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New Mass Spec Releases Target Single-Cell, Imaging Proteomics Work

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NEW YORK — While the COVID-19 pandemic has pushed this year's American Society for Mass Spectrometry meeting to the fall, vendors have kept to their traditional launch patterns, introducing this month a number of new mass spec instruments.

Among the launches are several new systems that could prove significant for proteomics, including new technologies from Sciex and Waters, and from Bruker the first mass spec platform marketed specifically for single-cell proteomics. Thermo Fisher Scientific released a new instrument aimed at small molecule work as well as a new version of its FAIMS device.

Bruker's single-cell instrument, called the Bruker timsTOF SCP, is a version of the company's timsTOF system optimized for high-sensitivity analyses of very small sample sizes, down to single cells. The platform is based on work done by Bruker in collaboration with the lab of Max Planck Institute of Biochemistry professor Matthias Mann, in which they modified a timsTOF Pro by introducing a brighter ion source and different ion optics elements to produce an ion current 4.7-fold higher than that of an unmodified instrument, allowing them to analyze single cells with high sensitivity.

Using the instrument, they managed to identify more than 1,200 proteins in single cells. According to Bruker, the new commercial version of the instrument offers fivefold higher ion transmission and has shown the ability to quantify roughly 1,500 proteins in single-cell samples.

As its name implies, the instrument is intended specifically for small sample sizes, said Gary Kruppa, vice president of proteomics at Bruker, noting that while it offers higher sensitivity at the low end, it does not have the dynamic range that would be required to effectively analyze both very small samples at that sensitivity level as well as larger samples.

The instrument allows for a label-free form of single-cell proteomics, which is an alternative to the isobaric labeling-based methods that have been most commonly used for single-cell measurements to date. In labeling-based methods, researchers label peptides from both the single-cell sample of interest and another, larger sample source (such as dozens or hundreds of cells of equivalent type) termed the "carrier proteome." By including the carrier proteome sample, they are able to ensure that even analytes present only at low abundance in the single-cell samples are present in relatively high abundance in the overall sample, making them more likely to be fragmented and detected by the mass spec.

Kruppa said that Bruker and its collaborators are also exploring isobaric labeling-based single-cell approaches on the timsTOF SCP instrument, noting that even if a carrier proteome weren't necessary to achieve the desired sensitivity, isobaric labeling would allow for higher throughput — a key concern for single-cell mass spec experiments. Additionally, he noted that the sensitivity of the instrument could allow it to use smaller collections of carrier cells to enhance an experiment's sensitivity while still managing good quantitation.

Besides proteomics labs, Bruker expects the platform will find a market at centers doing single-cell genomics and transcriptomics, many of which have been closely watching developments in single-cell proteomics, Kruppa said.

"We have seen that as the early [single-cell proteomics] labs have shown this is possible and started cranking out results, there has been tremendous interest from people doing single-cell genomics and single-cell RNA-seq in adding single-cell proteomics to their portfolio," he said. "A lot of places have set up single-cell research centers and they want to add proteomics."

Christopher Rose, a senior scientist at Genentech, said in an email that the introduction of the instrument was "very exciting" given its potential for improving analysis of small proteomic samples.

"There are many applications related to low-level samples where these advances could prove useful, including important translational experiment types such as the analysis of MHC-associated peptides," he said. "We are constantly being asked to analyze smaller and smaller cell populations, and the introduction of instruments with increased sensitivity always piques our interest."

Stefan Tenzer, a professor at the Institute for Immunology at the University Medical Center of the Johannes-Gutenberg University Mainz, said that his lab had acquired a timsTOF SCP and that he was interested in putting the instrument's high sensitivity toward phosphoproteomic analysis of very small cell populations.

Tenzer's lab also acquired Bruker's other new release, an updated version of the company's timsTOF Pro, the timsTOF Pro 2, which it is also using for phosphoproteomics work.

Tenzer said that, regarding phosphoproteomics, the timsTOF Pro 2's ion mobility capabilities provide "information that you cannot really get in that detail with other instrument types."

He highlighted in particular the case of isobaric phosphorylations, where a peptide may contain either of two different phosphorylation sites that are difficult to distinguish with traditional LC-MS workflows.

"The parent mass is exactly the same and they have very similar [LC] retention times, so you often get chimeric spectra of both phosphorylation site isomers," Tenzer said.

