Mass Photometry (MP)

For information on access fees, policies and getting started at the CMI, see the CMI Access Page.

MP at the CMI

Mass Photometry (MP) measures light scattering of single particles as they adsorb onto a glass microscope slide, and is used to rapidly determine mass, oligomeric state, and heterogeneity of a wide range of macromolecules and their complexes in solution, under equilibrium conditions and without the need for labels. Mass photometry can be used to determine masses of diverse macromolecules from 30 KDa to 5 MDa, including proteins, nucleic acids, lipids and small viruses, such as AAV.

Mass Photometry sample data.          Refeyn TwoMP

 

The CMI has a Refeyn TwoMP mass photometry instrument from Refeyn, Ltd, with Accurion vibration-isolation bench.

 

Key Features and Applications

Applications

  • Mass range: 30 kDa to 5000 kDa
    • Proteins/complexes
    • Nucleotides of up to ~5000 bp
    • Empty vs Full ratio for AAV and small viruses
  • Oligomeric state and heterogeneity
  • Equilibrium dissociation constant (KD), for high affinity binders (~nM)

Key Features

  • Small amount of sample required: typically a few μl at ~100 nM, measured at 10-20 nM
  • Single particle counting
  • Fast and easy to use

Mass Photometry Theory and Design

Built on the principles of interferometric scattering microscopy, Mass photometry measures the interference between the light scattered by a molecule in contact with the measurement surface and the light reflected by the surface. Data is collected as a short movie (1 minute) and then processed using ratiometric imaging, allowing weakly scattering single macromolecules to be distinguished from the high background signal.  For scattering particles greater than 30 KDa and smaller than 100 nm, the point spread function representing each molecule that touches the surface has a signal intensity that is directly proportional to mass and refractive index. The instrument is calibrated using molecular standards of known mass with refractive index similar to the analyte of interest (e.g. protein, RNA, or DNA). Calculated masses of single particles are plotted as a histogram and can be counted or fit to Gaussian distribution.

                           Ratiometric Contrast

Assay Buffers

  • Buffers should be filtered to remove dust and particulates which will scatter light.
  • Mass photometry is compatible with a range of buffers, but should avoid scattering particles when possible, including carrier proteins and detergents.
  • Detergents will cause background and at concentrations above the CMC, detergent micelles will scatter light. This makes measurement of membrane proteins very difficult.
    • Use the lowest concentration of detergent possible, preferably below the CMC. 
    • Under limited circumstances, membrane proteins in detergent have been analyzed by mass photometry, but scattering of the empty micelles may complicate interpretation.

Samples

  • The ideal concentration range for sample measurement is 10-20nM. Samples should be prepared at 100-200nM for best results, as you will typically perform an ~8-10x dilution when mixing your sample into the buffer droplet.

    • The Concentration Calculator in the AcquireMP Tools menu can help with this.

  • You may need to try multiple concentrations to find the optimal conditions for your protein.

    • If the concentration is too high, peaks will broaden, and particles may not be counted if they land too close to each other.

    • If the concentration is too low, you may not have enough events to fit a Gaussian curve and oligomers may dissociate.

  • Calibration standards should be measured in sample assay buffer.

Calibration Standards

  • Calibrate mass at least every 3 hours.

    • Calibrate in assay/sample buffer, as the refractive index of the solvent impacts the ratiometric contrast.

    • Mass standards must have a similar refractive index to your sample particles.

    • Use protein standards for protein samples, a DNA standard or ladder for DNA samples, etc.

  • The CMI provides a 1000X Protein Calibration Mix (10µL aliquots of 3µM Thyroglobulin 10µM BSA in PBS and 5% glycerol) stored in 10µL aliquots in the -20˚C freezer below the instrument. Perform two 1:10 dilutions of the calibration mix with your buffer. The first dilution (1:10) can be stored at 8˚C for ~7 days. The second dilution (1:100) is the working stock which you will dilute into the buffer droplet.

  • For, specific instructions on preparing DNA and RNA calibrants see the CMI’s Guide to Measuring Nucleic Acids.

Provided by the CMI:

  • Reusable Sample Well Cassettes (6-well silicone gaskets)
  • Immersion oil: Zeiss Immersol 518 F
  • Whatman® lens cleaning tissue, Grade 105
  • Isopropanol and ultra-pure water, for cleaning
  • No. 1.5H high precision glass coverslips (24x50 mm), Thorlabs CG15KH
  • 1000x Protein Calibration Mix (10µL aliquots of 3µM Thyroglobulin, 10µM BSA in PBS with 5% glycerol)

Purchased by the User:

  • Optional Pre-cleaned Sample slides: Refeyn Sample Carrier Slides
  • Optional Supplies (varies by experiment):
    • Poly-lysine: Sigma P4832 (for measuring nucleic acids)
    • Calibration standards for non-protein samples:
      • Invitrogen Low DNA Mass Ladder: Invitrogen 10068013
      • Millennium RNA Marker: ThermoFisher AM7150
      • Empty AAVs, high mass protein standard