1 of 24

Amy Chau, Ph.D.

Amy.chau@refeyn.com

Sr. Field Applications Scientist

Biomolecule characterizations with mass photometry

Accelerating discovery through innovation

2 of 24

Structural studies fail when heterogeneity is not detected by bulk measurements

2

“ Polydispersity of Aq880, not detectable in SEC profiles, is probably the reason why all structural approaches failed so far”

- Feyh et al, eLife 2021 (University of Marburg)

SEC could not resolve the heterogeneity in Aq880 protein samples

Mass (kDa)

MP revealed the heterogeneity present in Aq880

Counts

Accelerating discovery through innovation

Take home message:

Mass photometry measurements can prevent failure in structural studies by providing higher resolution insights on sample homogeneity.

Script:

Using different screening tools is important because sometimes traditional technologies in the toolbox like analytical size exclusion can miss heterogeneity in samples that lead to failure in structural experiments potentially wasting $1000s and days/weeks in cryoEM time
This is a published example of this where the metallonuclease protein, Aq880 appeared homogenous in analytical SEC when the protein samples were actually heterogenous
The authors noted that the inability to see the polydispersity in these protein samples with SEC was the likely reason all structural studies of this protein failed previously
The heterogeneity in the samples was only seen by a single particle screening technology, mass photometry
As we progress through the deck, I will show you more examples of how mass photometry measurements can inform downstream experiments in minutes using virtually no sample

3 of 24

Mass photometry is a quick method that directly measures the mass of single particles in native conditions from 30kDa to 5MDa

Introduction

3

Biomolecules near a slide surface are illuminated by a laser
MP contrast is the interference between the light reflected by the surface and the light scattered by the biomolecule
MP contrast scales linearly with the mass of the biomolecule

Acquisition

Mass histogram is generated from the measurement of thousands of single molecules

Output

Mass (kDa)

Counts

Accelerating discovery through innovation

Take home message:

Mass photometry is a newer tool for the structural biology toolbox that provides new information – the mass of single particles – not given by traditional screening technologies.

Script:

In case you’re not familiar with mass photometry it’s a newer tool for the structural biology toolbox that provides additional information – the mass of single particles – not given by traditional screening technologies.
Mass photometry directly measures the mass of single particles (no bulk averaging) in native conditions (no staining, in solution) using just nanograms of sample (saves precious samples) in minutes

It is a microscopy technique that measures the interference between light scattered by molecules near a slide surface and light reflected from the surface
The output is a mass histogram from thousands of single particle mass measurements showing the mass distribution in your samples

Mass photometry is compatible with a variety of sample types (proteins, nucleic acids and their complexes)
And mass photometry works over a wide mass range from 30 kDa to 5MDa not limited by column or gel exclusion limits

4 of 24

Mass photometry is “a desirable technique … before proceeding to negative-stain EM and cryo-EM” – cryoEM101.org

Introduction

4

1 minute acquisition

Uses 3 to 500ng

Easy experimental procedure

Intuitive data analysis

< $10 per measurement

Benchtop instrument

Accelerating discovery through innovation

5 of 24

Mass photometry measures every biomolecule and complex in native conditions from 30kDa to 5MDa

Introduction

5

Protein - nucleic acid complexes

Accelerating discovery through innovation

6 of 24

TwoMP-MassFluidix HC combo provides the best of both worlds

TwoMP

Low sample consumption (5-20 nM)
1 min acquisition, <5 min analysis
Low operation cost (< $5/sample)
Suitable for sample characterization, aggregation, formulation, high-affinity complex assessments

MassFluidix HC

Expands the concentration up to 50 μM
Rapid dilution (< 37 ms) keeps molecules intact and stable
Quick analysis (< 20 min from assembly to cleaning)
Suitable for concentration-dependent behavior, low-affinity complex, membrane proteins

Accelerating discovery through innovation

7 of 24

Accelerate structural biology studies with mass photometry at every step

7

Protein purification

Sample optimization

Structural analysis

Functional analysis

Check sample purity and homogeneity with single particle resolution

Screen conditions for optimization in minutes

Measure binding stoichiometry in native conditions

Gain novel insights into assembly mechanisms (e.g. cooperativity)

Accelerating discovery through innovation

Take home message:

Mass Photometry can provide new insights into sample homogeneity at every step in a structural biology study.

