Clinical and translational research is increasingly turning to molecular studies, such as proteomics and metabolomics, but research is limited by the availability of tools to catalog the many thousands of molecules in a single blood sample with high accuracy, sensitivity, and speed. Mass spectrometry can provide quantitative peptide-level information, but until recently has lacked the throughput to accommodate large-scale studies. Now, new instruments, sample preparation technologies, and data processing platforms are enabling researchers to collect proteomic and metabolomic data from patient populations on a scale previously unheard of.
“If you look at the history of mass spectrometry-based proteomics, the rate-limiting step has almost always been the mass spectrometer,” says Daniel Hermanson, PhD, director of product management at Thermo Fisher Scientific. Two years ago, Thermo introduced the Orbitrap Astral MS, which boosted throughput by about a factor of four, Hermanson says, enabling researchers to scan 180 samples per day. Now, the company has unveiled a new instrument, the Orbitrap Astral Zoom MS, which is not only faster but also more sensitive.
“It not only allows you to do these very high-throughput studies, but it brings additional depth and accurate and precise quantitation into play, which allows us to go much deeper, faster,” Hermanson says. The previous Orbitrap Astral MS can measure 180 proteomics samples per day and return results on around 8,000 different proteins. Using the same protocol, Hermanson says, the Orbitrap Astral Zoom MS can measure 8,600 proteins. “Alternatively, you could go to about 300 samples per day on Orbitrap Astral Zoom and get that 8000 protein number.”
The Orbitrap Astral Zoom MS achieves this improvement thanks to two big changes: an increased scan rate (from 200 Hz to 270 Hz) and pre-accumulation of ions. With pre-accumulation, the instrument adds an extra ion-capturing step in parallel. This provides more ions at the 200 Hz scan rate or a similar number of ions at the faster scan rate.
Having an instrument capable of running hundreds of samples per day won’t increase throughput if it simply shifts the bottleneck to the data processing step. On the software side, Thermo Fisher is increasing the capabilities of its Ardia Platform to efficiently manage and process data as it’s collected. “As soon as that raw data file is acquired, it starts processing in the data processing software,” Hermanson explains. “Now you’re not waiting for every sample to acquire before starting the processing.” The Ardia platform also aims to streamline collaboration by enabling the data to be read by third-party software, directly shared via a web browser interface, or deposited into public databases.
“It’s really an exciting time to be in proteomics,” Hermanson says. “It’s inspiring to see what people are able to do with this technology.” For example, he says, Yu-Ju Chen, PhD, distinguished research fellow at Academia Sinica in Taiwan, used the Orbitrap Astral Zoom mass spectrometer in her translational research focused on lung cancer screening. To screen 6,000 patients for protein biomarkers would have taken 1,000 days using existing mass spec instruments, but with the Orbitrap Astral Zoom, it would take 100 days. “That is so exciting that we can take proteomics into the scale that’s required to make a true impact in the real world,” Hermanson says.
Nanoparticles and the proteome
Once the scanning and data processing steps are sped up, the other potential bottleneck is sample preparation. While mass spectrometry proteomics can be applied to neat plasma, the tremendous dynamic range of proteins means that highly abundant proteins can mask the detection of rarer, but more biologically interesting, proteins. Immunodepletion methods are often used to enrich for low-abundance proteins, but these are too labor-intensive to scale and do not always produce reproducible results.
The ProteographTM product suite from Seer leverages unique properties of nanoparticles to rapidly and reproducibly extract peptides from a sample via a completely automated workflow. “The robustness and the reproducibility of these are incredible and suitable for doing large-scale studies,” says Omid Farokhzad, MD, CEO of Seer.
Proteograph begins with multiplexed, engineered nanoparticles designed to collect a broad range of proteins from a liquid sample, including plasma, urine, or cell lysate. Proteins in the sample accumulate around the nanoparticle, creating a shell called a protein corona, and this corona forms in a specific, predictable way. “These nanoparticles are designed specifically for maximum capture across the entire dynamic range,” Farokhzad says. “They capture the abundant proteins, but they also capture the least abundant proteins. Essentially, it compresses the dynamic range, but it does it quantitatively, and it does it reproducibly.” The entire process is automated, meaning that the user can put a biological sample directly into the instrument without any depletion or fractionation, and in about five hours, the peptides are ready to be analyzed in the mass spectrometer.
