IP of tyrosine phosphorylated peptides

[neil777]

Hi everyone
does anyone have experience of IP of tyrosine phosphorylated peptides. I have tried it several times and never get anything at the end of it. Any advice is much appreciated
best wishes
neil

[Infinity]

Indeed, I have a good experience with both IMAC and TiO2 for large scale phosphoproteomics but IP of peptides never ever worked for me. I'd be happy getting at least 50% of pY peptides at the end.

[edhuttlin]

We regularly do pTyr IP's, usually with good success. Which commercial antibody (and/or kit) are you using? Also, how much protein are you starting with? The key thing to remember is that pTyr is a very low abundance PTM (it's much lower in abundance than pSer and pThr), so you need to start with a ton of bulk protein in order to recover very much after the IP. To give you an idea, we always start with at least 10 mg of digested peptides prior to the IP (preferably more). Of course, the vast majority of these peptides are not bound. When we're done, the pTyr peptides that are eluted are enough for only one or two shots on our mass spectrometer. My guess is you're probably just not starting with enough material to obtain detectable levels of pTyr peptides in the end.

Here's a paper that describes the protocol we use:

http://www.mcponline.org/content/10/12/M111.009910.long

Good luck!

[Infinity]

Thank you for the protocol! I was using the same pY100 Abs from Cell Signaling that were already covalently immobilized on sepharose (cat #9419). Could you share the protocol you use for Abs immobilization?
In my experiments I was using just 1 mg of digested proteins just to make some tests that we based on peptide dot-blots and I also found that only very small part of pY peptides are retained on the antibodies, do you think it is normal?

[Santosh]

I agree with edhuttlin. Phosphoscan pTyr kit from CST works well for us to enrich pTyr-peptides. Yes, you do have to start with tons of proteins (usually 25-30mg) to get detectable levels of pTyr-peptides.

[neil777]

Hi thanks for the replies
I have tried 3 different agarose conjugated anti tyr antibodies, platinum 4g10 (millipore), py99 (santa cruz) and ptyr-100 (cell signalling).
The last experiment i tried using 10mg of peptides and i identified several hundred peptides, although none were tyrosine phosphorylated.
I have tried using some tyrosine phosphorylated peptide std and found that all 3 antibodies will selectively bind them. However only about 5% of the input tyrosine stds is recovered.
Any more advice greatly appreciated.

[Infinity]

Hi guys, let's try to solve this issue step by step. Please let me know if I'm doing something wrong.
1. sample prep: I always use phosphatase inhibitors, desalt and dry my peptides on speedvac (so there should be only peptides at the end, no salts or some stuff from lysis buffer)
2. Choice of antibodies: I always use pY100 from Cell Signaling that covalently immobilized on Sepharose
3. Choice of IP buffer: I saw many protocols where people use buffers with detergents (NP-40), EDTA, some other salts like MgCl2 even for IP of peptides. I think that it should be perfectly fine to go with TBS (NP-40 is to solubilize proteins, I do not need it for peptides, right?)
4. Proper Abseptide ratio. Currently I use recommended ratio in Cell Signaling protocol (which is designed for protein IP), could someone recommend the right ratio?
5. Incubation time: o/n @ 4C
6. Washing: extensive washing with TBS (5-7 times)
7. Elution: 1. 0.15% TFA; 2. 50% ACN 0.15% TFA (is there something special about TFA? Can it be replaced by higher concentration of FA?)

[Chris]

Hey all, I though I might add a quick tip that Forrest White's Lab uses for pTYR pulldowns. After the immuniprecipitation they will then do IMAC to further enrich the sample and get rid of any non-phosphorylated peptides. I have done a couple pTYR pulldowns and it was difficult for me to break a 50% enrichment, mostly due to the non-specific binding to the antibody. I think if I would have done IMAC afterword it would have cleaned up the sample immensely. Below is a paper reference that I quickly found that describes their use of IMAC after pulldown.

Kim J.E., White F.M. Quantitative Analysis of Phosphotryrosine Signaling Networks Triggered by CD3 and CD28 Costimulation in Jurkat Cells. The Journal of Immunology. 2006. 176 (5) 2833-2843.