Why do we treat the protein channel differently?

[shntnu]

Why do we treat the protein channel differently?

[shntnu]

Understood

For the sake of completeness and my future self, here's another Q I was pondering, with an obvious answer that hadn't quite sunk in until now.

In terms of profiling in general – not specific to predicting variant impact – how is the protein channel in Variant Painting different from the other channels, say ER?

By definition, the structure marked by the protein channel differs across perturbations. This is unlike with ER, where we are always observing the ER.

So when it comes to profiling with Variant Painting – the similarity between two perturbations in the protein channel (s_protein) is conceptually different from that in the ER channels (s_er), because

• s_protein measures the similarity across two different feature spaces (morphology of protein 1 vs morphology of protein 2), whereas
• s_er measures the similarity is in the same feature space (morphology of ER)

The only situation where s_protein is conceptually the same as s_er is when it is the same protein, and the two perturbations are variants or reference of the same gene.

So there is only one situation where it makes sense to use s_protein: when both perturbations are of the same gene, and in this case you can use it for doing whatever you like – comparing localization, clustering, predicting impact, etc.

But when you use it to compare perturbations across genes, it's unclear what s_protein is reporting, because the similarity is across two different spaces. At first, I thought s_protein is a reasonable way to compare localization of two different proteins, but then (in Variant Painting) you are doing so across two different perturbation states, not the same perturbation state, and that makes things confusing. Note that what I am saying here does not negate what we've done in https://pubmed.ncbi.nlm.nih.gov/37732209/ (Fig 1 below) because although we place all genes in the same map, we are only highlighting variant mislocalizations (compared to reference of the same gene), but not comparing localizations across genes.

[bethac07]

Unasked for nitpick - your statement

But when you use it to compare perturbations across genes, it's unclear what s_protein is reporting, because the similarity is across two different spaces. At first, I thought s_protein is a reasonable way to compare localization of two different proteins, but then (in Variant Painting) you are doing so across two different perturbation states, not the same perturbation state, and that makes things confusing.

only is true if we assume/know that expression of the tagged version of the gene is itself perturbing. That is more likely to be the case in an overexpression context than a knock-in context, but even in OE, based on TA-ORF and MorphMap we definitely know not all OEs (and maybe not even MOST OEs) are.

[AnneCarpenter]

That clarifies for me - I don't think generic neg controls are useful for direct comparison to samples in this experiment other than to establish the impact of plate layout effects and other technical variation.

For both protein channel and non protein channels, we really only want to know if the variant and reference differ.

It's more OBVIOUS for the protein channel, but the facts remain the same for Non-protein channels: these channels' morphology might be impacted by the presence of the reference protein expression and therefore the reference protein is the proper comparator for the variant of interest, we cannot use generic neg controls as the reference. I think I might've been confused by this before, thinking the non-protein channels would not be affected by overexpression of the protein of interest.

[shntnu]

only is true if we assume/know that expression of the tagged version of the gene is itself perturbing. That is more likely to be the case in an overexpression context than a knock-in context, but even in OE, based on TA-ORF and MorphMap we definitely know not all OEs (and maybe not even MOST OEs) are.

This is useful to keep in mind – thanks @bethac07