Hi everyone,
I am enriching phosphopeptides with TiO2 beads. I only use very manual like using eppendorf to combine with sample and TiO2 , then centrifuge to remove the supernatant and elute phosphopeptide by Ammonium Hydroxide solution and then analysis by LC MS/MS.
After analysis by LC MS/MS, The number of phosphopeptide didn't increase and around 15 phosphopeptide. I think it very low with reference that I read. And, the sample in the reference is Cell Line. Could the sample be responsible for the decrease in the number of phosphopeptides after TiO2 bead enrichment? Because I using protein hydrolysates.
Can someone knowledgeable in this field explain it to me? Thanks
Here are some checkpoints need to be corrected before making a spot-on suggestion;
Did you condition the beads?
What are the equilibration, washing, elution, and binding buffer compositions?
What is your sample matrix, Are there any competitors for nonspecific binding?
Do you have any positive control to assess the MS experiment in terms of phosphoproteome identification and detection capability?
Did you pay attention to potential phosphatase activity?
Did you use low-bind tubes?
Can you confirm that excluding all metal equipment either in sample prep or in LC configuration?
Best, İEA...
Hi,
I would recomment EasyPhos. It is quite easy as it is indicated, however, requires extra tools to perform. I don'T have a profound lab experince, however, I get more than 10000 phosphosites per sample in our cell line.
Thanks your reply
With my experiment, i don’t use cell line, i use the sample is crude protein extract.
Is that the reason the phosphopeptide is not active?
Which steps were included when producing the crude protein extract? You had indicated that it is protein hydrolysates...information about the treatment is needed to say whether it could be caused by the sample ingredient or the lab process (such as...how the proteins were isolated, extracted, and digested, which buffers, containers, and reagents were used, etc...)
The sample will first be defatted with hexane and then subjected to enzymatic hydrolysis. With digestive enzymes, I use solutions depending on the enzyme. Then I use ultrafiltration to filter proteins MWs
The procedure I am running is that the ratio of protein to TiO2 particles is 1:80 and the concentration of the sample when dissolved with conditioning buffer is 0.125mg/mL. Currently, I want to take the amount of sample after enrichment to check the tests, still keep the ratio with the sample concentration as above and just increase it all together like sample, TiO2 particles, buffer?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides