I have my own optimized protocol for standard protein using 1 pmol, but I need a suitable protocol for my protein extraction with amount 2 mg of protein.
Hi Eric,
as a general rule you will just need to scale up the volumes of reagents that you use. We also perform the enrichment in "batch mode," ie. we perform the reaction in eppendorf tubes not the standard tip setup. This has the advantage that it is easier to deal with larger volumes and we also increase the incubation times. For binding and elution we use 10 min and washing is around 5 min. Search any of the papers by Martin R Larsen for info. I'd recommend the following if you haven't already got them;
S. S. Jensen and M. R. Larsen. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun.Mass Spectrom 21 (22):3635-3645, 2007.
Kasper Engholm-Keller, Thomas Aarup Hansen, Giuseppe Palmisano, and Martin R. Larsen. Multidimensional Strategy for Sensitive Phosphoproteomics Incorporating Protein Prefractionation Combined with SIMAC, HILIC, and TiO2 Chromatography Applied to Proximal EGF Signalling. J.Proteome Res. 10 (12):5383-5397, 2011.
Kasper Engholm-Keller, Pernille Birck, Joachim Storling, Flemming Pociot, Thomas Mandrup-Poulsen, and Martin R. Larsen. TiSH - a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC. Journal of Proteomics, 2012. 10.1016/j.jprot.2012.08.007
Kasper Engholm-Keller and Martin R. Larsen. Technologies and challenges in large-scale phosphoproteomics. Proteomics, 2013. 10.1002/pmic.201200484
Hope that helps,
Peter
Hi Eric,
I agree with Peter and recommend another detailed protocol from Thingholm, Larsen:
Thingholm, TE et al., Mol Cell Proteomics 7, 661 (Apr, 2008). PMID 18039691.
The ratio of TiO2 to peptide seems to be important (0.6 mg per 100 µg, recommended in Engholm-Keller 2012). Too much TiO2 will increase binding of unphosphorylated peptides. I used to do the binding in batch mode and used a column for slow elution.I also recommend the combination of IMAC and TiO2 ("SIMAC", see Thingholm, TE et al., Methods Mol Biol 527, 67 (2009). PMID 19241006).
Good luck
Marc
Peter G Hains Hi Peter, What type the sample you use ? Cell or Hydrolysate protein? Could you describe how do you do enrichment?
I doing enrichmentenrichment phosphopeptide from hydrolysate protein but the number of phosphopeptide collected very low aroung 5 phosphopeptide
I use both cell line and tissue homogenates that have been digested with Lys-C and trypsin. I refer you again to the citations above. A Google Scholar search will also reveal numerous alternative methods. Phosphopeptide enrichment can be difficult. Start with standards, spike them into lysates and make sure you have the technique down before doing "real" samples.
Thank you your suggest. I also read the article you suggested but I have some unclear ideas. I would like ask you 2 question:
1. For binding, when you incubate sample with TiO2 beads, You shake the mixture in high speech or low speech?
2. After you enrichment phosphopeptide by Elute . How do you do to remove TiO2 in phosphopetide solution? Because If you use TiO2-kit, it have column to remove TiO2. But you perform the reaction in the eppendorf tubes.
1. Shaking is low speed, maybe 400-800 rpm
2. Give the resin a gentle spin to pellet it and carefully remove the supernatant.
Thank you for you answer.
What kind of TiO2 do you use and where did you buy it from?
We bought a column and popped it open to access the resin; Titansphere TiO2 (GL Sciences, Tokyo, Japan). I'm sure there are plenty of resins you can use.
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