "That's exactly the purpose."

Mann also gave as examples of areas where the new instrument and technique could prove useful the analysis of phosphoproteins in small amounts of material or analysis of formalin-fixed paraffin-embedded cancer samples."

"If the promise holds up, I don't see any areas where the approach should not be beneficial," he said.

According to Thakur the timsTOF Pro is currently available for order and will begin shipping by the end of the quarter.

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