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April 9, 2013
Mass Spec Continues Its Conquests
You know, mass spectrometry has been gradually taking over the world. Well, maybe not your world, but mine (and that of a lot of biopharma/biophysical researchers). There are just so many things that you can do with modern instrumentation that the assays and techniques just keep on coming.
This paper from a recent Angewandte Chemie is a good example. They're looking at post-translational modifications of proteins, which has always been a big field, and shows no signs of getting any smaller. The specific example here is SIRT1, an old friend to readers of this site, and the MALDI-based assay reported is a nice alternative to the fluorescence-based assays in that area, which have (notoriously) been shown to cause artifacts. The mass spec can directly detect deacetylation of a 16-mer histone H4 peptide - no labels needed.
The authors then screened a library of about 5500 natural product compounds (5 compounds per well in 384-well plates). As they showed, though, the hit rates observed would support higher pool numbers, and they successfully tested mixtures of up to 30 compounds at a time. Several structures were found to be micromolar inhibitors of the deacetylation reaction. None of these look very interesting or important per se, although some of them may find use as tool compounds. But the levels of detection and the throughput make me think that this might be a very useful technique for screening a fragment library.
Interestingly, they were also able to run the assay in the other direction, looking at acetylation of the histone protein, and discovered a new inhibitor of that process as well. These results prompted the authors to speculate that their assay conditions would be useful for a whole range of protein-modifying targets, and they may well be right.
So if this is such a good idea, why hasn't it been done before? The answer is that it has, especially if you go beyond the "open literature" and into the patents. Here, for example, is a 2009 application from Sirtris (who else?) on deacetylation/acetylation mass spec assays. And here's a paper (PDF) from 2009 (also in Angewandte) that used shorter peptides (6-mers) to profile enzymes of this type as well. There are many other assays of this sort that have been reported, or worked out inside various biopharma companies for their own uses. But this latest paper serves to show people (or remind them) that you can do such things on realistic substrates, with good reproducibility and throughput, and without having to think for a moment about coupled assays, scintillation plates, fluorescence windows, tagged proteins, and all the other typical details. Other things being equal, the more label-free your assay conditions, the better off you are. And other things are getting closer equal all the time.
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