A classical method to prove phosphorylation is to use PKA to phosphorylate your candidate peptide, and run MALDI again for any gain of 80 Da from its parental mass.  You need to clean up the mix prior to spotting.

Hi, 

MALDI can indeed detect phosphopeptides (I run them sometimes).

What it cannot tell you is WHERE the phospho site is, unless you do a fragmentation experiment.

Unfortunately, MALDI systems commercially available will cleave the side chain modification (not always, but most of the time).

The easiest way I know of is to perform an Electron Transfer Dissociation (ETD) or Electron Capture Dissociation (ECD) experiment.

These are fragmentation methods for mass spectrometry which are an alternative to classic CID.

The advantage for both is that they KEEP the side chain modification. So you end up with a tandem MS/MS spectrum where some fragments are shifted by the MW of the side chain modification. This way, you can pinpoint the modification site.

I do phosphoproteomics and glycoproteomics on an ion trap with ETD and it works pretty well. 

Hi Shanmuka,

What was the source of your human protein?

If you can get unphosphorylated protein and PKA, you can do the reaction, subject your protein to tryptic digestion and look for phosphopeptides. Compare peptides with protein not phosphorylated. If your protein is sufficiently clean, the source is immaterial, trypic digestion, followed by ientification of the peptides should do the trick. I will send you our recent work on something related.

Good luck

Sujay

A mechanism of global shape-dependent recognition and phosphorylation of filamin by protein kinase A. Ithychanda SS, Fang X, Mohan ML, Zhu L, Tirupula KC, Naga Prasad SV, Wang YX, Karnik SS, Qin J. J Biol Chem. 2015 Mar 27;290(13):8527-38

thanks for your suggestion Sujay

The Protein was overexpressed by pcDNA vector with His tag. We have also purified the protein in bacterial system with his tag but under denaturating conditions (8M Urea). So my question is whether incubation of the bacterial purified protein with PKA solve the purpose?

..let me look into the paper and will reply back

Thanks Ke-Wei Zhao, Fabrizio Donnarumma ...

Hi Shanmuka,

Now you have a much more tricky issue. The bacterial expressed protein may not be properly folded. My guess is that, it is an aggregate. Have you tried running it on a gel filtration column to check its oligomerization status after renaturing? You may try the PKA reaction, it may work, but no guaranties here. What is the site if I may ask? (RxS or RxxS or RRxxS). If your pcDNA expressed protein is pure enough, tryptic digestion and peptide identification should work. Alternately you can use H-89, an inhibitor of PKA during growth and see if phosphorylation is reduced at these sites. There will be technical issues that you need to figure out here (cells will not be happy growing with H-89). Is there an antibody that recognizes this phosphorylation? 

Hope this is useful, good luck

Sujay

Hi Sujay,

No i have not checked for the gel exclusion after renturation.

The site I am suspecting contains RXXS.

There is no position specific antibody available.

Y I had an idea about PKA inhibitor study.

Any way thanx for the info once agin

Hi Shanmuka,

Let's assume that your site is phosphorylated. But it may not be a functional phopshorylation. The articles by Landry CR addresses this issue. My reading of the literature is that "a majority of phosphorylations are for no purpose". Biology is noisy and creates lots of waste along the way. Physiologically relevant phosphorylations will be controlled by conformation, concentration and localization. Hope your's is one too. A couple of articles by Landry and others that highlight the issue are below.

Regards

Sujay

Extracting insight from noisy cellular networks. Landry CR, Levy ED, Abd Rabbo D, Tarassov K, Michnick SW. Cell. 2013 Nov 21;155(5):983-9.

Weak functional constraints on phosphoproteomes. Landry CR, Levy ED, Michnick SW. Trends Genet. 2009 May;25(5):193-7.

"a majority of phosphorylations are for no purpose"

I would like to disagree with the following.

The author claim that there is no purpose. This can be stated ONLY in the case we have a comprehensive knowledge of ALL biochemical processes and their roles.

Considering we still know little of the whole picture, defining those phosphorylation sites as "for no purpose" it is not correct.

I believe it is correct to state that " for the majority of these phosphorylation site, the biological meaning is still unknown".

Very stimulating discussion.  I tend to agree with Fabrizio's opinion, as the majority of genome sequences were viewed as "meaningless" introns and other junks when the human genome project completed years ago, and we are beginning to understand some of these "junks".  It may also true in studying the stars without light?

Good to see some of you pushing me here. Let me add here that gene knock out studies have already shown that many genes that were once thought very non disposable have only mild or no affects. Knock out mice of some LIM domain proteins(3 genes totally absent) have no major problems at all. Once we decide that every phosphorylation is somehow important in some context, we may end up chasing phenomena that are minuscule or irrelevant. The lack of proper tools makes it harder to find clear cut answers. For example, it is my opinion that the use of D and E amino acids to mimic phosphoserine/threonine is not always appropriate. But an emormous amount of literature gets built around it that are very difficult to falsify. 

I am not sure if my answers are convincing. The papers by Landry CR and others that I pointed earlier are a good read. 

Cheers All