The instrument can process 80 samples at a time. When Seer launched in 2017, Farokhzad says, the largest deep-plasma proteomic work ever published analyzed just 48 samples with coverage of 1850 proteins. Today, Seer has multiple customers using Proteograph to process thousands of samples with coverage of more than 8000 proteins.
For example, in 2020, Seer spun out PrognomiQ, a multiomics company pursuing early cancer detection, using data generated by Proteograph to identify biomarkers. In a paper in medRxiv, PrognomiQ reported that their lung cancer early detection blood test achieved 89% sensitivity and 89% specificity, using biomarkers derived from DNA, RNA, proteins, and metabolites.
“They did unbiased proteomics to a depth of about 13,000 proteins in their study looking at 2500 individuals to identify biomarkers of cancer,” Farokhzad says, adding that the test is expected to launch in late 2025. “That test is the first clinical application of our platform as a commercially available product.”
More than proteins
Increasingly, scientists are turning to small molecule analysis for biomedical insights, increasing demand for instruments that can catalogue metabolites accurately and at scale. Earlier this year, SCIEX, an operating company of Danaher Corporation, launched its ZenoTOF 8600 system, which improves sensitivity by up to a factor of ten over the previous model. Additionally, “it offers a unique combination of state-of-the-art technologies that expand its application to all the different omics workflows,” says Katherine Tran, senior manager of global market development for proteomics at SCIEX. “The thing with the ZenoTOF is it’s so versatile.”
The ZenoTOF 8600 system starts with the triple quadrupole front-end ion source and ion guide that SCIEX is known for and combines it with a Zeno trap-enabled QTOF and a new optical detector. It also includes Mass Guard, a feature that uses T-bar electrodes to filter out contaminating ions and create a cleaner ion beam, and tunable electron activated dissociation (EAD), which provides a range of different free electron-based fragmentation mechanisms within one device. Finally, the ZenoTOF 8600 system includes ZT Scan DIA 2.0, which covers a larger mass range than the previous version. “Together, the extended mass range provided by ZT Scan DIA 2.0 and tunable, higher-energy EAD cell enable comprehensive analysis of both small and large molecules, such as immunopeptides and glycopeptides,” Tran says. It’s also fast: with ZT scan DIA 2.0, the instrument can achieve a scan rate of 858 Hz, “making it the fastest QTOF system for high resolution accurate mass,” Tran says.
Tran adds that the company has not neglected the user experience. “At the end of the day, it’s not just the improvements in the instrument that will benefit a user, it’s the usability of it as well,” she says. “Enhanced functionality in SCIEX OS acquisition software improves the user experience by providing real-time data quality metrics, method optimization, and system performance monitoring.”
Small molecules, big impact
Bruker Corporation is also moving into the metabolomics space, expanding on its trapped ion mobility spectrometry (TIMS) technology to create the timsMetaboTM platform, specifically designed to tackle small molecule identification and quantification.
“TIMS technology has done very well in proteomics, and the company wants to extend that proficiency across the omics space,” says Matthew Lewis, PhD, vice president of metabolomics and lipidomics at Bruker. “Through changes to the hardware combined with new acquisition modes, we’ve been able to extend the mobility range that we can transmit, and that enhances greatly the sensitivity of the system for small molecule applications.”
Lewis says that TIMS technology has key benefits that make it well-suited to small molecule analysis. Metabolites, as a group, are much more chemically diverse than proteins. TIMS provides high resolution of molecules of similar or identical mass, allowing more selective measurement of quantities and cleaner MS/MS fingerprints. It also provides collisional cross section (CCS) measurements, an orthogonal measurement by which to validate an annotation. “CCS values are intrinsically, accurately predictable, unlike MS/MS fragmentation patterns, unlike LC retention times,” Lewis says. “We can now say we think the annotation is correct based on mass, retention time, isotopic pattern, fidelity, all these things, and then we validate it by comparing the measured CCS to either a library or a predicted value.”
Another unique feature of TIMS is that it orders the ions by CCS measurement and by mobility. “As a consequence of that, all the downstream components have some knowledge of what’s coming and when,” Lewis says. The timsMetabo includes the new Athena Ion Processor, which can use this knowledge of the incoming ions to optimize their transmission to the TOF, boosting sensitivity.
“The timsMetabo comes from a dedicated push to better serve the small molecule communities of metabolomics, untargeted screening, [and] bioanalysis,” Lewis says. “My hope for this instrument is that people use it as a routine workhorse.”
