[proteomics] Checking Isoelectric Forms of Proteins in 2D Gels

  • From: Mavi Gozler <mavigozler@xxxxxxxxx>
  • To: proteomics@xxxxxxxxxxxxx
  • Date: Thu, 14 Dec 2006 05:20:04 -0800 (PST)

When you see what appears to be proteins of identical MW in a 2D gel but appear 
to have many isoforms that differ in their isoelectric point (pI), you might 
want to assume these possibilities:

1) a protein with multiple glycosylation sites that feature sialylated 
oligosaccharides (these are negatively charged sugars)

2) a protein with multiple phosphorylation sites which are phosphorylated

In the case of (1), you can possibly pre-treat the protein (or protein mixture) 
with a deglycosylating enzyme (perhaps PNGase F, also called N-glycanase) or 
with a de-sialylating enzyme (neuraminidase) and see if the multiple spots 
disappear into one spot on the 2-D gel.

In the case of (2), you should be able to dephosphorylate the protein in some 
way.  There are protein phosphatase specific for serines/threonine type 
phosphorylation (PP1, PP2a, PP2b, etc.) and some specific for tyrosine 
phosphorylation, but these are often quite expensive, and perhaps insufficient 
as a preparative treatment.  A less expensive treatment is with alkaline 
phosphatase, but that may not be specific for protein.  It is reported to 
dephosphorylate peptides,  but as for intact proteins of fairly 
average-to-large MW, it might require extensive denaturation of the 
polypeptide, including having urea present while the alkaline phosphatase (ALP) 
hydrolyzes the phosphates.  There are urea-resistant and heat-stable ALP 
enzymes reported in the literature, in particular in increased activity from 
the neutrophils of pregnant women.  Other kinds of dephosphorylation include 
chemical means (beta elimination reactions to form alkenes).


Everyone is raving about the all-new Yahoo! Mail beta.

Other related posts:

  • » [proteomics] Checking Isoelectric Forms of Proteins in 2D Gels