[proteomics] Phosphatase for Preparative Work on Intact Proteins

  • From: Mavi Gozler <mavigozler@xxxxxxxxx>
  • To: proteomics@xxxxxxxxxxxxx
  • Date: Fri, 15 Dec 2006 04:23:34 -0800 (PST)

A problem came up in which we would like to dephosphorylate all proteins in a 
mixture of the typical complexity of extracts.

I would like to do enzymatic treatment, probably with a broadly specific 
phosphatase that at least dephosphorylates the serines and threonines, as I 
doubt there is any tyrosine phosphorylation to worry about (these are proteins 
actually secreted into a body fluid).

Some alkaline phosphatases (ALPs) will dephosphorylate phosphopeptides less 
than 4000-5000 Da, and perhaps larger polypeptides too.  For example, on-MALDI 
target dephosphorylation of peptides reported by Torres et al (J Proteome Res 
4: 1628, 2005) although we want to do it simply in the standard conical tube.

Peleg et al (Prenat Diag 19: 224, 1999) reported that ALP 
extracted from neutrophils taken from whole blood of pregnant females has 
significant heat stability and resistance to urea (up to 3.7 M for the better 
part of an hour), and they tried to use this feature as a screening test for 
mothers possibly carrying Downs fetuses.

I believe that an ALP or similar phosphatase that retains activity in the 
presence of denaturants would be the ideal enzyme to dephosphorylate intact 
(and unfolded) proteins.  The protein phosphatases (PP1, 2a, 2b, etc) for both 
serine/threonine and tyrosine residues are not sold in quantities for 
preparative work (enough to treat thorough between 100-500 ug total protein), 
and would probably be prohibitively expensive.  I have found nothing in the 
literature in which someone reported using these particular protein 
phosphatases for work on that scale.

Sigma-Aldrich and others (NEB) sell a number of products but I don't see any 
description of them as working on intact proteins.
 To maximize the possibility that the enzyme will dephosphorylate intact 
proteins of large size (MW), I am strongly thinking that the proteins will have 
to be denatured...possibly by heat denaturation that may like cause 
precipitants to come out which I would like to avoid, or by inclusion of urea, 
and thus my interest in a phosphatase that resists the effects of urea if I go 
that route.

The alternative to enzymatic dephosphorylation would be chemical:  use a strong 
base (hydroxide) to cause beta-elimination to form dehydroalanine forms of Ser 
and Thr residues, and then stabilize the product by following with something 
like 2-mercaptoethanol to form a hydroxylated thioether (I have yet to get 
around to finding a published method for this).

Ideas?  Information?


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