Ensure Sample Quality With Rapid, High-Resolution Protein Characterization

APPLICATION NOTE Need help? Get in touch here Rapid, high-resolution protein characterization with MyMassMP www.refeyn.com APPLICATION NOTE APPLICATION NOTE Need help? Get in touch here Abstract Understanding protein structure and function requires high-quality samples and assays performed under optimal buffer conditions. This application note uses wellcharacterized proteins to demonstrate how the mass photometer MyMass™ quickly assesses sample purity and homogeneity using nanograms of sample. MyMassMP measurements work across a broad mass range (55 kDa– 3 MDa) and in a range of different buffers, and take just one minute. Employing mass photometry technology, MyMassMP measures the mass of single proteins in solution. With its speed, ease of use, and broad applicability, MyMassMP offers a powerful alternative to SDS-PAGE, SEC, and DLS for routine sample evaluation. Introduction Developing an in-depth understanding of protein structure and function requires high-quality samples and multiple assays performed under varied conditions. It is valuable to determine how buffer, temperature and other conditions affect sample purity and stability. For techniques such as cryo-electron microscopy (cryo- EM), native mass spectrometry (MS), surface plasmon resonance (SPR), and biolayer interferometry (BLI), there is a need to ensure samples have optimal purity and homogeneity. While this type of information serves as a vital foundation for almost any structure and function characterization work, it can be difficult to obtain due to the limitations of widely used techniques. SDS-PAGE, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), for instance, are all bulk methods with limited resolution relative to single-molecule techniques. DLS measurements are dominated by larger particles, which can mask the presence of smaller species in heterogeneous samples. The conditions in SDS-PAGE can disrupt complex formation and oligomerization, and in SEC, column-buffer interactions and conformational heterogeneity can be significant challenges. With all three methods, it can be difficult to effectively characterize heterogeneous samples. Mass photometry (MP) is a technique that provides singlemolecule resolution, with measurements in native buffer conditions. It provides a result in under five minutes – making it at least 15–20x faster than SDS-PAGE and SEC – and uses little sample. Compatible with a wide range of buffers with varying levels of salts and pH, it facilitates rapid screening of buffer conditions. Using single-molecule mass measurement, it readily identifies heterogeneous samples – resolving different oligomeric species and complex formation. In this application note, we demonstrate how the mass photometer MyMass™ provides valuable insights into sample purity and homogeneity over a broad mass range and a variety of buffers, with speed and accuracy. First, it shows how changes in pH alter the ratio of thyroglobulin monomers to dimers. Second, it reveals differences in the purity of two commercially available catalase enzyme products. Third, it clearly reports how the presence of urea and nucleotides affects the stabiity of the 14-mer chaperonin GroEL. With its ease of use, rapid data acquisition (one minute), and low sample requirements (30 μL at 10 nM), MyMassMP enables fast, reliable sample assessment without compromising accuracy. Good resolution and a broad accessible mass range (55 kDa to 3 MDa) allow characterization of diverse biomolecular systems under native buffer conditions, making MyMassMP a powerful tool for quick sample checks and an attractive alternative to SDS-PAGE, SEC, and DLS. At a glance • The mass photometer MyMass™ assesses sample purity and homogeneity in minutes, using just nanograms of sample. • MyMassMP works across a broad mass range (55 kDa–3 MDa). • This application note uses well-characterized proteins to demonstrate how MyMassMP can quickly check sample purity and help optimize buffer conditions. • MyMassMP leverages mass photometry to measure biomolecules at the single-molecule level in native buffer conditions. • MyMassMP is ideal for checking sample quality or optimzing buffer conditions before challenging or costly analytical techniques such as cryo-EM, native MS, SPR, or BLI. • MyMassMP is easy to use and versatile, offering a fast, reliable alternative to SDS-PAGE, SEC, and DLS for routine sample evaluation. APPLICATION NOTE Need help? Get in touch here Measuring thyroglobulin sample purity and pH sensitivity The