Script:

Mass Photometry can provide new insights into sample homogeneity at every step in a structural biology study.
We’ll next look at published examples of how mass photometry can be used at each step of a structural biology study

At the first step of sample preparation during protein production and purification, mass photometry can check sample purity and homogeneity with single particle resolution
In the next step during sample optimization when screening different buffer conditions (e.g. salt, pH) or crosslinker concentrations, mass photometry can measure samples in minutes using just nanograms saving time and sample for downstream analysis
Finally, mass photometry can complement structural and functional experiments by providing insights into binding stoichiometry and assembly mechanisms (e.g. cooperativity)

8 of 24

Accelerate structural biology studies with mass photometry at every step

8

Protein purification

Sample optimization

Structural analysis

Functional analysis

Check sample purity and homogeneity with single particle resolution

Accelerating discovery through innovation

9 of 24

Optimize purification in minutes with mass photometry

Figures from: Sonn-Segev et al, Nat Comm 2020 (Kukura Lab, University of Oxford)

9

Mass photometry is a rapid alternative to nsEM for fraction purity screening

SDS-PAGE only showed the subunits and could not show the intact complex (data not shown)
MP and nsEM agreed which fraction had the highest abundance of fully assembled APC/C complex

Purification of large protein complexes is often a multistep process (e.g. for APC/C)

Fractions from different steps of purification were assessed by SDS-PAGE, MP and nsEM

MP

nsEM

MP ( ) and nsEM ( ) agree on APC/C mole fraction

Protein purification

Sample optimization

Structural analysis

Functional analysis

Accelerating discovery through innovation

Take home message:

Mass photometry measures sample purity and homogeneity with single particle resolution in minutes, making it a rapid alternative to nsEM during protein purification.

Script:

Moving into published examples of how mass photometry can support structural studies, firstly at the protein production and purification stage
Protein purification of large protein complexes is often a multistep process
Authors took fractions from different steps of the purification and measured them by SDS-PAGE nsEM and MP

It is impossible to tell if the APC/C complex is fully assembled from SDS-PAGE which can only show the subunits are present
So, traditionally additional time-consuming and expensive methods would be required to assess complex assembly like negative stain EM

But here we see that MP can provide this information (in agreement with nsEM) in minutes in contrast to nsEM that would have taken at least a few hours
Demonstrating that MP is a rapid alternative to nsEM for fraction purity screening

10 of 24

Optimize membrane protein purification with single particle resolution

App Note data courtesy of

10

SDS-PAGE and DLS did not show clear differences in homogeneity between the fractions
MP showed that Fraction A was more homogenous and further confirmed the mass of the population was the desired ~110kDa

DLS

MP

Mass photometry revealed the most homogenous fraction using nanograms of sample

Membrane protein preparations are difficult to purify and stabilize (e.g. GPCRs)

Protein purification

Sample optimization

Structural analysis

Functional analysis

SEC purified fractions of a GPCR embedded in Native^MP copolymer nanodiscs were checked for quality

95

55

26

kDa

Fraction

A

B

SDS-PAGE

Fraction B

PDI = 0.19

Fraction A

PDI = 0.29

Fraction B

Fraction A

Accelerating discovery through innovation

Take home message:

Mass photometry conserves sample while rapidly screening quality for difficult to produce sample types (e.g. membrane proteins)

Script:

Membrane protein samples are precious as preparations are difficult to purify and stabilize
In this collaboration with Cube Biotech, two SEC purified fractions of a GPCR embedded in copolymer nanodiscs were checked for quality by SDS-PAGE, DLS and MP
The bulk measurement techniques, SDS-PAGE and DLS, did not show clear differences in homogeneity between the fractions
It was only with single particle measurements by MP that homogeneity differences were observed between the fractions
MP identified that Fraction A was more homogenous and appropriate for downstream analysis in minutes using at least 10x less sample than SDS-PAGE and DLS.

