[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?
SMH
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