protein thyroglobulin (Tg), produced by the thyroid gland, stores and transports thyroid hormones. Tg exists as a non-covalent dimer of ~660 kDa (human) and ~670 kDa (bovine). As a large and structurally heterogeneous glycoprotein with multiple glycosylation and iodination sites, it can be difficult to characterize by conventional methods. We used MyMassMP to assess the purity and homogeneity of commercially available bovine Tg. According to the analysis, the most abundant population (73% of fitted particles) had a mass close to the expected 670 kDa (Fig. 1). Lower-mass species were also detected, at 144 and 336 kDa, while higher-mass species were observed at very low abundance, below the threshold for fitting. Mass photometry signals with a mass below 55 kDa were not counted, as they cannot be reliably differentiated from background noise. The 336 kDa species likely represents the Tg monomer, while the 144 kDa species may correspond to degradation products or impurities (e.g. antibodies). The results show that the measurement is accurate and provides an overview of sample composition. The dimer peaks, at 663 kDa, are within 2% of the expected value; the monomer peaks, at 336 kDa, are within 1%. This accuracy is particularly notable because Tg – as a large glycoprotein – tends to display significant structural heterogeneity, making it difficult to analyze with chromatographic methods, such as gel electrophoresis and SEC. It may even be too large to enter the chromatographic matrix (PAGE) or elute near the void volume with low resolution (SEC). However, as MP accommodates a wide mass range and is not sensitive to shape, it can readily characterize large, structurally heterogeneous molecules like Tg. As the ratio of monomeric to dimeric Tg is responsive to changes in acidity,1 we next evaluated the effect of pH changes on the Tg monomer:dimer ratios we observed with MyMassMP. We measured the samples immediately after dilution into buffer of pH 3, 4.6, 5.8, or 7. Dimeric Tg remained the more abundant Tg species at pH 4.6, 5.8, and 7, with about 80% dimers and 20% monomers for all of three pH values (Fig. 2). However, at pH 3, the ratio shifted and monomers became more abundant – with 45% dimers and 55% monomers (Fig. 2). For all four pH values, the relative abundance of dimers remained unchanged after 1 h and even 24 h of incubation at the new pH (not shown). The mass of each peak (based on the mean of a Gaussian fit) was not affected by pH. These results confirm that the Tg dimer interface is immediately disrupted in very acidic conditions. By revealing the relative abundance of the dimers and monomers – and detecting additional species present – the analysis provides valuable details about sample purity as well as sensitivity to buffer conditions. Figure 1. MyMassMP reveals abundance of Tg monomers and dimers, and presence of additional species. Three peaks were detected, including the main expected dimer peak at 663 kDa and lower-mass peaks at 144, and 336 kDa. Analysis was performed on 40 ng of Tg in PBS (pH 7.4) at a concentration of 1.33 μg/mL. Inset: Standard output from MyMassMP, showing the output of MyMassMP measurements: Mass (kDa), standard deviation of each Gaussian fitted peak (σ), and relative abundance of each population (%). Figure 2. MyMassMP reveals sensitivity of Tg monomer: dimer ratio to pH. Tg was diluted to 1.33 μg/mL in buffer of the indicated pH and measured immediately on MyMassMP. As in Fig. 1, multiple peaks were detected. The Tg monomer (labeled “A”) and dimer (labeled “B”) peaks were selected for quantification. Peak Mass (kDa) σ (kDa) % of fitted A 144 24 11% B 336 28 16% C 663 42 73% APPLICATION NOTE Need help? Get in touch here Relating catalase sample purity to catalytic activity Many functional studies rely on reagents of biological origin, which can vary from batch to batch. Enzymes are a prime example, and their quality is often quantified in terms of their enzymatic units. However, what lies behind varying levels of catalytic activity across different batches or purification procedures is not always known. One example of an enzyme that is widely used in the medical and food industries is catalase, which breaks down hydrogen peroxide into water and oxygen. Active catalase is a tetramer of ~240 kDa. To explore whether MyMassMP could provide insights into differences in catalytic activity, we used it to assess two different commercially available catalase products, one with low catalytic activity (2 KU per mg of protein) and one with higher catalytic activity (10 KU/mg). MyMassMP