tag:blogger.com,1999:blog-1136153439451224584.post8170312548026946004..comments2024-03-27T06:45:59.174-07:00Comments on Practical Fragments: That's One Way How to Do ItDr. Teddy Zhttp://www.blogger.com/profile/07288045760981372367noreply@blogger.comBlogger8125tag:blogger.com,1999:blog-1136153439451224584.post-14893200742634665392014-01-21T14:58:16.701-08:002014-01-21T14:58:16.701-08:00I'm not sure that there is a simple answer to ...I'm not sure that there is a simple answer to this. The fragments that we reported in the Vom paper were from our first library. Many of those compounds wouldn't make it into cocktails now, and weeding out the badly behaved compounds using no-protein controls is clearly a good way to get rid of some of the false positives as Darren said.<br /><br />However, we still see cases where fragments that have good experimentally measured aqueous solubility and do not give signals in STD spectra of the mixtures without protein behave as bad actors. We don't yet have enough data to indicate whether detergent makes a systematic difference to these.<br /><br />I'm not sure that it would be wise to treat a detergent as a benign additive that simply prevents aggregation, so whether to add detergent and which detergent to add is probably dictated by the target.Martin Snoreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-11827241707465984562014-01-13T04:34:35.746-08:002014-01-13T04:34:35.746-08:00Teddy Z
As far as I'm aware, mass spec can t...Teddy Z <br /><br />As far as I'm aware, mass spec can tolerate detergents, you just need to use synthetic ones that have a single peak rather than giving you a smeary mess (octylglucoside being the one we mostly use for screening).<br />Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-88779333416099178072014-01-10T12:50:51.498-08:002014-01-10T12:50:51.498-08:00I think everyone agrees that aggregation is a crit...I think everyone agrees that aggregation is a critical factor in fragment screening, and can be more prevalent in the absence of detergent. But there is nothing about STD-NMR more inherently sensitive to aggregating compounds than other methods testing hydrophobic compounds at high concentrations in aqueous buffers. Moreover, there are drawbacks to using protonated detergents for STD-NMR, which Teddy, my team & I discussed while writing this protocol:<br /><br />1) Detergents will show up in the aliphatic spectral region, potentially masking the only proton signals for certain fragments, such as those 3D fragments of the future;<br />2) Depending on their chemical structure, detergents may experience direct irradiation by the saturation pulse, creating potential for positive % STD due to binding events with the detergent and not the protein...detergents which bind the protein may have STD enhancement as well, and in turn indirectly generate false positives;<br />3) In certain cases, detergents can compete for fragment binding sites, preventing fragments from binding in important spots; <br />4) At sufficiently high concentrations, detergents lead to reduced NMR signal strength and lower quality data;<br />5) Some protein targets are incompatible with anything remotely denaturing.<br /><br />With such complicating factors (and limited space for text), we felt it was not suitable to present in our method for introductory screening. Instead, we chose another solution: test your library. Screen the compounds under the same/very similar conditions by STD-NMR minus the protein, and eliminate false positives that way. Saves you much time and frustration, rather than following up on the false positives post-screen, as was presented in Vom's 2013 AJC paper.<br /><br />Note that, in the same study (Table 3), detergent only eliminated 1 bad actor at high concentration of fragment (1.0 mM ± Tween), while simply reducing the fragment concentration (1.0 mM -> 300 µM) eliminated 6 false positives. We have found reduced concentration to be similarly superior to detergents for removing bad actors, and presented those instructions in the method.<br /><br />I'm not saying detergents should be left out of the realm of buffer agents one tries when screening. But when it comes to minimizing downstream problems, at least for the type of work that I do, I tend to put vetting the library first, and working up the best conditions on a per-target basis, detergent or no detergent. <br /><br />- dbDarren B.http://www.embios.comnoreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-3355188840507862492014-01-10T09:26:12.064-08:002014-01-10T09:26:12.064-08:00One interesting example is the rise in Mass-Spec m...One interesting example is the rise in Mass-Spec methods. The cannot tolerate detergents at all. So, should we be more worried if they are used?Dr. Teddy Zhttps://www.blogger.com/profile/07288045760981372367noreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-61992487910957147002014-01-09T19:46:13.896-08:002014-01-09T19:46:13.896-08:00That's my feeling too. Since the Current Proto...That's my feeling too. Since the Current Protocols paper does not mention detergent, which one(s) work best for NMR?Dan Erlansonhttps://www.blogger.com/profile/07927082337051189270noreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-90915201331703064952014-01-09T06:51:01.506-08:002014-01-09T06:51:01.506-08:00I think detergent should only be left out of any a...I think detergent should only be left out of any assay in exceptional cases. Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-59202452739657951252014-01-08T22:40:27.858-08:002014-01-08T22:40:27.858-08:00I would say it is very important, for any assay fo...I would say it is very important, for any assay format.Dr. Teddy Zhttps://www.blogger.com/profile/07288045760981372367noreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-30137442394366836262014-01-07T06:46:39.951-08:002014-01-07T06:46:39.951-08:00This is a really nice resource for folks getting i...This is a really nice resource for folks getting into STD NMR, but I did have one question. It seems that the technique is particularly susceptible to <a href="http://practicalfragments.blogspot.com/2013/12/fragments-in-australia.html" rel="nofollow">aggregating compounds</a>, so what do folks think of routinely running STD NMR in the presence of detergent?Dan Erlansonhttps://www.blogger.com/profile/07927082337051189270noreply@blogger.com