Single-Molecule Mass Measurement — Determine Stoichiometry, Oligomeric State & Binding in Minutes, Using Nanograms of Sample
Creative Proteomics offers mass photometry (MP) services based on interferometric scattering microscopy (iSCAT) for label-free, single-molecule mass determination of biomolecules and their complexes in solution. No tags, no immobilization, no chromatography — just a direct readout of how many species are present, what they weigh, and how they assemble.
What Mass Photometry Reveals:
See every molecule. Understand your sample. Make decisions in minutes, not days.
Mass photometry (MP) is a label-free, single-molecule technique that measures the mass of individual biomolecules in solution by detecting the light they scatter as they land on a glass coverslip. Based on interferometric scattering microscopy (iSCAT), the technique quantifies the interference between light scattered by a molecule and light reflected from the glass-water interface — a signal that scales linearly with molecular mass.
Unlike ensemble-averaging techniques, mass photometry counts individual molecules one by one. In a single 2–5 minute measurement, you see the complete mass distribution of every species in your sample: monomer, dimer, higher-order oligomers, bound complexes, aggregates, and contaminants — all resolved simultaneously. No labels. No chromatography. No immobilization. Just a drop of your sample on a coverslip.
Mass photometry bridges a critical gap in biophysical analysis: it provides mass information at single-molecule resolution using orders of magnitude less sample than traditional methods like SEC-MALS or AUC, and does so in minutes rather than hours. For interaction analysis, it answers the most fundamental question: what is actually in my sample, and what is it bound to?
If you need to know what is in your sample and what it is bound to — before investing days in SPR, ITC, or structural studies — mass photometry gives you the answer in one rapid measurement.
Single-Molecule Resolution — See Every Species in Your Sample
Mass photometry counts individual molecules, resolving coexisting species that ensemble techniques average together. Detect monomers, dimers, tetramers, ligand-bound and free forms, aggregates, and contaminants — all in a single 2-minute acquisition — with sensitivity down to <1% relative abundance.
Minimal Sample, Maximum Speed
Each measurement requires only 2–20 µL of sample at 10–100 nM — less than 1 ng of protein. Results are available in minutes. This makes mass photometry ideal for precious early-stage samples, rapid screening across conditions, and projects where sample quantity is the limiting factor.
| Parameter | Capability |
| Mass Range | 30 kDa – 5 MDa |
| Mass Precision | ±2% (for well-resolved peaks) |
| Concentration Range | 100 pM – 100 nM |
Label-Free, Immobilization-Free, In-Solution Measurement
No fluorescent tags. No surface chemistry. No chromatography column. Proteins are measured as they exist in solution, landing freely on a clean glass surface. This eliminates the immobilization artifacts of SPR, the shear forces of SEC-MALS, and the labeling perturbations of fluorescence methods — revealing true native-state behavior.
From Rapid Screen to Quantitative Answer
Use mass photometry as a first-pass QC tool to verify sample quality before crystallography or cryo-EM. Screen buffer conditions for optimal complex stability. Determine binding stoichiometry and estimate Kd for high-affinity interactions. The same instrument, the same workflow — just different questions.
Mass photometry is a versatile first-pass and validation tool across the biomolecular interaction workflow. Each service below leverages the technique's unique combination of single-molecule counting, minimal sample consumption, and rapid turnaround to answer questions that would be impractical with more labor-intensive methods.
Sample Preparation & Dilution
Samples are diluted to the optimal concentration range (typically 10–100 nM) in a compatible buffer. Mass photometry tolerates a wide range of buffers including PBS, Tris, HEPES, with moderate salt, common detergents, and glycerol. Filtration through 0.22 µm is recommended to remove particulates.
Coverslip Loading
A clean glass coverslip is placed in the Refeyn mass photometer. 2–20 µL of diluted sample is pipetted directly onto the coverslip. Molecules freely diffuse and land on the glass surface. No flow system, no immobilization chemistry, no chromatography column.
Single-Molecule iSCAT Acquisition
The instrument records interferometric scattering events from thousands of individual molecules as they land. Each molecule produces a step-like increase in contrast proportional to its mass. A typical acquisition captures 2–5 minutes of landing events for robust statistics.
Mass Calibration & Distribution Analysis
Landing events are converted to mass values using a calibration curve from protein standards of known mass. The resulting mass histogram reveals all populations present: monomer, dimer, higher-order oligomers, bound complexes, aggregates, and contaminants.
Peak Fitting & Quantification
Gaussian peaks are fitted to each population in the mass histogram. Relative abundances, mean masses, and mass precision are calculated. For binding studies, the bound-to-free ratio at each concentration is used to estimate Kd.
