Comparison of Plasma Proteome Analysis Methods

Blood plasma is a convenient and rich source of protein biomarkers for disease detection. Biocompare recently interviewed experts from three companies to compare two common methods for analyzing the plasma proteome: mass spectrometry and a targeted immunoassay approach.

Advances in mass spectrometry

Rebecca Rutherford, Ph.D., is the Director of Product Management at Seer, while Aaron Gajadhar, Ph.D., is the Associate Director of Strategic Applications. They explain that a major challenge with plasma proteomics is the dynamic range problem that arises when proteins vary in concentration by up to 10 orders of magnitude. In mass spectrometry, this variation complicates downstream analysis with a single detector.

To deal with the dynamic range issue, Seer’s Proteograph™ Product Suite harnesses the company’s proprietary nanoparticles to enrich proteins. This boosts the detectability of low-abundance proteins and removes the need for time-consuming fractionation and depletion steps.

Seer’s technology has been the basis for several recent publications, including one in Nature Communications. Rutherford and Gajadhar further note that the company’s nanoparticle-based workflow now enables the accurate identification of more than 6,000 proteins from plasma. Furthermore, a single Proteograph instrument can enable the measurement of up to 10,000 samples per year with the right configuration.

Another challenge in mass spectrometry involves improving throughput (or the speed of analysis) without compromising selectivity or sensitivity. Torsten Mueller and Pierre-Olivier Schmit are Business Development Managers for Proteomics at Bruker Daltonics. They explain that Bruker has developed a timsTOF platform for a new approach called four-dimensional (4D) proteomics.

“4D proteomics is an exciting way to study peptides and proteins,” they say. “Fundamentally, 4D proteomics involves measuring an additional dimension beyond traditional mass spectrometry.” More specifically, the measurement of a peptide ion’s mobility allows for better characterization.

Mueller and Schmit explain that Bruker’s instruments combine trapped ion mobility spectrometry (TIMS) with time-of-flight (TOF) mass analysis in a technique called timsTOF. While the physics behind 4D proteomics is complex, Mueller and Schmit explain the basic principle of how it works. “Every measured peptide is encoded with its collisional cross section on top of its retention time, mass-to-charge ratio, and fragmentation pattern. These metrics are then used to gain higher confidence in identifying and characterizing a peptide.”

Mueller and Schmit further stress that Bruker’s technology is easy to use and does not require a specialist technician. “We have built-in methods to obtain high-level, reproducible results with the first injection.” Furthermore, they note that the cost per sample has been decreasing with more recent generations of Bruker instruments.

Mass spectrometry for proteoform exploration

A major advantage of mass spectrometry over antibody-based methods is its ability to readily distinguish between different proteoforms―the different molecular forms in which the protein product of a single gene can be found. Examples of proteoforms include splice variants, post-translational modification patterns, and endogenous proteolysis products.

“When it comes to clinical studies, the capacity of 4D-proteomics to increase the number of proteoforms being distinguished really enhances its potential for discovery,” explain Mueller and Schmit. “And beyond discovery, mass spectrometry is a great way to further qualify and characterize potential targets of interest.”

In contrast to mass spectrometry, targeted immunoassay platforms require antibodies to bind to their target protein and generate a signal. Therefore, only those proteins that are specifically targeted can be measured. Furthermore, because different proteoforms share the same epitope (or site to which the antibody binds), they cannot always be distinguished with immunoassay approaches.

Targeted immunoassays

Michael Gonzales, Ph.D., is the Vice President of Global Marketing at Olink Proteomics. He says that Olink developed proximity extension assay (PEA) technology to simultaneously measure many proteins and samples using minute volumes of plasma or other biological matrices.

Gonzales explains that each protein is detected with a matched pair of antibodies coupled to complementary DNA oligonucleotides that must hybridize before the signal is read out―using next-generation sequencing or quantitative PCR. The oligonucleotides act as a DNA barcode, enabling accurate protein identification in a multiplex format. He notes that Olink platforms deliver a specificity of 99.5%, irrespective of protein size or abundance.

The convenience of Olink’s approach

Compared with mass spectrometry, Gonzalez stresses that Olink’s platform is ideal for measuring plasma proteins at lower abundances. For example, he says that the Olink Explore HT platform can now measure over 5,400 proteins spanning 10 orders of magnitude in concentration with minimal sample processing.

Gonzales notes that Olink’s platform can analyze up to 344 samples in a single run. Furthermore, the technology is also easily scalable to projects of any size―from hundreds to millions of samples. “Mass spectrometry, on the other hand, often has a more limited multiplexing capability. For instance, most commercial labeling kits will only allow for the simultaneous analysis of 18 samples or less.” Olink also offers convenient pre-made biomarker panels, some of which measure the absolute concentrations of plasma proteins.

Another advantage of Olink’s technology over mass spectrometry involves sample preparation. “Preparing plasma for Olink analysis is straightforward, quick, and easy, eliminating the need for fractionation or depletion.” In contrast, Gonzales notes that bottom-up mass spectrometry often requires the time-consuming steps of protein denaturation―including reduction, alkylation, digestion, and clean-up. He further stresses that such procedures may introduce additional variation in the results.

A final advantage of Olink involves smaller sample volumes. While mass spectrometry typically requires 10 to 200 µL of plasma, Olink biomarker panels use as little as 1 to 2 µL. “These minute volumes are important to preserve precious clinical sample collections. They are also ideal for repeated sampling from the same animal over time to monitor disease progression.”

Complementary advantages

Ultimately, everyone agreed about the potential advantages of combining mass spectrometry and Olink’s targeted approach for biomarker research. Gonzales points out a 2021 study demonstrating that the two techniques detected very different plasma proteins with limited overlap. “In the end, Olink technology and mass spectrometry have distinct and complementary advantages for plasma proteomics,” concludes Gonzales.

References

Blume JE, Manning WC, Troiano G, et al. Rapid, deep and precise profiling of the plasma proteome with multi-nanoparticle protein corona. Nat Commun. 2020;11(1):3662.

Huang T, Wang J, Stukalov A, et al. Protein coronas on functionalized nanoparticles enable quantitative and precise large-scale deep plasma proteomics. bioRxiv. 2023:2023.08.28.555225. doi: 10.1101/2023.08.28.555225. Preprint.

Petrera A, von Toerne C, Behler J, et al. Multiplatform approach for plasma proteomics: complementarity of Olink proximity extension assay technology to mass spectrometry-based protein profiling. J Proteome Res. 2021;20(1):751-762.