Accelerate hit discovery with
Affinity Selection-Mass Spectrometry

AS-MS is first and foremost a binding assay, and specifically, we are looking for noncovalent binders. Right? AS-MS can also be used in a low throughput mode or a high throughput mode depending on how you have it set up for your particular system. The general workflow for AS-MS goes like so. Right? So we'll take a target protein and then throw it into a mixture of compounds. Right? This is something we call a multiplexed compound library. So we'll let this sample incubate for a specific amount of time, and then when it's ready, we will inject it onto a two dimensional LCMS. The first dimension of of LCMS is usually size exclusion. Right? And what we'll do here is we will cut out our protein compound peak and then send it over to the second dimension, which is usually reverse phase chromatography. This protein compound complex will dissociate because, again, we're under reverse phase conditions, so high temperature, low pH, aqueous organic mixtures. And then lastly, we have hit identification at the mass spec. Right? So this is direct detection of our binders.

So if there's anything to take away from this talk, it's this. Virscidian is working super hard to streamline our AS-MS analysis pipeline to get an easier to use product into our clients' hands and position these in such a way that our automation improvements can still be applied to screening data, as well as more in-depth analyses like dose response data.

We can still interrogate the crosshits. So, for example, here in sample position a twelve, this is our original hit, but what we can see is that this signal is also observed across almost the entire plate. And if we pull up these chromatograms side by side, their peak shapes, their peak intensities, peak areas, and retention times are all super similar to one another. This is what we call a cross hit. It's a hit that originates in one well but is also observed in other wells. And since they look so similar to one another, we can't trust them.

So rather than sorry. Rather than, you know, look through well by well for every single sample, what we can do next is perform crosshit filtering. And really in a nutshell, crosshit filtering is eliminating false positives and removing background noise so that any hits remaining in our hit list are high confidence. Okay. So here is our hit list before cross hit filtering. And then once we click on this filter button right here, right, this recording was, it's being shown in real time. Right? So just in a few seconds, Crosshit filtering is completed, which is much, much faster than our previous versions of ASMS. And to reiterate here, we have a fairly large number of compounds in our hit list. List. But previously, pre filtering, we didn't know which of these hits were good and which ones were bad. After crosshit filtering, which only took a few seconds, right, our hit list now reflects just these strong hits shown in dark green, which I'm highlighting here.

We can also apply all of these automation improvements on top of our KD analysis. So we still have to generate a compound list, create a submission within SMS, generate that sample list, and then acquire the data. And we have a new panel now called our DRC results panel, so that's dose response curve results panel. Here over on the left are the, what we call the KD results summary. Right? So we have four separate DRCs here. As you click through everything, right, the visualization of the plate map and chromatogram and spectra get updated. Over here on the right half of this this DRC panel are the individual items, the individual injections that make up this particular series. Right? So these are all the varying concentrations of our injection. If we go ahead and display multiple chromatograms at the same time, right, we can even see a binding curve start to appear based purely on just a chromatographic visualization. Right? But kind of the real component of our KD analysis is down here in the bottom with these plots. So we are plotting peak area and peak intensity information, as well as calculating the Kd value, the dissociation constant value, for this particular protein and compound. We calculate a Bmax value, as well as various statistics like r squared values for your data. Right. So now this KD data can be used as a confirmation tool after hit identification, and we can use KD analysis to rank order which of these compounds is the best binder.