showed that both catalase products had prominent peaks at the expected tetramer mass of ~240 kDa. However, the product with higher catalytic activity/ mg of protein clearly showed a higher abundance of tetrameric catalase than the lower-activity product (Fig. 3). In the higher-activity product, the tetramer accounted for 55% of the species in fitted peaks; in the loweractivity product, it was 34% (Fig. 3 insets). In addition, in the lower-activity product, the mass of the lowermass species was 87 kDa, which does not correspond to catalase monomer or dimers – suggesting that this preparation contained impurities. The catalase measurements show that the overview of sample composition from MyMassMP is valuable for detecting impurities and ensuring that a sample is suitable for downstream applications or further functional studies. Figure 3. MyMassMP analysis of catalase products reveals differences in sample purity that correspond to differences in catalytic activity. MyMassMP analysis of commercially available catalase products with A) lower and B) higher enzymatic activity revealed a greater abundance of tetrameric catalase in the higher-activity product. 72 ng of each catalase was measured in PBS. Insets: Standard output from MyMassMP, showing the output of MyMassMP measurements: Mass (kDa), standard deviation of each Gaussian fitted peak (σ), and relative abundance of each population (%). Catalase tetramer schematic modified from Yancheva et al. (2025).3 Peak Mass (kDa) σ (kDa) % of fitted A 87 23 66% B 229 28 34% Peak Mass (kDa) σ (kDa) % of fitted A 60 21 45% B 240 22 55% APPLICATION NOTE Need help? Get in touch here Observing how buffer conditions affect GroEL stability The bacterial chaperone GroEL forms an ~800 kDa double-ringed barrel-shaped complex composed of 14 identical subunits. GroEL is also an ATPase, and although its structure is stable without bound nucleotides, the presence of ATP or other adenine nucleotides makes its oligomeric assembly more easily disrupted by agents like urea.2 GroEL is challenging to purify and characterize for several reasons. As a molecular chaperone, it frequently co-purifies with unfolded or partially folded substrate proteins, complicating the assessment of sample purity. In addition, GroEL lacks tryptophan residues, making its concentration difficult to quantify using absorbance at 280 nm (A280). As a result, A280-based concentration estimates can substantially underestimate the true protein concentration and enrichment during purification is hard to monitor. Limitations of common characterization techniques arise with GroEL, but are overcome by MP. SDS-PAGE disrupts non-covalent interactions, preventing observation of its native oligomeric assemblies. Native PAGE preserves oligomerization but is challenging for very large complexes such as GroEL, as the low-percentage gels required are fragile and migration becomes difficult to reproduce. SEC maintains oligomeric structure but typically relies on UV detection; because GroEL has negligible absorbance at 280 nm, it is often poorly detected unless additional labeling or alternative detection methods are used. MP avoids these challenges as it has a wide mass range, does not require labels, and uses native buffer. MyMassMP characterization of GroEL samples diluted to 25 nM showed three main populations, corresponding to 14-mers, 7-mers, and monomers (Fig. 4). The presence of these different peaks indicates that some disassembly of the 14-mer occured, but the amount of monomer present cannot be reliably determined from this measurement due to the monomer mass (~57 kDa) being at the instrument’s lower limit of detection (55 kDa). To study GroEL-assisted folding of proteins, target proteins are often unfolded using denaturants such as urea. However, it is important to work under urea concentrations that do not destabilize the GroEL 14- mer, and make it non-functional. We therefore explored whether MyMassMP could check this stability. We used MyMassMP to assess GroEL stability when exposed to urea (1 M) in the presence of the nucleotides Mg-ATP and Mg-ADP (Fig. 5). It showed that the 14-mer Figure 4. MyMassMP reveals GroEL disassembly after dilution in PBS. At GroEL was diluted to 25 nM (monomer) in PBS, then measured after 90 min. The three main peaks correspond to monomers (A), 7-mers (B) and 14- mers (C). remained mostly stable in the presence of urea without nucleotides, even after 2 h incubation. However, the 14-mer had clearly disassembled after 2 h incubation in the presence of the nucleotides, and Mg-ADP was associated