Report Delivery & Interpretation
You receive annotated mass histograms, tabulated population assignments, binding curves with estimated Kd (when applicable), and a written interpretation summarizing the key findings — ready for publication or project decision-making.
Refeyn TwoMP Mass Photometer
– Interferometric scattering microscopy (iSCAT) detection
– Mass range: 30 kDa – 5 MDa with ±2% mass precision
– Concentration range: 100 pM – 100 nM
– Sample volume: 2–20 µL per measurement
– Acquisition time: 2–5 minutes per sample; up to 50 measurements per day
The Refeyn TwoMP platform provides the sensitivity and mass range needed for comprehensive biomolecular interaction analysis. For projects requiring microfluidic rapid dilution to capture weak or transient interactions, the MassFluidix HC add-on is available upon consultation.
| Parameter | Recommended Specifications |
| Sample Types | Purified proteins, protein complexes, antibodies, protein–ligand mixtures, protein–DNA/RNA complexes, membrane proteins (in detergent or nanodisc), viral vectors, AAV capsids |
| Purity | ≥ 90% recommended; lower purity acceptable if the target mass is distinguishable from contaminants. Mass photometry reveals impurities directly. |
| Concentration | 10–100 nM (optimal); concentration optimization performed during initial measurement |
| Volume Required | 2–20 µL per measurement; 10–50 µL total recommended for replicates and optimization |
| Amount Required | <1 ng protein per measurement; ~100 ng – 1 µg total for a full characterization |
| Buffer Compatibility | PBS, Tris, HEPES, phosphate buffers with up to 500 mM salt. Compatible with common detergents (DDM, LMNG, CHAPS), glycerol (≤5%), and reducing agents (DTT, TCEP) |
| To Avoid | Large particulate contamination (filter through 0.22 µm recommended); high concentrations of carrier proteins (BSA) that produce background |
| Membrane Proteins | Compatible with detergent micelles, amphipols, and nanodiscs. The mass contribution of the detergent or lipid is included in the measured mass |
| Shipping | Ship on dry ice or ice packs. Include buffer composition and concentration information |
Mass photometry has the most flexible buffer compatibility of any single-molecule biophysical technique. If your protein is stable in a standard biochemical buffer, it is almost certainly compatible. Contact us for a free feasibility assessment if you have questions about your specific buffer system.
Each biophysical technique answers different questions about your interaction. The table below helps you choose — and in many cases, the best answer is to use mass photometry as a rapid first screen, then go deeper with the appropriate follow-up method.
| Feature | Mass Photometry | SPR | ITC | SEC-MALS |
| Primary Information | Mass distribution, oligomeric state, stoichiometry, Kd (nM) | Kinetic rates (ka, kd), equilibrium Kd | Thermodynamics (ΔH, ΔS, ΔG), stoichiometry, Kd | Absolute molar mass per peak, oligomeric state |
| Principle | Single-molecule iSCAT — counts individual molecules landing on glass | Surface plasmon resonance — refractive index change at sensor surface | Heat of binding — direct calorimetric measurement | Light scattering + concentration — absolute mass from first principles |
| Immobilization / Column | ||||
| Sample Per Measurement | <1 ng; 2–20 µL at 10–100 nM | 1–10 µg; ~30 µL per concentration | 10–100 µg; 300 µL at 5–50 µM | 50–200 µg; 5–100 µL per injection |
| Time Per Measurement | 2–5 minutes | 30–60 min per series | 1–2 hours per titration | 20–30 min per SEC run |
| Mass Range | 30 kDa – 5 MDa | >10 kDa | >150 Da | ~5 kDa – 5 MDa |
| Kd Range | nM (high affinity) | nM – mM | nM – µM | µM – mM |
| Kinetics (ka, kd) | ||||
| Thermodynamics | ||||
| Heterogeneity Detection | ||||
| Best Used For | Rapid QC, oligomeric state, stoichiometry screening, high-affinity Kd | Detailed kinetic profiling (ka/kd), concentration screening | Full thermodynamic characterization, method validation | Absolute molar mass per species, conjugate analysis |
Mass photometry is a powerful screening and characterization tool, but it is not a replacement for every biophysical technique:
| If Your Question Is... | Mass Photometry Limitation | Consider Instead |
| I need kinetic rate constants (ka, kd). | Measures equilibrium populations, not real-time kinetics | SPR or BLI |
| I need thermodynamic parameters (ΔH, ΔS, ΔG). | Does not measure heat | ITC |
| My complex is below 30 kDa or mass difference between species is <5%. | Below the resolution limit of iSCAT detection | AUC or Native ESI-MS |
| My interaction has a Kd in the µM–mM range (weak binding). | Weak complexes dissociate at the low concentrations required for single-molecule counting | ITC or MST |
| I need atomic-level structural information about the binding interface. | Provides mass and stoichiometry, not residue-level contacts | XL-MS, HDX-MS, or Cryo-EM |
The best workflow: screen by mass photometry in minutes → confirm stoichiometry and purity → invest in deeper kinetic (SPR), thermodynamic (ITC), or structural (XL-MS, Cryo-EM) analysis with confidence that your sample is well-behaved. Contact our team to design an integrated characterization strategy.
