Hi all,
I have recently done a pilot protein extraction from primary lung basal cells with phosphopeptide enrichment using the protocol described by Humphrey et. al, and found 2433 phosphopeptides with 97% enrichment. I was wondering what other people have found as an expected number of phosphopeptides from basal cells.
Many thanks,
Sam
Humphrey, S. J., Karayel, O., James, D. E., & Mann, M. (2018). High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform. Nature Protocols, 13(9), 1897–1916. https://doi.org/10.1038/s41596-018-0014-9
Hello Sam Siljee
The EasyPhos approach is great. Humphrey et al. wrote a very clear protocol but as you know, every cell or tissue type provides varying phosphopeptide numbers. If your enrichment was 97%, that's great.
We've improved our phosphopeptide numbers by including a Fe-NTA enrichment after the titanium dioxide enrichment as outlined in Thermo's "SMOAC" approach.
Consider implementing the additional phosphopeptide enrichment.
Good luck!
Best,
Peter
Thank you Peter Allen Faull
Thank you for your suggestion to use Fe-NTA enrichment.
I have since found a paper by Kitata et al where they found very high numbers of phosphosites using a data-independent acquisition, up to 36,350 from a cell line. I would be very pleased to identify that many!
http://www.nature.com/articles/s41467-021-22759-z
Best regards,
Sam
Dear Sam,
The number of phophopeptides found in an enrichment protocol depend on the amount of sample that you use, if you found these ~2000 proteins from
Hi Dr. Siljee,
It is not the same histology but I think to compare, Friedrich et al. have done phosphoproteomics from FFPE in Lung adenocarcinoma and squamous cell carninoma using DDA and TMT. I add the name of the article. Hope it is useful as a mention in your discussion.
Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories | Nature Communications
Dear Martin E Barrios-Llerena,
Thank you for your helpful feedback!
I have since played around with different search strategies and re-run the samples following calibration of the mass spectrometer with improved results.
I've found the adjusting the fragment mass tolerance in ProteomeDiscoverer is particularly critical. Lower tolerance not only speeds up the search, but actually gives far more PSMs. Likely due to reduced decoy identification. More starting material (~200ug) also gives better results.
I've now identified 5933 phosphosites from 2257 proteins which I'm pretty pleased with.
I've not yet tried fractionation, but I will keep that in mind!
Next step is to try DIA for quantification
Sam
Dear Fuat Kaan Aras,
Thank you for the paper, it looks like an interesting read, and indeed worth mentioning in my literature review.
They did identify quite a few more proteins/phosphosites than I did, however I agree with you that histology is not the same as a cell line, and I believe that there are significantly more proteins found in whole tissues than cell lines.
I'm not discouraged!
Sam
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