Avoidance of missed cleavages, methionine, cysteine ddickerson  2020-07-20 08:58
 

I understand that proteases with high numbers of missed cleavages are to be avoided if using Skyline. Also, peptides which contain methionines or cysteines, which can be post-translationally modified, are also to be avoided. I was asked to work on a project which involved trypsin+Glu-C digested peptides (with high numbers of missed cleavages), and also many peptides which contained methionine and/or cysteine. A lab mate had already done some work on samples of such peptides using DDA, and he asked me to do the PRM. The samples were in quadruplicate. My results gave similar results to his, and many of the peptides detected by PRM had coefficients of variation <.05 for the 4 repeats, and the differential expression volcano plots showed many peptides had significant p-values. I used 9 maximum missed cleavages for digestion and included a background proteome with 9 max missed as well. The results seem plausible, and the corresponding trypsin-only digests gave far fewer results. Can you please confirm whether I need to throw away this data.
 
 
Nick Shulman responded:  2020-07-20 13:59
I have never heard anything about avoiding cysteines for quantification. (There might be a valid reason for this, but I have never heard it mentioned). In the experiments that we do in our lab, we nearly always treat the samples with carbamidomethyl so that the cysteines to not for disulfide bonds with each other.

The reason to avoid things like missed cleavages and oxidizable methionines is that you may observe a change in the quantity of such a variant, but it will have been caused by a change in sample prep, and not reflective of an interesting biological condition.

If you would like, you could send us your Skyline document and we could tell you whether anything looks suspicious.

In Skyline, you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here:
https://skyline.ms/files.url

-- Nick
 
Brendan MacLean responded:  2020-07-20 15:31
I think if you return to the presentation, can you provide the link to what you are watching? I think you will find that the presenter is saying these are things you might consider when you are trying to design a "quantotypic" assay for a protein, but the end result is that considering everything various people have suggested at various times (like avoiding Methionine) might leave you with nothing to measure.

You also need to consider whether you are trying to find peptides which are good surrogates for protein quantity or whether you may actually be trying to measure specific peptides themselves or even PTMs. Skyline has been an indispensable tool to many for PTM studies, where the same type of exclusions suggested for protein measurements really do not work.

If you have solid IDs and reproducibility for missed cleavages under your experimental conditions, then by all means use those peptides as your targets. I believe these were just generalized suggestions, based on the most common proteomics experimental protocols, not meant to be prescriptive.

I saw a very nice set of experiments with Skyline on neuropeptides, which are small enough to measure with no protease cleavage at all. They had higher charge states and required CE optimization due to issues predicting optimal CE for high charge state fragmentation.

So, definitely consider your experiment and adapt as necessary. Thanks for posting to the Skyline support board.

--Brendan