Frontiers in Protein Quantitation

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Michael Scott Long received his Ph.D. in analytical chemistry from Penn State. Nature, the Washington State Academy of Sciences, and many others have published his scientific writing and science journalism. He lives in Richland, Washington.

June 13, 2024

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Many bioanalyses depend on reliably measuring the protein concentration in aqueous samples—such as targeting the human kinome (Gomez et al., 2024), identifying biomarkers in the cancer cell secretome (Almeida–Marques et al., 2024), and evaluating the success of multiple myeloma therapy (Wijnands et al., 2024). The impact of protein quantitation is perhaps best indicated by its global market size, which reached USD 2.8 billion in 2023 and is estimated to reach USD 5.6 billion by 2032 (IMARC Group, 2024).

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How can you sort through the advantages and disadvantages of each approach to protein quantitation, and in so doing optimize it for your research program? Here, we provide representative commercial solutions and recent research advances in method and tool development.

Colorimetric assays simplify protein quantitation

Mark Pawlicki, Senior Product Manager at MilliporeSigma, summarizes the challenges of protein quantitation and the opportunities provided by the well-known bicinchoninic acid (BCA) assay: “The complexity of quantifying protein in a sample is often taken for granted. While UV absorbance is common and easy, there are many factors that can lead to inaccurate and imprecise results. Things like non-protein interference, amino acid content, pH, and solubility are some of the top confounding factors.”

According to Pawlicki, “The benefit of using a BSA-based method, the technology standardized for protein quantification in the QuantiPro BCA kit, is that it interacts with the amide of the peptide backbone of the protein. This way there is a linear relationship between color development and actual protein quantity, and amino acid side chain reactivity is not a major factor. Additionally, the standards that come along with the QuantiPro kit allow researchers to build their own curve and verify findings against precisely formulated protein standards.”

Modern chromatographic columns facilitate antibody analysis

More than 100 monoclonal antibodies (mAbs) are FDA-approved as human therapeutics (Lyu et al., 2022), yet it’s challenging to engineer the mAb characteristics that lead to successful clinical trials (Jhajj et al., 2023). Optimized chromatographic tools will help such efforts. Steve Murphy, Workflows and Applications Manager at Agilent Technologies, says: “Bio-Monolith protein A and protein G affinity inline columns play a crucial role in protein and mAb quantitation applications. These columns are designed for measuring mAb titers, assessing biopharmaceutical production processes, and purifying small quantities of mAbs for subsequent characterization.”

Murphy continues: “The workflow includes sample capture, impurity removal, elution, and quantification. These columns offer high throughput, robust recovery, IgG subclass separation, flow-rate independence, and rapid transport. They are compatible with HPLC and UHPLC systems, making them indispensable tools for accurate mAb quantitation. AssayMAP protein A and protein G cartridges are used with the AssayMAP Bravo automated sample prep system for off-line mAb purification. The AssayMAP platform can purify mAbs from one to ninety-six samples in parallel for rapid and quantitative purification, to enable high-throughput LC/MS-based quantification and characterization.”

Imaging mass cytometry accelerates proteome research

Imaging mass cytometry (IMC) is an especially versatile approach to studying the proteome. “Quantifying protein expression is crucial because it allows us to understand the functional state of cells, identify biomarkers for diseases, and develop targeted treatments with high precision and efficacy. The ability of IMC to detect 40-plus proteins at once accelerates these applications. It can also detect protein and RNA transcripts together for an even more comprehensive look into functional states and secreted proteins,” Clinton Hupple, Director of Product Management (Imaging) at Standard Biotools, explains.

In this context, Hupple continues: “The Hyperion XTi imaging system is a multiplexed tissue imager that uses metal-tagged antibodies, instead of fluorophores, to simultaneously acquire 40-plus protein and RNA markers, without autofluorescence interference. This provides a unique opportunity to garner the most data out of limited samples with the clarity and precision of images that have little to no background. By leveraging the different modes on Hyperion XTi, researchers can quickly visualize tissue heterogeneity—without having to manage time-consuming cycles, spectral unmixing, or tissue degradation—and can then conduct deeper quantitative analysis on targeted regions.”

Hupple adds: “IMC used with RNAscope enables the study of multiple chemokines—leveraging the sensitivity of IMC, even for medium- and low-expressed genes, due to a lack of background staining issues. Detecting both RNA and proteins on the same slide provides an abundance of data about what interactions are occurring and where. Tools like the RNAscope along with IMC provide a thorough examination of the dynamic networks of cells and other factors that influence their behavior, shedding light on specific targets that could lead to the development of effective therapies.”

Mass spectrometry is easier than ever before for protein quantitation

An expensive and time-consuming limitation of using mass spectrometry (MS) for absolute protein quantitation is the need to create isotope-labeled peptide standards. Fnu et al. (2024) reported a possible solution: labeling peptides with the electroactive reagent monocarboxymethylene blue NHS ester, followed by coulometric MS analysis. In this approach, a redox reaction lowers the intensity of the MS spectrum in a manner that is quantitatively proportional to the amount of the protein. The error in cytochrome c quantitation was −2% (11.6 pmol detected vs. 11.9 pmol added), whereas that of beta-casein quantitation was nearly −26% (18.8 pmol detected vs. 25.3 pmol added; reportedly due to incomplete tryptic digestion).

Proteins often exhibit post-translational modifications (PTMs) and other variations—collectively termed proteoforms—in a manner that modifies protein function. However, it can be difficult to comprehensively assay proteoforms by MS and thus reliably assess their biological implications. Accordingly, Huang et al. (2024) developed an MS methodology based on parallel reaction monitoring that has low-femtomole sensitivity: a limit of quantitation of 2.18 fmol for ubiquitin, 3.43 fmol for myoglobin, and 12.6 fmol for carbonic anhydrase. Furthermore, in an analysis of human peripheral blood mononuclear cells, a panel of 24 immunoproteoforms—over half of which contained PTMs—exhibited expression trends that are consistent with a previous study.

Protein quantitation is foundational to many bioanalyses and is a critical laboratory need. However, each method and tool has advantages and disadvantages (Song et al., 2023). For example, whereas MS can detect urinary glycopeptides that correspond to ovarian cancer with 100% sensitivity and nearly 92% specificity (Aitekenov et al., 2021), such instrumentation is expensive. It is critical to understand the corresponding science to choose the protein quantitation workflow that best meets your needs. Speak with an industry specialist to avoid common pitfalls and make choices that are most appropriate for your application.