Phosphopeptides on OFFGEL system

Experiment:  Cells were treated with a general phosphatase inhibitor for 1 hour, lysed and the protein digested, all the while using a phosphatase inhibitor cocktail.
The resulting phosphopeptides were separated on an OFFGEL fractionator using low-resolution strips, pH 3-10.  The fractions were cleaned up using zip-tips and were ran on an LTQ  to determine if/where phosphopeptides aggregate during OFFGEL isoelectric focusing.

As predicted, the phosphopeptides seem to aggregate in the acidic range, with the exception of a large number of them that appeared in fraction 7, near neutrality.  It is important to note that this was a single run per fraction as we're simply working on optimizing this procedure.  The most interesting part to me is the fact that we were seeing unique phosphopeptides all the way to pH 10.

The Orbitrap XL is finally here!  The service engineer has been working on it all day.  He'll be back tomorrow on Monday to finish the install.  Today hasn't been the greatest day for science, but a great day for moving refrigerators!

58th ASMS in SLC

ASMS begins in SLC next week.  Unfortunately, due to the job switch there was no way for me to justify going this year.  Two of my coworkers will be attending and I can't wait to hear what happened there this year.

Filter aided capture and elution (FACE)

This very understated paper in JPR may be the biggest advance in phosphoproteomics since the neutral loss scan.  This method outlines a new method, dubbed FACE, for Filter Aided Capture and Elution.
The process:
1) Digest all of your peptides
2) Incubate your peptides with an anti-phospho- antibody (they used 3G10 anti-phosphotyrosine)
3) Spin your peptides through a size-exclusion column.  The unbound peptides flow right through, leaving only the antibody and bound peptides
4) Wash to remove all unbound peptides
5) Elute your phosphotyrosine peptides using acid.

I see almost unlimited promise in this method.  I asked around and there are anti-phosphoserine and -threonine antibodies, but they are generally regarded as no good.
I will be trying this method very soon, considering phosphoproteomics is what I was hired for.

This blog

With the successful completion of my first postdoctoral fellowship in academia in sight, I've decided to pursue a second fellowship in the private sector.  My goal is to explore as much of science as I can, in order to see if my goal of being a professor is still the best route for me.  I have three job interviews scheduled for the coming month, one with a pharmaceutical company, one with a government lab, and one with a private company contracting at a government facility.  All three of these jobs will involve working with applying my skills to new chemotherapy agents.  Since these will be patented drugs, very little of my work will be published.
The goal of this blog is to show that although I am not publishing, I am still keeping current on newest advances in my field -- both at the level of machines and software, and with the current literature.  It also keeps me writing, which is something that I don't want to get out of the habit of doing.
If you have any comments about the material in this blog, please email me at: orsburn@vt.edu
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