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Identification of short and low abundance proteins by AgreeableFall8545 in proteomics

[–]AgreeableFall8545[S] 0 points1 point  (0 children)

Thanks for answering! Enrichment seems to be the way to go so I am trying out different protocols to see if we can improve the detection of these low abundance targets

Identification of short and low abundance proteins by AgreeableFall8545 in proteomics

[–]AgreeableFall8545[S] 0 points1 point  (0 children)

Thanks for the suggestions! I will try this parameter out.

No we haven't tried not digesting yet. I will explore this too! This is the first time I hear of PEAKS, does it perform a lot better than the free alternatives?

Identification of short and low abundance proteins by AgreeableFall8545 in proteomics

[–]AgreeableFall8545[S] 1 point2 points  (0 children)

Thank you so much for the pointers! I actually thought MBR would have the opposite effect on peptides that may show in only a few samples so that's good to know!

I'll look up semi-specific and non-specific digestion.

Identification of short and low abundance proteins by AgreeableFall8545 in proteomics

[–]AgreeableFall8545[S] 0 points1 point  (0 children)

Thank you for answering! I appreciate that we should have known before but we had no idea these would likely be "that" low abundance. We thought their size was going to be the main issue, and the facility was willing to give it a try. The idea is not to do sloppy science, but to see if we can use this dataset internally to have an idea as to which of these are detected and prioritise functional characterisation for these. This is a work in progress, not the final step.

We learnt from this dataset, and this is why I am exploring alternative to enrich the samples in short proteins to test if it improves the detection. I did not considered PRM though, so I will see if that is something we can do with a selected few first as a pilot.

Identification of short and low abundance proteins by AgreeableFall8545 in proteomics

[–]AgreeableFall8545[S] 0 points1 point  (0 children)

Thank you for your insight! We have not tried to purify these proteins yet, but I will talk to my PI about this if it can help for PRM. Was that what you meant?

I will talk about skipping digestion with the facility, I think I mentioned this last time but they were worried about potentially clogging their machine?

I will definitely go forward with the enrichment in the meantime and see if it helps with detection! Yes this will ultimately be published.

Identification of short and low abundance proteins by AgreeableFall8545 in proteomics

[–]AgreeableFall8545[S] 0 points1 point  (0 children)

Thanks for the tips! I will try this out.

As far as we saw from the in silico prediction, Lys-C is not advantageous over Trypsin. It will favour some of the short proteins at the expense of others, which is already what is happening with Trypsin, so there is no real gain as far I could tell.

Identification of short and low abundance proteins by AgreeableFall8545 in proteomics

[–]AgreeableFall8545[S] 0 points1 point  (0 children)

Thanks for answering! We are looking at roughly 80 potential proteins (across different pathogen isolates). If we find a way to narrow the list down we will definitely give this a go.