Is subtracting negative mass events from the positive histogram a valid baseline correction in mass photometry?
Hi everyone,
I was recently suggested to perform baseline correction by subtracting the negative mass events from the positive histogram (or using the negative side to correct the positive side) before interpreting the protein peaks.
Is this an accepted approach in mass photometry data analysis, or should the negative mass events simply be considered background while interpreting only the positive protein peaks?
Has this type of baseline subtraction ever been used or validated in any published MP study? If so, could you please point me to the relevant reference?
I'd appreciate any guidance on the recommended best practice. Thanks!
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Dear Sathya
Thank you for your question. My name is Bhaskar Barnwal and I am the senior field application scientist for Refeyn based out of Singapore.
As the molecules land on the glass coverslip (binding event), it scatters light, creating a contrast—or dark spot (negative contrast)—on the imaging sensor. The intensity of this contrast directly correlates with the mass of the particle: the darker the spot, the higher the particle’s mass. Conversely, when a previously bound molecule detaches and diffuses back into the solution (unbinding event), it appears as a white spot (positive contrast) that is registered as a negative mass on the -X axis. Most of the proteins will bind well to the coverslip and you will see fewer unbinding events in comparison to the binding events unless you run samples like apoferritin which show significant unbinding. To answer your question, in short, we do not do any kind of blank or baseline correction by subtracting negative mass events from positive side. The histogram data (mass vs counts) is directly taken from DiscoverMP software and can be presented or published.
Before running your protein samples, it is always recommended to run your buffer/additives to ensure there is little to no noise due to your buffers. Here, the unbinding data helps us interpret the noise from the buffer and additives. When you run a buffer/additive then you might see the mirror-image peak – with equivalent, ‘negative’ apparent mass and the same height – as you see in picture below in case of PBS (low-count, low-mass noise). If you get higher noise due to the additives then you can run your samples in it provided the mass of your protein sample is higher and is not affected by the noise as you can see in picture below (mirror-image noise due to buffer and a protein peak at 160 kDa). If there is any interference due to additive, you need to dilute your sample in buffer without additive to suppress the noise.
We have a very nice application note on detergents which will give you more information about mirror-image peaks and noise due to different detergents. If you don’t mind, you can provide email id so I can forward you that for your reference and can guide you better on your experiments. You can always reach out to me at bhaskar.barnwal@refeyn.com. Please let us know if we can help you further. Thanks!
Regards
Bhaskar