TwoMP

Description

Specifications

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. 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 Ratiometric Contrast of each particle (or point spread function representing each molecule that touches
the surface) has a signal intensity that is directly proportional to mass and refractive index. To correlate ratiometric contrast to mass, the Refeyn TwoMP 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.

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

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

Limitations
• Limited mass and concentration range
• Difficult/impossible to work with membrane proteins and detergents
• No good standards for protein conjugates and mixed-typed complexes

Sample requirements

Sample Preparation and Experimental Design

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 10-20 nM. Samples should be prepared
at 100-200 nM 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 about every 1 hour.
• 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 two different 1000x protein calibration mixes stored in 10 μl 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.
• BSA-TG 1000x Protein Calibration Mix: 10 μl aliquots of 3 μM Thyroglobulin 10 μM BSA in
PBS and 5% glycerol
• BAM-TG 1000x Protein Calibration Mix: 10 μl aliquots of 3 μM Thyroglobulin 10 μM betaamylase
in PBS and 5% glycerol
• For, specific instructions on preparing DNA and RNA calibrants see the CMI’s Guide to Measuring
Nucleic Acids.
Experimental Design Tips
• PROTECT THE OBJECTIVE, by cleaning carefully and regularly.
• You will find focus using a buffer droplet of 15-18 μl and then mix your sample in the droplet.
CMI Getting Started Guide
cmi.hms.harvard.edu • Kelly Arnett, PhD, Director
CMI • Harvard Medical School • 240 Longwood Ave • Building C, room 303 • Boston, MA 02115
4
o You may need to optimize the buffer and the dilution factor.
o Filter buffers and samples to remove particulates and aggregates that may scatter.
• If measuring samples with mass close to the lower limit of 30 kDa, position the objective slightly off
center to limit interference from back reflection.
• DNA and RNA will not adhere to glass. Carrier slides must be coated with poly-L-lysine.
• Large proteins (>1 MDa) may not adhere to glass due to their size). This may be remediated with
poly-L-lysine.
• In normal measurement mode (used for proteins and nucleic acids), the Refeyn TwoMP has three
pre-defined image sizes. They are small, regular, and large. Choose your image size according to
the expected sample mass. Always calibrate with the same image size with which you plan to
collect data.
• Regular is the default image size and is recommended for most samples with particle
masses below 1 MDa.
• Large is recommended for samples with high particle mass (>1 MDa), as it allows for the
detection of more events in a single frame. While increasing the image size improves
statistics, it also decreases the signal-to-noise ratio, thereby decreasing mass sensitivity for
smaller particles.
• The recommended maximum event counts for each image size are as follows:
▪ Small: 3,000 events
▪ Regular: 6,000 events
▪ Large: 30,000 events
General Care and Maintenance
• The Refeyn TwoMP instrument will generally be powered off.
• To avoid overheating, the instrument should not be powered on for more than 12
continuous hours.
• Allow 45-60 min to equilibrate the temperature after turning on the power. This is a good time to
prepare samples.
• Keep the lid to the optics compartment (located on the top of the instrument) closed to minimize
dust in the instrument.
• The objective must be cleaned thoroughly between coverslips, or any time you pause for more
than 30 minutes, and very thoroughly after use. DO NOT LEAVE IMMERSION OIL ON THE
OBJECTIVE OR YOU WILL DAMAGE IT PERMANANTLY!

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