Tuesday, March 15, 2016

Proteomics of MRSA blasted with antibiotic combo!

MRSA (methicillin resistant Staph aureus) is one of a bunch of scary-as-heck superbugs out there that are resistant to just about every drug we can throw at them.

Several recent studies tout the efficacy of so-called "resistant breakers," which are multi-drug combinations that lend efficacy to traditional antibiotics (cause...we'll probably have new antibiotics in 20 years or so...).

In a very similar tilt, this new paper from Xiaofen Lu et al., takes a look at an effective drug combo at a proteomic level in MRSA.  The drugs in this study are plain-old oxacillin and a subtly altered derivative of erythromycin.

In general, the -cillin drugs block the action of Penicillin Binding Proteins (PBPs). These proteins catalyze the formation of short peptide crossbridges that hold the long glycan strands of bacterial cell walls together. Erythromycin...well...um...even though we've been using it for 60 or so years...it... kills bacteria of some kinds? We're not really sure how. But it is pretty well established that it doesn't directly affect the bacterial cell wall.

Check out the electron microscopy at the top, though!  The panels go like this 1) MRSA chillin' 2) MRSA chillin' 3) MRSA chillin' 4) MRSA FREAKING OUT!!!!

So we've got a drug combo that kills this scary stuff. Lets do some proteomics!

So they harvested some peptides using normal techniques for bacteria and loaded it on an LTQ Velos and used spectral counting for quantification. Look, I've got exactly zero problems with spectral counting if it works for you. Are there more sophisticated quantification methods? Sure! Should you use spectral counting on a high resolution/accurate mass instrument? Probably not without really thinking about your experimental design (particularly your instrument parameters - as most Orbitraps instrument methods are designed in such a way, by default, to lower the efficacy of spectral counting techniques  -- not on purpose, but because you give up sample depth by increasing spectral repetition - and they are designed with sample-depth in mind). [P.S. There are ways to improve the efficacy of spectral counting on instruments like the Q Exactive that are designed, by default to "ignore isotopes" and not repeat the fragmentation of the same ions. But you will be giving up coverage. If it is worth it for you I can show you how some groups I've worked with are currently doing it, though I just gave you two big hints]

If you've got a fast ion trap and large amounts of material and good software for spectral counting statistics -- spectral count away!

Just look at the results this group got. Hundreds of statistically significant differentially expressed proteins! [Okay. Is "differentially" not a word? Cause Blogger thinks its not a word. But I'm going to use it in every post, so there.]

For data processing, this group used OMSSA, threw out any proteins with less than 5 spectral counts, and did downstream analysis with MatLab. Q-RT-PCR was used to validate the observations. In the end, they find a big change in the abundance of a major player in cell wall formation and a disruption in the bacteria's ability to respond to oxidative stress.

All in all, this is a really nice study. Great model, good use of a work horse of an instrument and some good downstream analysis that reveals some surprising new information about one of the deadliest things out there!  Highly recommended (and open access!)

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