with slightly more disassembly than Mg-ATP. Although the loss of 14-mer could not be correlated to an increase in monomers (due to the monomer being at the instrument’s detection limit), disassembly was confirmed in a separate measurement on a TwoMP mass photometer (not shown). This type of information is valuable for researchers using GroEL and urea in folding assays, as it provides certainty about the combination of urea concentration and buffer conditions needed to preserve GroEL stability and function. APPLICATION NOTE Need help? Get in touch here Figure 5. MyMassMP analysis reveals how urea-induced disassembly dynamics of GroEL are affected by the presence of Mg-ATP or Mg-ADP. MyMassMP histograms of GroEL exposed to urea under control conditions or in the presence of Mg-ATP or Mg-ADP – either A. immediately after or B. 2 h after the introduction of urea. Conclusion MP offers a fast, label-free way to characterize proteins and other biomolecules and particles in solution at the single-molecule level. It measures sample purity and heterogeneity, and more under native conditions. The MyMassMP instrument builds on these advantages to deliver rapid, high-resolution analysis through an easy-touse benchtop instrument. Each measurement takes just one minute and requires minimal sample (30 μL at 10 nM). Unlike SDS-PAGE, which denatures proteins and disrupts oligomers, MP preserves native assemblies. Compared with SEC, it avoids slow column-based separation, eliminates potential sample–matrix interactions, and provides direct mass measurements rather than relying on elution volume. DLS, as a bulk technique, averages over all particles and often lacks the resolution to detect minor populations, whereas MP identifies individual molecules, revealing subtle differences in oligomer composition and purity. In this application note, MyMassMP was used to effectively characterize samples of three different proteins – providing useful insights for assessing sample purity and optimizing buffer conditions. For the large, heterogeneous glycoprotein thyroglobulin, it revealed monomer–dimer ratios and pH-dependence; for the the widely used enzyme catalase, it showed differences in purity across different commercial preparations, which corresponded to differences in catalytic activity; and for the 14-mer GroEL, it reported how urea and the nucleotides Mg-ATP and Mg-ADP affected stability. MyMassMP assesses sample purity and homogeneity in minutes, using nanograms of sample. It helps researchers quickly determine whether samples are suitable for downstream functional or structural studies, optimize experimental conditions, and ultimately save time and resources. APPLICATION NOTE Need help? Get in touch here Materials and methods MyMassMP measurements MassGlass™ UC sample carrier slides and cassettes were assembled as recommended by the manufacturer (Refeyn Ltd.). The MyMass instrument was calibrated with Refeyn’s MassFerence™ P1 protein calibrant. Samples were diluted in the indicated buffers to the indicated concentrations and 30 μL of sample was run on MyMassMP following the manufacturer’s instructions. Thyroglobulin sample preparation Citrate and Na2HPO4 were combined at different ratios to prepare buffers of different pH, and NaCl was added to a final concentration of 150 mM. Tg (Sigma, 1 mg/mL in PBS) was diluted to a final concentration of 1.33 μg/mL for measurement in the buffer indicated. Catalase sample preparation Catalase 2 KU/mg and 10 KU/mg (Sigma) were resuspended in PBS at 1 mg/mL. They were diluted to 2.4 μg/mL (~10 nM tetramer/ 40 nM monomer) in PBS prior to measurement. GroEL sample preparation GroEL (Sigma) 1 mg/mL was diluted to 0.15 mg/mL in either: • PBS (control) • PBS with 10 mM MgCl2, 1 mM ATP • PBS with 10 mM MgCl2, 1 mM ADP 10 μL of GroEL 0.15 mg/mL was combined with 4 M urea (final urea concentration 1 M) and incubated on ice for the indicated time. Irrespective of pretreatment, samples were diluted to 1.5 μg/mL GroEl (~25 nM monomer) in PBS at room temperature for measurement. Follow us on LinkedIn ©2026 Refeyn Ltd. Unit 9, Trade City, Sandy Lane West, Oxford OX4 6FF, United Kingdom For information on products, demos and ordering, email orders@refeyn.com Refeyn, MyMass, MassFerence, and MassGlass are trademarks of Refeyn Ltd. v01-Mar26 References 1. Schneider, A. B. & Edelhoch, H. 1970. J. Biol. Chem.. https://doi.org/10.1016/S0021-9258(18)63348-4 2. Lissin, N. M. 1995. FEBS Lett. https://doi.org/10.1016/0014-5793(95)00151-x 3. Yancheva, Y.D. et al. 2025. Nano Lett. https://doi.org/10.1021/acs.nanolett.4c05792