11 of 24

Accelerate structural biology studies with mass photometry at every step

11

Screen conditions for optimization in minutes

Protein purification

Sample optimization

Structural analysis

Functional analysis

Accelerating discovery through innovation

12 of 24

Optimize buffer conditions in minutes with mass photometry

App Note data measured in house

12

Mass photometry enabled rapid buffer pH screening using nanograms of sample

MP showed that dimeric thyroglobulin disassembles in buffers more acidic than pH 4.6

Changing buffer conditions can improve the stability and homogeneity of protein complexes or oligomeric state

MP was used to screen buffer pH conditions that stabilized dimeric thyroglobulin (~660kDa)

15%

85%

19%

45%

55%

81%

16%

84%

Protein purification

Sample optimization

Structural analysis

Functional analysis

Accelerating discovery through innovation

13 of 24

Optimize buffer conditions in minutes with mass photometry

Figures from: Sonn-Segev et al, Nat Comm 2020 (Kukura Lab, University of Oxford)

13

Mass photometry is a rapid, label-free alternative to nsEM for buffer optimization

nsEM

MP

MP agreed with nsEM and showed 4 protein complexes at each salt concentration tested
In addition, MP revealed complexes containing the cofactor not resolved by nsEM

Different salt concentrations (0-500 mM) were used to find the desired and stable protein complex

Changing buffer conditions can improve the stability and homogeneity of complexes with their cofactors

Increasing salt

Protein purification

Sample optimization

Structural analysis

Functional analysis

Not resolved by nsEM

Accelerating discovery through innovation

14 of 24

Accelerate structural biology studies with mass photometry at every step

14

Measure binding stoichiometry in native conditions

Gain novel insights into assembly mechanisms (e.g. cooperativity)

Protein purification

Sample optimization

Structural analysis

Functional analysis

Accelerating discovery through innovation

15 of 24

Reveal mechanism of binding and cooperative behaviour in minutes

Figures from: Pillai et al, Nature 2024 (Baker lab, University of Washington)

15

Mass photometry showed cooperative binding and binding stoichiometry of in silico designed proteins in minutes

nsEM showed the conformation changed upon ligand binding
Mass photometry revealed the cooperativity of ligand bind with an intuitive readout

The cooperative binding protein shows only all or nothing binding
The non-cooperative binding protein shows intermediate stoichiometries

Authors designed proteins in silico to switch conformation upon binding ligand.
Selection was based on cooperativity (all or nothing binding)

MP

Protein purification

Sample optimization

Structural analysis

Functional analysis

Cooperative binding:

Non-cooperative binding:

Y4

X3

nsEM

+P

Accelerating discovery through innovation

Take home message:

MP reveals insights into assembly mechanisms in terms of protein binding stoichiometry and cooperativity within minutes

Script:

Now looking at published examples of how mass photometry complements structural and functional experiments by providing new insights into binding behaviour
In this study, done in the lab of David Baker (2024 Nobel Prize in Chemistry), authors designed proteins in silico to switch conformation upon binding ligand

Selection of the desired protein was based on cooperativity of binding – ability to bind the ligand in an all or nothing manner

nsEM showed that the conformation changed upon ligand binding (X3 to Y4)
They then used MP to test the cooperativity of the ligand binding to the designed protein with a negative control (non-cooperative binding protein)

The cooperative protein shows only all or nothing binding (only having 3:0 and 4:4 populations upon ligand addition)
The non-cooperative protein shows intermediate stoichiometries (containing a mix of 4:0, 4:1, 4:2, 4:3 and 4:4 populations upon ligand addition)

Mass photometry showed the binding cooperativity and stoichiometry of the in silico designed proteins within minutes

16 of 24

Confirm in silico predictions of protein complex assembly in minutes

Figures from: Drobnic et al, Nat Microbiol, 2025 (Imperial College London)

16

Protein purification

Sample optimization

Structural analysis

Functional analysis

AlphaFold was used to predict how components of a large motor protein complex assemble, e.g.:

Protein PflA binds with PflB via residues 16-168

Experimental validation of the in silico predictions was done by MP and cryoEM

MP agreed with cryoEM data and showed that:-

PflA ( ) and PflB ( ) heterodimerize
PflA and PflB cannot heterodimerize when PflA does not have the 16-168 residues

No Heterodimer

Heterodimer

Mass photometry rapidly validated in silico predictions of protein binding

cryoEM

MP

Loss of PflB

Accelerating discovery through innovation

17 of 24

How do we ensure we get good quality data?

