tag:blogger.com,1999:blog-1136153439451224584.post5970839843347010247..comments2024-03-27T06:45:59.174-07:00Comments on Practical Fragments: Benchmarking native mass spectrometryDr. Teddy Zhttp://www.blogger.com/profile/07288045760981372367noreply@blogger.comBlogger3125tag:blogger.com,1999:blog-1136153439451224584.post-89301995609152274542015-06-09T05:56:28.564-07:002015-06-09T05:56:28.564-07:00Thanks Teddy and Valerie. I do like the idea of na...Thanks Teddy and Valerie. I do like the idea of native MS, I just don't recall seeing many examples where it excels in true discovery mode as opposed to proof of concept.<br /><br />It's true that native MS does give you protein QC, though this can be a limitation if you're working with, for example, glycosylated protein.<br /><br />Also, you can get stoichiometry with SPR, though it requires more effort. That said, I wonder how often apparent stoichiometry observed with native MS is actually just non-specific binding.<br /><br />I did notice that while the <a href="http://www.ldorganisation.com/v2/produits.php?langue=english&cle_menus=1238915905" rel="nofollow">NovAliX biophysics conference</a> going on right now has two scheduled presentations on <a href="http://practicalfragments.blogspot.com/2014/08/hydrogendeuterium-exchange-mass.html" rel="nofollow">HDX MS</a>, there do not appear to be any on native MS (though I don't have access to the poster titles).Dan Erlansonhttps://www.blogger.com/profile/07927082337051189270noreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-42378532891873476482015-06-09T03:00:18.723-07:002015-06-09T03:00:18.723-07:00Non-specific adduction occurring during electrospr...Non-specific adduction occurring during electrospray has been largely documented by Klassen's group (see a nice review from Kitova et al. published in JASMS (2012) "Reliable determination of Protein-Ligand interactions by direct ESI-MS measurements. Are we there yet?"). Artefactual adduction of compound to protein during solvent evaporation, concentration and fission of the charged dropplets typically show Poisson-like distribution. This phenomenum is prone to occur with large excess of compound in incubation mixtures. <br />Therefore screening for low affinity hits, in the three digit µM range for example (typically fragment molecules), using native MS, absolutely requires high affinity tool molecule to be available to distinguish true hits from false positives. From our experience a screen of 1500 fragment molecules (MW ~ 200) against a nuclear hormone receptor target produced about 3.5% validated hits. Binding stoichiometry and reversibility as well as an estimation of the binding affinity range were also directly available from mass spectra. Screen was done side by side against the apo receptor and against the receptor saturated with the tool compound.<br />Regarding false negative due to in source dissociation of the complex, the group of John Klassen has described additives and indirect approach that may be beneficial (see above review).Valerie Vivatnoreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-67824684951443748582015-06-08T11:05:53.545-07:002015-06-08T11:05:53.545-07:00I really really like native MS. I have seen it wo...I really really like native MS. I have seen it work where SPR outright fails. There are some tricks to the trade of course. It has some significant advantages over the other direct detect biophysical methods: protein QC every experiment and the ability to do stoichiometry for example.Dr. Teddy Zhttps://www.blogger.com/profile/07288045760981372367noreply@blogger.com