"We've seen that a number of these [isobaric] phosphopeptides are actually pretty nicely separated in the gas phase by ion mobility," he said. "That's a huge benefit in terms of both coverage and confidence in our identification that we actually have the right phosphosite."

Tenzer said that he could not directly compare the timsTOF Pro 2 to the original timsTOF Pro since his lab did not have the older device, but he noted that one major advantage of the Pro 2 is the higher capacity of its trapped ion mobility spectrometry, or TIMS, device.

"You can put more ions in the TIMS device, and that allows you to have more ions in your spectra, which gives you more sensitivity in the end," he said.

Tenzer said his lab has also been using the Pro 2 for immunopeptidomics work identifying MHC Class I and II ligands.

"There, actually, ion mobility is very beneficial, because the problem with immunopeptidomic samples is that the peptides occupy a pretty tight mass range, and many of these MHC Class I ligands are very close together in their m/z values," he said. "Chromatography-wise they are not so easy to separate, so the ion mobility helps a lot to get deeper into the immunopeptidome."

Kruppa cited the Pro 2's new TIMS device as one of the main areas of improvement versus the previous edition of the instrument, highlighting its higher ion capacity. He noted that while the original timsTOF Pro could measure around 4,500 proteins in cell digest using a 90-minute LC gradient, the Pro 2 can measure around 6,000 proteins in a 60-minute gradient.

Waters, Sciex, Thermo Fisher

Waters and Sciex both introduced new instruments featuring additions to their TOF technologies. Waters' new Select Series MRT instrument features what the company calls its multi-reflecting time-of-flight technology, essentially a structure that lengthens the flight path of ions within the TOF system to improve its resolution without sacrificing speed. According to the company, the instrument can provide 200,000 resolution at speeds of up to 10Hz. By way of comparison, Waters' Xevo G2-XS QTOF offers resolution of around 40,000.

In TOF instruments, "the resolution and mass accuracy gets better the longer the [ion] flight tube," said Jeff Mazzeo, vice president of scientific operations and marketing at Waters. However, he added, "if you try to make it too long then you are going to have a massive instrument that fills up your lab."

The MRT system addresses this by reflecting the ions multiple times within the instrument so as to increase the flight path by roughly an order of magnitude without increasing the size of the instrument.

The Select Series MRT is intended primarily for mass spec imaging, Mazzeo said. The instrument includes both DESI and MALDI sources, allowing researchers to use both ionization modalities. And because DESI is a non-destructive ionization technology, researchers can use it first to collect data and follow that with MALDI analysis of the same sample.

"With DESI we can see things like small molecules, metabolites, lipids, et cetera, whereas with MALDI you see the larger things like peptides and proteins," he said. When combined with the MRT technology, this enables the identification of "many more different components in a tissue sample and with that very high mass resolution and mass accuracy," he added.

He said the system provides a roughly three-fold improvement in mass resolution and two-fold improvement in mass accuracy compared to existing TOF imaging systems.

Sciex also introduced new technology in its new TOF system, the ZenoTOF 7600. The instrument features what the company has termed its Zeno trap, which allows researchers to accumulate ions that can then be injected in pulses into the TOF analyzer. This lets the instrument analyze a higher percentage of ions generated from a sample (greater than 90 percent according to Sciex), which the company said produces sensitivity gains of between five- and 20-fold and yields up to 40 percent more protein identifications in proteomic experiments.

The system also features a new fragmentation technology called electron-activated dissociation, which the company said allows for more finely tuned free electron-based fragmentation, letting users better tailor their fragmentation to the type of molecules they are analyzing.

Thermo Fisher Scientific also released this month a new mass spec instrument, its Orbitrap IQ-X Tribrid system, though the device is focused on small molecule analysis as opposed to proteomics.

The company also released a new version of its FAIMS system, which has seen uptake among proteomics researchers. The system, called the FAIMS Pro Duo interface, makes FAIMS more compatible with higher LC flow rates, said Andreas Huhmer, senior director of omics marketing at Thermo Fisher.

While this extension is primarily focused on areas like metabolomics and other small molecule work where higher LC flow rates are common, it also fits into the move within proteomics toward the use of higher flow rates, Huhmer said.

"If you think about translational research studies where you are trying to analyze a cohort of, say, 1,000 or 2,000 samples, you want a very stable operation over a long period of time, and in that case you don't want nanoflow [LC], you typically want microliter flow rates," he said. "And that is where the new FAIMS Pro Duo really makes a difference."

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