Open andyzim111 opened 2 years ago
I don't think this percentage is low... The localisation score DIA-NN reports corresponds to confidence that all phosphosites on the peptide are correct. I don't trust confidence scores reported by Spectronaut, there hasn't been any validation of these published.
Best, Vadim
Thanks for your reply.
I noticed that multiple phosphorylated peptides has only one PTM localization confidence in DIA-NN reports, and then which one phosphosite does the confidence correspond to?
Localisation score in DIA-NN indicates the probability that all phosphosites on the respective precursor are shown correctly, i.e. if shown unmodified, then are unmodified, and if shown modified, then a re modified.
I got it. The localisation score shows the likeliness that the whole phosphopeptide bearing modifications is present or not, this kind of like "peptidoform".
I don't think this percentage is low... The localisation score DIA-NN reports corresponds to confidence that all phosphosites on the peptide are correct. I don't trust confidence scores reported by Spectronaut, there hasn't been any validation of these published.
Best, Vadim
Here is the paper: https://www.nature.com/articles/s41467-020-14609-1
You know about it, you cited it in your own work. The localization algorithm is explained in detail in the supplementary and it was validated in this work alone using 3 different methods.
Hi Oliver @oliver-bernhardt, of course I've read the paper, was highly interesting, very nice contribution by you and the team! I trust some things in Spectronaut, but I've no idea how well your localisation algorithm actually works, as I don't think the validation in that paper is sufficient.
Therein it's validated on a single dataset of ~200 detectable spike-in peptides, and
So instead of: "there hasn't been any validation of these published."
you should rather say that you don't agree on how it was validated.
Well, if a 'validation' experiment does not really validate the algorithm, I would not call it 'validation'. Does not mean it's not useful: it does show that filtering based on localisation score can be used to give confidence in localisation. But it does not prove that the confidence rate reported by Spectronaut is accurate.
while we are at the topic, can you point me to the publication where the PTM.Site.Confidence and the PTM.Qvalue reported in DIA-NN is described and validated?
PTM.Q.Value is described and validated in our ubiquitin paper https://www.nature.com/articles/s41467-021-25454-1, supp. fig. 10. Which also includes a benchmark for Spectronaut, btw. Although the implementation is now somewhat different. We did not publish benchmarks for site confidence.
Just to clarify but that means that the recommendation is to use PTM.Q.Value for localization filtering? Since PTM.Site.Confidence is not validated right now.
All recommendations are in DIA-NN docs. Not all validations have been published.
Hi, Vadim
I recently want to test DIA-NN function of phosphosite localization. I downloaded the raw MS data which included 166 synthetic phosphopeptides spiked in yeast lysate and the spectral library generated by spectronaut, which is published in Nature Communications entitled "A data-independent acquisition-based global phosphoproteomics system enables deep profiling".
Based on the provided spectral library that contained 166 phosphopeptides, DIA-NN identified 103 phosphopeptides with PTM location confidence >0.75 out of total 166 phosphopeptides.
Considering relatively low percentage (103/166) phosphopeptides with confident localization, I wonder what goes wrong with my analysis.
In the study, the authors used spectronaut to analyze the same data, resulting in 157 class I phosphopeptides.
Thanks. 41467_2021_22759_MOESM5_ESM.xlsx spectronaut_library_DIA-NN.txt Sythetic phosphopeptides.txt