Phosphopeptide enrichment: Fe-NTA vs TiO2

As I reported in a previous entry, we didn't have any luck obtaining phosphopeptides with the Pierce Gallium Swell Disk cartridges (except for enriching acetylated peptides).  This is a bit of a consensus, mentioning this kit actually brought an unintentional smirk from one of the company's employees.
Therefore, we were hesitant to try Pierce's new columns, the supposedly complementary Fe-NTA IMAC columns and the TiO2 spin columns.  We took a gamble on them because they were very easy to use, and this postdoc is wearing far too many hats to try packing his own gel-loading pipette tips with enrichment resin.

The preliminary results are nice, but not exactly mind-blowing:

Yes, the results are complementary.  Yes, they resulted in more than 600 unique phosphopeptides.  I would be completely satisfied by these two enrichment methods (that are very easy, btw), if the filter aided capture and elution (FACE) technique hadn't produced over 1,000 unique phosphopeptides on its own.  And as easy as these two techniques are, the FACE technique is easier and cheaper.  Fortunately, the results are completely complementary with FACE as over 90% of the phosphopeptides found by TiO2 and IMAC are phospho-serine and phospho-threonine, while 95% of the phosphopeptides from FACE are the rare (and incredibly important) phospho-tyrosines.

Summary:  If you want to get a great phosphopeptide enrichment, perform FACE, then TiO2 on the flowthrough followed by IMAC enrichment of that flowthrough.

Gallium enrichment of acetylated peptides

I've been spending the weekend reviewing some data from a while ago for something I meant to evaluate further.  Pierce used to sell a phosphopeptide enrichment kit that used 'gallium swell disks' to capture and selectively elute phosphopeptides.  When I first used this kit last year I remember being surprised by the number of acetylated peptides.  Looking at the data further suggests that this kit may actually be better at enriching acetylated peptides than phosphorylated ones.  Too bad they don't make it anymore, because it might be a nice tool if one were going after the acetylome.

Imaging mass spectrometry for antibiotic discovery

I am absolutely blown away by this work out of Peter Dorrestein's lab.  This work is some of the most simple and elegant MS work that I have seen in years.
This is what they do, essentially:
1) They grow a lawn of methicillin resistant staph aureus (MRSA)
2) Then they take some bacteria and draw a "t" across the surface of the MRSA culture
3) They look to see if the new bacteria inhibits the MRSA growth in any way.
4) If it does, they ionize the area of the zone of inhibition and analyze the ions by MS/MS.
Of course, its a bit more complicated than than, but this is essentially the plan.

I saw Dr. Dorrestein speak on this subject a while ago, but I've only recently been able to catch up on the literature and their current work, including a compound they've discovered that is significantly more potent than the most powerful antibiotics we currently have in use.
I sincerely suggest that you take a look at some of his papers.  I think that you'll be impressed, and perhaps feel a little dumb for not thinking of it yourself....
For more of this lab's great work, click here.

MCP changes policy on raw data

As of February 22, authors submitting to MCP are no longer required to place their RAW data in a publicly accessible database.  I don't know why the journal has made this decision, but I don't like it.  Its simply too easy in this field to fabricate data.  MCP still 'strongly encourages' the deposition of data, but I would prefer that it was mandatory unless particular stipulating conditions, such as patent rights were involved.  Even in those cases I feel that the data should be deposited once those stipulations have expired or been resolved.

Handbook of Basic Mass Spectrometry

My first full-length book is out.  The handbook of basic mass spectrometry is my attempt to simplify this field down to what biologists and medical technologists need to know.  You don't need a physics degree to do a mass spec experiment and you shouldn't need one to understand the machine you are using.  I hope that this helps make the field of mass spectrometry a little less daunting to potential practitioners out there.

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
Also, please see my disclaimer statement.