Advances in Quantitative Proteomics

In recent years, quantitative proteomics has emerged as a crucial field for elucidating cellular processes and developing new biomarkers. This article highlights the innovative efforts of three leading companies—Thermo Fisher Scientific, Waters Corporation, and Abselion—as they expand the horizons of this rapidly evolving discipline.

New instruments for biomarker research

Aaron Robitaille, Ph.D., Director of Marketing for Mass Spectrometry at Thermo Fisher Scientific, notes that the term proteomics was first coined by Australian scientist Marc Wilkins in 1994. “Quantitative proteomics has experienced exponential growth since then and continues to rapidly expand, driven by both mass spectrometry and complementary non-mass spectrometry methods.”

He notes that mass spectrometry-based proteomics remains the gold standard for quantitative accuracy and precision, facilitating the measurement of proteins, peptides, proteoforms, and post-translational modifications.

Robitaille highlights Thermo Fisher's recent launch of two new instruments, which he says have “significantly disrupted the proteomics status quo.” First, the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer enables discovery-based proteomics. “In its first year alone, the Orbitrap Astral MS generated ten times more publications than previous instrument launches, highlighting how this groundbreaking technology has accelerated scientific research for customers.”

Meanwhile, the Thermo Scientific™ Stellar™ mass spectrometer is designed for targeted peptide verification and validation. Advantages of the Stellar platform include lower costs, massive throughput, and enhanced sensitivity and specificity. Robitaille stresses how the Stellar measures up to 8,000 peptide targets in 60-minute gradients. “Customers are now able to target complete signaling pathways—like the mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) pathways—in both their activated and non-activated forms.”

“The enhanced sample throughput capabilities of these two mass spectrometers allow customers to increase their sample and study sizes while reducing instrument analysis time,” he says. This ultimately accelerates scientific progress by boosting the statistical power needed to identify new biomarkers, particularly in precision medicine research.

In addition to these new instruments, Robitaille adds that Thermo Fisher has recently introduced its Thermo Scientific™ TMTpro™ 32plex isobaric label reagents, which increase the multiplexing capabilities in quantitative proteomics. “These reagents enable the simultaneous analysis of up to 32 samples in a single liquid chromatography-mass spectrometry (LC-MS) injection using Orbitrap instrumentation.” This represents an increase from the previous limit of 18 samples.

Unveiling new biomarkers

Robert Plumb, Ph.D., is a scientific advisor for Waters Corporation, while Lee Gethings, Ph.D., is a consulting scientist. They highlight the company’s Xevo™ TQ Absolute™ Mass Spectrometer. “This high-sensitivity tandem-quadrupole mass spectrometer has been specifically developed to deliver robust, high-sensitivity analysis over five orders of magnitude of linear dynamic range—making it ideal for measuring low-abundance peptides and proteins.”

Plus, the Xevo TQ Absolute Mass Spectrometer is very environmentally friendly. “The platform’s compact footprint and state-of-the-art design uses approximately 50% less electricity and gas and produces 50% less heat than other high-performance tandem-quadrupole mass spectrometers on the market,” they emphasize.

Plumb and Gethings explain that another problem plaguing LC-MS is the non-specific binding of proteins and peptides to LC columns, preventing reliable and reproducible analysis.

To address this challenge, Waters has developed the ACQUITY™ Premier System for ultra-performance liquid chromatography and ACQUITY Premier Columns with associated consumables. “These products incorporate a novel technology—called MaxPeak™ High Performance Surfaces—that mitigates non-specific binding to the transition metals in LC systems and glass and plastic components in LC-MS consumables.”

On the sample preparation side of things, they note that Waters offers the ProteinWorks™ Auto-eXpress Digest Kit for simplified, fast, high-throughput protein digestion. “The kits, which are particularly applicable for plasma and serum-based samples, contain all the necessary reagents required for protein digestion. They provide scientists with the opportunity to prepare samples for quantification using surrogate peptides.”

Of additional interest to biomarker researchers, Waters has developed a ready-to-use LC-MS/MS assay for quantifying over 75 endogenous kinases via signature peptides. Sensitivities are as low as 100 pg (using ACQUITY Premier System and the Xevo TQ-XS™ Mass Spectrometer).¹ Plumb and Gethings note how kinase enzymes are now considered to be significant biomarkers for different human diseases, including renal and liver cancer.

Finally, Plumb and Gethings highlight some exciting recent use cases of the company’s technology. For instance, in a 2024 Nature paper, researchers at the University of Cambridge harnessed the Xevo TQ-XS system (coupled with a Waters UPLC™ System) to identify GDF15 as the hormone responsible for nausea during pregnancy.²

A novel platform for real-time measurements

Ruizhi Wang, CEO of Abselion, explains how the company’s Amperia™ platform allows for the real-time quantification of proteins—including therapeutic antibodies and capsids associated with adeno-associated viruses (AAVs). “At Abselion, our goal is to simplify protein quantification so that scientists can accelerate the development of biotherapeutics.”

Wang notes that quantification methods like mass spectrometry require extensive hands-on time from experienced operators. Protein quantification also often requires sample storage and the transfer of samples to centralized laboratories for automated analysis.

In contrast, the Amperia platform removes these analytical delays by providing automated quantification directly from unpurified samples in standard microplates. Results—which are highly sensitive and specific—are available in as little as 1 minute per sample.

The Amperia platform is ideal for measuring protein concentrations in cell culture supernatants (no filtering is required) and lysates. “For antibody and AAV measurements, pre-coated sensors are available, and customers don’t need to source additional reagents,” he adds. “We provide everything.”

Wang emphasizes how the Amperia system is built on the company’s proprietary Redox Electrochemical Detection technology. Electrical sensors with four measurement probes are dipped into the sample wells, enabling parallel measurements. The assay takes place directly on the sensor tip, where protein binding triggers an electrical signal read by the device.

The direct analysis of crude samples eliminates the need for time-consuming purification and complex sample preparation. Therefore, the system is ideal for applications requiring frequent quantification and rapid answers—such as identifying antibody-producing clones during cell line optimization or analyzing viral vector titers for gene therapy. “Our quantification method is accessible to anyone in the lab comfortable with basic pipetting into a standard 96-well plate,” Wang adds.

The system is compact and robust, and no calibration or maintenance is required between runs. Finally, unlike most mass spectrometry-based methods, Amperia can be deployed anywhere—including laboratory benches or beside bioreactors.

References

1. Daly et al. 2023. Endogenous kinase quantification assay combining low-flow LC and tandem quadrupole (QqQ) mass spectrometer. Waters Kinase Application Note, September 2023. Reference: 72008020.

2. Fejzo M, Rocha N, Cimino I, et al. GDF15 is linked to maternal risk of nausea and vomiting during pregnancy. Nature. 2024 Jan; 625(7996):760-767.