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### Phosphoproteome enrichment and Phosphorylation stoichiometry measurement

Posted: **Wed Apr 15, 2015 11:45 pm**

by **Ironman**

Hi,

Our group published two papers which are related to phosphopeptides purification and phosphorylation stoichiometry measurement recently. This maybe of interest to some of you

Sequential IMAC for phosphopeptide enrichment
Phosphorylation stoichiometry measurement
Comments/questions are welcome

Thanks

Posted: **Thu Apr 16, 2015 7:06 am**

by **Infinity**

I really liked the second one (stoichiometry). At the same time I think there is one problem with majority of the approached that measure stoichiometry. We all know that it could be very tricky to spot small changes even with quite precise SILAC quantitation. Let's say we obtained FC=0.1 (log2-transformed). Is it a real 0.1 change or simply a technical variability??? In my experience even if we mix the same sample 1:1 (light:Heavy SILAC) and analyze distribution of log2(FC) we would see that it is somewhere between -1 and 1 (OK, let's say that under ideal conditions it will be -0.5 to 0.5). So in a real experiment measurement of 0.1 can be just a technical variation. From this we either do multiple biological replicates (and I'm speaking of much more than 3 to reveal such a small changes with t-test) or use only relatively high/low FC measurements. By now you might be wondering how it is connected with stoichiometry measurements? The answer is that everything with stoichiometry less than 30-40% would be in that "technical error" interval, so all these techniques will be precise only for high stoichiometries (and probably you would see it if experiments will be performed in replicates).

I'm not criticising the paper, it is really good. I just want to bring your attention to one of the problems that still remains in this field.

Posted: **Thu Apr 16, 2015 5:31 pm**

by **Ironman**

Hi Infinity

Thanks for your comments^^. It is really good to get comment here after publication.

We also notice the limitation of this method. The small fold-change which represented the low stoichiometry may not be accurate.

Therefore, the different stoichiometries have been categorized into four region such as 1. <10% 2. 10-30% 3. 30-75% 4. >75% in Supplementary Table 1. But, just like you said, the method currently can be precise for high stoichiometries not for low stoichiometries. We will try to solve this problem for measurement of low stoichiometries in the near future^^. Thanks for your comment.

Posted: **Thu Apr 23, 2015 12:08 pm**

by **Infinity**

I have a question regarding sequential elution from IMAC. What is the difference between venn diagramms Suppl. Figure 3 and Figure 3. Why you have much lower complementarity between Ga and Fe IMAC in Supplementary figure?

Posted: **Thu Apr 23, 2015 8:38 pm**

by **Ironman**

Hi Infinity

In this work, we evaluated the performance of Ga-IMAC and Fe-IMAC by using both parallel and sequential IMAC enrichment.

For Suppl. 3, the tryptic peptides would be individually purified by Ga-IMAC and Fe-IMAC.

For Figure 3, the tryptic peptides would be sequentially purified by 1st Ga-IMAC and 2nd Fe-IMAC.

Based on the property of metal ions, the weaker Ga ions can catch the the multiple and acidic A.A. containing phosphopeptides and then the rest phosphopeptides can be purified by Fe ions.