17

Before the

Measurement

During the

Measurement

After the

Measurement

Temperature

Instrument warm-up

Immersion oil

Focus-finding & stability

Glass surface

Calibrations

Concentration & binding events

Buffer system

Mass shifts

Reproducibility

Analysis

Accelerating discovery through innovation

18 of 24

MP bases its analysis on single-molecule resolution

If concentration is optimal:

✓ Good separation & identifiable binding events for our software

✓ Sufficient statistics to correctly identify your sample distribution

If concentration is too high/low:

Peaks are poorly resolved (higher σ-values)
Lower-mass species will be underestimated or go undetected

* MP measurement in a regular field-of-view

18

Before measurement

After measurement

Concentration & binding events

During measurement

56

112

224

194 counts

636 counts

2123 counts

5277 counts

11220 counts

6860 counts

6570 counts

This measurement contains high activity, possibly due to high sample concentration or noise

Accelerating discovery through innovation

19 of 24

Non-standard buffer additives can add noise to a measurement

Filtration through 0.2 µm PES filters can help reduce noise (recommendation: vacuum filtration)
Non-standard additives can add to (existing) buffer noise

* See Refeyn AppNote “Mass photometry with detergents”

19

During measurement

Before measurement

After measurement

Component	Dilution buffer tolerance	Sample buffer tolerance
Glycerol	Avoid if possible	Final concentration < 5%
Sucrose	Avoid if possible	Final concentration < 0.1%
Detergents	Avoid if possible	Sample-specific; see Refeyn AppNote *
High salt (> 1M)	Avoid if possible (may increase low mass noise)	Yes
Metal ions (Mg²⁺, Zn²⁺, etc.)	Most (dilute fresh & test threshold via manual measurement)

Buffer system

96 binding events

@37 kDa

Accelerating discovery through innovation

20 of 24

Temperature affects particle mass drift

20

Temperature

Before measurement

After measurement

During measurement

No mass shift or shoulder

Slight mass shift and shoulder

Larger mass shift and shoulder

Accelerating discovery through innovation

21 of 24

Peak-to-peak separation is determined by FWHM

Peak resolution is determined by full width at half maximum (FWHM).
Typically, FWHM can be estimated as 2.35σ for Gaussian distribution

TwoMP resolution at FWHM: 25 kDa at 66 kDa, 60 kDa at 660 kDa

Analysis

Before measurement

After measurement

During measurement

∆M < FWHM

∆M > FWHM

∆M >> FWHM

General rule of thumb, 10 < σ < ±10% of mean

Accelerating discovery through innovation

22 of 24

Treat the objective with care

Clean the objective when changing MassGlass slides & at the end of the day

Use lens-cleaning tissue and molecular grade isopropanol to clean the objective

In-between MassGlass slides, clean once to remove residual immersion oil
At the end of the measurement session, clean as many times as needed to completely clean the lens

DO NOT use Kimwipes or molecular grade methanol/ ethanol

22

Cleaning protocol

Before measurement

After measurement

During measurement

Accelerating discovery through innovation

23 of 24

Our products

Company overview

23

SamuxMP|

SamuxMP Auto

TwoMP|

TwoMPAuto

KaritroMP

Service contracts for all instruments

Consumable kits

MassFerence�P1 & P2

Package for GMP

(SamuxMP)

Additional Discover^MP licenses

MyMass

MassFluidix HC

Accelerating discovery through innovation

24 of 24

Contact details at Refeyn – <customer name>

24

General Support

support@refeyn.com

Applications support is free of charge for all customers

Applications Support

Amy Chau, Ph.D.

Amy.chau@refeyn.com

Applications support is free of charge for all customers

Sales Orders

orders@refeyn.com

Quotes need to be arranged by local representative

Technical Sales Specialist

Marcus Page

Marcus.page@refeyn.com

Accelerating discovery through innovation