I am performing whole cell phosphoproteomics. Following cell lysis, I have trypsinized and desalted 10mg of cell lysate, which I am using for phosphoenrichment. The enrichment is antibody based, binds to phosphorylated tyrosines. However, we are unable to detect phosphopeptides by mass spectrometry following enrichment. One concern raised was that the starting protein is low. What is a good amount of lysate to begin with?
Whilst I have not done pTyr Ab pulldowns, I would not have thought 10 mg was a limiting factor here. Many phospho-studies use 1 mg to start with, but they are analysing the more common/abundant, pSer and pThr. If you have not already done so, I suggest you look at the abundant literature to see what others have successfully done. I’d also very carefully look at your pulldown conditions. I assume you have phosphatase inhibitors in your buffers?
Thank you for your answer. The buffer used during enrichment has sodium orthovanadate as the inhibitor. 10-20mg of starting protein is recommended for detection of phosphotyrosines via mass spectrometry. Is there anywhere else I may be going wrong?
The obvious experiment is to run a positive control to test your systems. This will assess the entire pipeline from pulldown to identification. If you still do not see anything, then you know you’ve got issues. My guess is finding a control for pTyr could be challenging. At the least, I’d check the MS side of things and run some controls there. Beta-casein is an easy choice, but I’d argue it’s too easy. You could just try a cell lysate and do a standard IMAC, or TiO2 phospho purification. No, it’s not pTyr, but at least you know you can detect phosphopeptides with your LC-MS/MS setup, so better than nothing in my view.
Late answer but TiO2 and FeNTA are not suitable for phosphotyrosines( i.e I quantified 43 phosphotyrosines by a sample from which I quantified nearly 9000 S/T phosphosites so much less than 1% with TiO2) If you don't have sample quantity problems(not working on clinical data) pY100 antibodies are the best for phosphotyrosines. Not really an expert but if you can quantify more than 300 phosphotyrosines its a success.
- Badges
- Science method