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Harnessing the Power of Mass Photometry for Protein Characterization

Discover how mass photometry is transforming applications from analyzing complex protein interactions to optimizing antibody production

by
Tomás de Garay

Tomás de Garay has years of hands-on experience with mass photometry and is currently product manager with Refeyn. As a technology expert, he has the goal of shaping mass photometry...

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Whether you’re developing antibodies or investigating protein-protein interactions, having access to rapid, accurate protein characterization tools is invaluable. While conventional bioanalytical techniques often put a strain on time and sample volume, technological advancements are making it faster and easier to characterize proteins and other macromolecules. 

Mass photometry is a novel single-molecule analysis technology that offers a swift and sensitive solution for a broad range of macromolecule characterization applications. From resolving complex equilibria to uncovering protein oligomerization, the technology has potential for protein research and manufacturing.

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What is mass photometry?

Mass photometry measures the molecular mass of biomolecules by quantifying light scattering from individual particles as they adsorb onto a glass microscope slide. When a molecule in solution contacts the glass slide, it creates interference between the reflected light and the scattered light. The magnitude of this interference is directly proportional to the molecular mass; allowing for precise mass determination with no need for labels or tags, and minimal sample requirements of 5 to 20 µL of sample at low nanomolar concentrations. Figure 1 illustrates the principle of the technology.

 The principle of mass photometry.
Figure 1: The principle of mass photometry.
Credit: Refeyn

This approach can be harnessed to rapidly resolve the mass, oligomeric state, and heterogeneity of various macromolecules and their complexes in solution and under equilibrium conditions within two to five minutes. The speed and simplicity of mass photometry, coupled with its high sensitivity, makes the technology well-suited for a versatile range of protein characterization applications. 

Delving into these application areas can be a valuable way to explore this novel bioanalytical technology further. Here, we discuss three of the major application areas in which mass photometry can bring advantages over conventional characterization techniques:

1. Analysis of complexes

Oligomerization, the process by which proteins self-assemble into specific quaternary structures, regularly modulates protein functionality. This self-assembly is fundamental for many proteins to carry out their biological functions effectively, facilitating processes like molecular recognition, signal transduction, and catalytic activities. 

Understanding the dynamics and behaviors of these oligomeric states is essential when seeking to gain a deeper insight into protein function and regulation. Mass photometry offers rapid, high-precision characterization of these oligomeric behaviors. The technology can detect species representing less than one percent of the molecules in a sample, providing a window into the diversity of oligomeric states and their impact on protein function. This high resolution makes the approach particularly beneficial for identifying and analyzing transient and low-abundance complexes that are often missed by less sensitive methods.

Figure 2 illustrates the oligomerization dynamics of a target protein under various conditions. It demonstrates how mass photometry can identify distinct monomer, dimer, and tetramer complexes, revealing how environmental factors affect protein assembly. Approached conventionally, researchers often use analytical ultracentrifugation (AUC) to interrogate protein oligomerization, but compared with mass photometry, the technique brings technical complexity, a time-consuming workflow, and a requirement for large sample volumes. Furthermore, since the concentrations in mass photometry experiments are nanomolar, akin to physiological conditions, the observed oligomeric states more closely reflect the protein's natural behavior in vivo.

2. Sample characterization

Antibodies are essential tools with countless applications spanning from research to therapeutics. Antibody production is a delicate process in which having access to tools that can assess sample purity and quality at various stages of production is crucial. Detecting antibody fragmentation and aggregation events is especially important during production, as these can have a significant impact on the quality, efficacy, and safety of the final product. 

A mass histogram of a protein-only sample, with monomers and dimers present. B: Calcium chloride (present in excess) induced the formation of tetramers and reduced the number of dimers. C: Adding a saturating concentration of EDTA reversed this effect, resulting in the disappearance of the tetrameric species.
A mass histogram of a protein-only sample, with monomers and dimers present. B: Calcium chloride (present in excess) induced the formation of tetramers and reduced the number of dimers. C: Adding a saturating concentration of EDTA reversed this effect, resulting in the disappearance of the tetrameric species.
Refeyn

Mass photometry provides a valuable tool for characterizing antibody aggregation and fragmentation. It can detect contaminants down to picomolar concentrations and performs well across a wide range of buffers and experimental conditions, minimizing the need for buffer adjustments. Unlike most conventional methods for antibody characterization, such as AUC or size exclusion chromatography (SEC), it enables a rapid turnaround time of a few minutes. This allows therapeutics developers to conveniently monitor antibody samples at multiple stages and use little sample. This is important when dealing with such a high-value product. 

Unlike traditional methods such as size exclusion chromatography with multi-angle light scattering (SEC-MALS), which provides a population-averaged mass measurement following physical separation of populations, mass photometry provides a precise single-molecule mass measurement at equilibrium. The technique requires significantly less sample volume and delivers results rapidly, presenting a more convenient and reliable tool for in-process monitoring and decision-making.

3. Interaction studies

Protein-protein interactions are critical for numerous cellular processes, from signaling to differentiation, transport, and much more. Studying protein interactions however, brings inherent challenges since they often involve multiple components and complex, dynamic equilibria. Many of the resulting protein complexes are transient or exist at very low concentrations, making them difficult to detect and quantify with traditional analytical methods like Western blotting.

By analyzing individual molecules, mass photometry can detect and quantify the relative abundance of each species present in a protein-protein interaction at equilibrium. This enables users to identify protein complexes formed, determine their stoichiometry, and evaluate the strength of interactions via calculation of the dissociation constant (KD). The ability to observe protein interactions in their native state without the need for labeling or extensive sample preparation brings value to a broad range of molecular biology applications. 

To advance its detection range further, the technique can be enhanced with the addition of a microfluidic device that rapidly dilutes samples prior to measurement. This significantly broadens the range of sample concentrations suitable for investigation by mass photometry. It increases the upper sample concentration limit from the nanomolar to the micromolar range, enabling applications such as the characterization of low-affinity interactions. By facilitating the exploration of transient and weak interactions, this capability provides a more comprehensive understanding of protein behavior and equilibrium dynamics.

A novel tool for protein research and beyond

Mass photometry is a novel bioanalytical technique with true potential. Covered here are just a few of the numerous applications supported by mass photometry. The technology is universally applicable for large molecules, above 40kD in size, for DNA analysis or for characterization of viral vectors for gene therapy. 

For example, supporting cell and gene therapy development, mass photometry can be used to analyze adeno-associated viruses (AAVs), the most popular vector platform for gene therapies. It provides rapid, detailed measurements of AAV capsid populations, including empty, full, and partially filled capsids, as well as aggregates and contaminants. In addition, a recently introduced and closely related technology, macro mass photometry, can be used to analyze two other widely used viral vectors—adenoviruses and lentiviruses. 

By enabling rapid, sensitive, and label-free protein analysis with minimal sample requirements, and as further new applications are found, the technology is paving the way for new discoveries in disease mechanisms, therapeutic development, synthetic biology and beyond.