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LC-MS: The Answer to Advancing Targeted Protein Analysis

Novel mass spectrometry methods facilitate accurate, sensitive, and reproducible protein analysis

Kerry Hassell, PhD

Kerry Hassell, PhD is a content manager for the clinical research and toxicology vertical market at Thermo Fisher Scientific. She has more than 14 years of experience as an analytical chemist and specializes in small and large molecule LC-MS/MS workflows on all Thermo Scientific’s mass spectrometry portfolio including triple quadrupole and Orbitrap™ mass spectrometers. Kerry’s previous experience in the Thermo Fisher demonstration laboratory provides her with deep insights into customer needs, helping her to solve complex problems using LC-MS/MS. Prior to joining Thermo Fisher Scientific, Kerry worked as a postdoctoral fellow at Northeastern University, working on hydrogen exchange experiments. Kerry’s doctoral work was performed at Purdue University, training under professor Scott McLuckey and developing experiments using ion/ion reactions. As a content manager, she is focused on sharing insightful content with the clinical research and toxicology scientific community as Thermo Fisher Scientific seeks to make the world healthier, cleaner, and safer.

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Published:Aug 20, 2021
|3 min read
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The use of proteins—in or as therapeutics—is expanding our ability to treat and cure complex diseases. Since 2011, the U.S. Food and Drug Administration (FDA) has approved 62 recombinant therapeutic proteins through its Center for Drug Evaluation and Research (CDER) and Center for Biologics Evaluation and Review (CBER). 

With proteins playing an increasing role within therapeutic development, accurate characterization is essential to ensure efficacy and patient safety. Analysis of proteins and biotherapeutics is necessary across a wide range of applications, including pharmaceutical research, development and production, and for analyses in clinical research laboratories ranging from biomarker identification, diagnostics, and biotherapeutic monitoring. Traditional approaches such as ligand-binding assays (LBA) are commonly used to analyze biotherapeutics, however liquid chromatography-mass spectrometry (LC-MS) is becoming an increasingly accepted technology and has established itself as the gold standard for the quantitation of macromolecular drugs, therapeutics, and biomarkers. Here, I discuss how LC-MS is enhancing protein analysis to meet the demands of the modern clinical laboratory.

Limitations of ligand-binding assays 

Quantitative analysis of proteins is one of the most commonly performed analytical steps in biochemistry. It is essential for the quality assurance (QA) and quality control (QC) of products, as well as pharmacokinetic and pharmacodynamic studies in clinical laboratories. LBAs are typically used to analyze the distribution and biotherapeutic effects of protein-based products in the human body, providing high sensitivity, high throughput, and excellent reproducibility in plasma-based testing. 

There are, however, a number of notable limitations of LBAs. They can be matrix- and assay-selective, which reduces flexibility, and validated methods only exist for a limited number of monoclonal antibodies (mAbs). Furthermore, new method development is a costly and time-consuming process as highly selective antibodies must be produced.

While LBAs undoubtedly play an important role in clinical laboratories, as market and regulatory requirements increase, there is a clear need for additional technology to overcome these issues.

LC-MS methods offer new capabilities


LC-MS provides a next-generation solution that addresses many of the limitations associated with LBAs, and offers impressive new capabilities in protein analyses. With LC-MS, protein-based biotherapeutics and protein markers are analyzed using their corresponding signature peptides. These peptides are produced through a controlled digestion process and can be easily identified and quantified at very low concentrations. 

MS-based targeted protein assays offer two compelling advantages over immunoassays. First, they provide scientists with the ability to systematically design a specific assay for any protein (or post-translationally modified proteins) without the requirement for an antibody. And, second, they enable the monitoring and quantitation of multiple peptides in a single analysis. Thus, scientists can improve the speed and efficiency of their work, while also being able to review data retrospectively. This can be especially useful when tracking a patient’s therapeutic response as it makes it possible to monitor how different protein levels change over time. 

The LC-MS analytical process allows unmatched sensitivity and accuracy in the detection and quantitation of signature peptides. Using MS provides excellent flexibility, enabling scientists to analyze many different proteins with consideration given to targeted peptide selection, sample preparation, and choice of monitoring mode, which can all be tailored for specific requirements.

Transforming the landscape of protein measurement

In light of the rising prevalence of infectious diseases worldwide, and the increasing complexity of the biotherapeutics being used to combat these diseases, there is a higher demand for improved specificity, selectivity, and sensitivity for targeted protein biomarkers used in the development of such biotherapeutics. These improvements are needed to provide confidence in results, reduce false positives or negatives, and ensure an effective QA/QC process for biotherapeutic production. Additionally, laboratories need to be able to quickly add and remove targets of interest and easily adapt their methods to keep up with emerging trends and new demands.

The use of MS technology is gaining traction and likely to become a key technology in clinical laboratories. Using LC-MS, scientists can more easily perform accurate, sensitive, and reproducible analysis of proteins for applications ranging from biomarker identification, QA/QC of biotherapeutics, and quantitation of a therapeutic for patient monitoring. By enhancing these processes, LC-MS will support the safeguarding of patient health and lead to improvements in care.