Clean, Quantitative, Publication-Ready Data — in Minutes, Not Days
Each mass photometry project delivers a complete, interpreted data package. The emphasis is on clarity and quantitative rigor — you see exactly what was in your sample and what it means.
Annotated Mass Distribution Histograms
Mass histograms showing all detected populations with Gaussian peak fits. Each peak is labeled with its fitted mass, relative abundance, and assignment.
Multi-Condition Equilibrium Shift Overlay
Overlaid mass histograms showing how the oligomer equilibrium shifts across conditions — for example, calcium titration driving monomer → dimer → tetramer transitions. Each condition is color-coded, revealing dynamic behavior at a glance.
Binding Curves & Affinity Estimation
Fraction bound vs. concentration plots with fitted binding isotherms and estimated Kd. Multiple replicate measurements shown with error bars.
How does mass photometry differ from traditional mass spectrometry?
Traditional mass spectrometry (including Native ESI-MS) measures mass-to-charge ratio (m/z) of ionized molecules in vacuum. Mass photometry measures the mass of individual molecules in solution by detecting their light scattering as they land on a glass surface — no ionization, no vacuum, and no charge-state deconvolution required. The two techniques are complementary: mass photometry for rapid screening, native ESI-MS for detailed stoichiometry with ligand resolution.
What mass range and precision can mass photometry achieve?
Mass photometry detects biomolecules from approximately 30 kDa to 5 MDa with a mass precision of approximately ±2% for well-resolved peaks. Species whose masses differ by approximately 25–30 kDa or more can be distinguished. For example, a 150 kDa monomer and its 300 kDa dimer are easily resolved.
How much sample do I need to provide?
Mass photometry requires remarkably little sample. Each measurement uses 2–20 µL of sample at 10–100 nM concentration — less than 1 ng of protein. For a complete characterization including multiple conditions, replicates, and concentration series, we typically request 10–50 µL of your sample at the highest available concentration. This is orders of magnitude less than SPR, ITC, or SEC-MALS.
Can mass photometry measure weak or transient interactions?
Standard mass photometry works best for high-affinity interactions (nM Kd range) because the low nanomolar concentrations required for single-molecule counting favor bound complexes. For weaker interactions (µM–mM Kd), complexes dissociate at these low concentrations. The MassFluidix HC microfluidic add-on enables rapid dilution from µM to nM on a millisecond timescale, capturing weakly bound complexes before they dissociate. SPR or ITC may be more appropriate for weak interactions if microfluidic MP is not available.
Is mass photometry compatible with membrane proteins?
Yes. Mass photometry is compatible with membrane proteins solubilized in detergent micelles, amphipols, or reconstituted into nanodiscs. The measured mass includes the protein and its associated detergent or lipid. With appropriate controls (empty micelle or empty nanodisc measurements), the protein-only mass can be estimated.
How does mass photometry compare to DLS for sample quality control?
DLS measures hydrodynamic radius and is exquisitely sensitive to aggregates (a few large particles dominate the signal), but it cannot resolve individual oligomeric species of similar size. Mass photometry directly resolves monomers, dimers, tetramers, etc. as distinct mass peaks, providing far more detailed compositional information. DLS is faster and simpler for routine aggregation screening; mass photometry is the better choice when you need to know exactly which oligomeric states are present and in what proportions.
Can mass photometry analyze samples directly from cell lysate or serum?
Mass photometry requires that the target species be distinguishable from background. Complex mixtures like cell lysates or serum contain thousands of proteins at varying concentrations, creating a dense background that obscures individual species. The technique is best suited to purified or partially purified samples. If you are working with complex mixtures, we recommend prior enrichment (affinity purification, SEC fractionation) before mass photometry analysis.
How do I prepare my sample for mass photometry?
Most samples require only dilution to the appropriate concentration range (10–100 nM) in a clean buffer. We recommend filtering all buffers through a 0.22 µm membrane to remove particulate contamination. No labeling, immobilization, or buffer exchange is required for standard biochemical buffers. We provide detailed preparation guidelines during project consultation and can perform buffer optimization as part of the service if needed.
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