Corante

About this Author
DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

Chemistry and Drug Data: Drugbank
Emolecules
ChemSpider
Chempedia Lab
Synthetic Pages
Organic Chemistry Portal
PubChem
Not Voodoo
DailyMed
Druglib
Clinicaltrials.gov

Chemistry and Pharma Blogs:
Org Prep Daily
The Haystack
Kilomentor
A New Merck, Reviewed
Liberal Arts Chemistry
Electron Pusher
All Things Metathesis
C&E News Blogs
Chemiotics II
Chemical Space
Noel O'Blog
In Vivo Blog
Terra Sigilatta
BBSRC/Douglas Kell
ChemBark
Realizations in Biostatistics
Chemjobber
Pharmalot
ChemSpider Blog
Pharmagossip
Med-Chemist
Organic Chem - Education & Industry
Pharma Strategy Blog
No Name No Slogan
Practical Fragments
SimBioSys
The Curious Wavefunction
Natural Product Man
Fragment Literature
Chemistry World Blog
Synthetic Nature
Chemistry Blog
Synthesizing Ideas
Business|Bytes|Genes|Molecules
Eye on FDA
Chemical Forums
Depth-First
Symyx Blog
Sceptical Chymist
Lamentations on Chemistry
Computational Organic Chemistry
Mining Drugs
Henry Rzepa


Science Blogs and News:
Bad Science
The Loom
Uncertain Principles
Fierce Biotech
Blogs for Industry
Omics! Omics!
Young Female Scientist
Notional Slurry
Nobel Intent
SciTech Daily
Science Blog
FuturePundit
Aetiology
Gene Expression (I)
Gene Expression (II)
Sciencebase
Pharyngula
Adventures in Ethics and Science
Transterrestrial Musings
Slashdot Science
Cosmic Variance
Biology News Net


Medical Blogs
DB's Medical Rants
Science-Based Medicine
GruntDoc
Respectful Insolence
Diabetes Mine


Economics and Business
Marginal Revolution
The Volokh Conspiracy
Knowledge Problem


Politics / Current Events
Virginia Postrel
Instapundit
Belmont Club
Mickey Kaus


Belles Lettres
Uncouth Reflections
Arts and Letters Daily
In the Pipeline: Don't miss Derek Lowe's excellent commentary on drug discovery and the pharma industry in general at In the Pipeline

In the Pipeline

« Hexacyclinol Rides Again | Main | The Sun Shines at Pfizer »

July 25, 2006

Back To Life

Email This Entry

Posted by Derek

It's been a little while since I updated everyone on my long-running series of experiments, but I do have some news. At last report, I'd set up a large crucial experiment, and actually seemed to get it to work in the notorious Vial Thirty-Three. Of course, when you get something remarkable to happen in the lab, the first thing to do is see if you can make it happen again, and that's where the trouble started. The first repeat was rather nasty, and the second one was no better. I was baffled, since the first run had looked so promising.

My colleague Joanne, who was analyzing these samples for me, was puzzled, too, but she at least knew something to do about it. There's a huge benefit to working with people who know what they're doing. She took that third run and ran the samples for me again, this time in a much longer gradient on the LC/MS. (For the non-chemists out there, this means that the purification part of the method was extended, spreading out the various components of the mixture more). The control vials looked just like they had in the first run - not much, which is what controls are supposed to look like. The experimental vials had looked the same way, though, but with this new run my data appeared as if the results had come out from behind a cloud. Suddenly it looked like the first run again!

They'd been in there all along, as it turns out, and a cloud of ion suppression is what they were hidden by. This is a real problem with mass spec methods using mixtures of proteins (and the stuff that keeps them happy). There are a lot of reasons for this, only some of which are well understood, but having your analytes disappear and reappear unpredictably on you is apparently a widely shared experience.

I tried to see if there was some single component in my brew that I could leave out and thus fix my problem, but I should have saved my effort. That rarely seems to be successful - the real solution, as would have been clear to a real chemical biologist, is to run things the way you have to, and then clean up your samples before they go into the machine. The best way to do that is probably solid-phase extraction (SPE), which entails loaded your mixture onto some sort of powdered polymeric stuff which binds the analyte you care about. That lets you wash all the gunk out of the system, and then you use a different wash to elute the good stuff.

Here's an older review that illustrates the principle. These days, there are dozens of competing SPE technologies from all the major lab vendors. I evaluated a set of the more popular ones by setting up a row of dummy experiments - all my proteinaceous stuff, spiked with a constant amount of my desired product. All of them improved things, but one in particular (the Waters Oasis MCX, for those curious) seemed to do the best job, although I'm sure that there are others that would work as well. The method I worked out for it was the most complicated of all the ones I tried, but it's probably washing out the most sludge, too, because I'm getting ten- to twenty-fold more signal than I did before.

So, late last week I set up my first "Vial Thirty-Three" experiment again and worked it up with the SPE. It reproduced perfectly, to my great happiness, which takes me right back to the edge of things. Before I left the lab on Monday, I set up another run, this time with six different control and experimental arms, in duplicate, the most comprehensive look at this effect I've ever taken. I'm working it up today. Results in a couple of days, most likely. I'll keep everyone informed.

Comments (7) + TrackBacks (0) | Category: Birth of an Idea


COMMENTS

1. grad on July 25, 2006 7:58 AM writes...

I'm an experimental chemist, but honestly, this seems more exciting that anything I'm working on. I think its actually the lack of any knowledge of what your doing, so just the excitment comes through.

Good luck.

(Isn't the company name Waters rather than water?)

Permalink to Comment

2. Dave on July 25, 2006 8:44 AM writes...

Sounds quite promising, congrats!

I'm currently working on a tantalizing yet maddening project myself and can only hope to sort this one out in the end...

Permalink to Comment

3. Derek Lowe on July 25, 2006 8:50 AM writes...

Fixed, grad, along with a few other typos. Thanks!

Permalink to Comment

4. Mark on July 25, 2006 11:14 AM writes...

I am glad you kept at it until the mystery of the anlytical part was solved.

Why does it seem that on the brink of discovery there is often an eqpt. malfunction or other system error to overcome.

Definitely pays to be persistent, patient and always pondering in this business.

Cant wait to read the next post in this series!

Permalink to Comment

5. otey2 on July 25, 2006 11:56 AM writes...

Don't even think about not keeping us informed of developments.

Permalink to Comment

6. Hap on July 25, 2006 12:48 PM writes...

Do you have to control for the SPE as well as your standard control variables (to make sure the solid phase isn't binding what you care about or something that affects it?)?

Permalink to Comment

7. Derek Lowe on July 25, 2006 2:21 PM writes...

That's one of the things I was looking at in my method-search experiments. Starting each test reaction with the same amounts of the exact stuff that I'll be looking for in the real experiments gave me head-to-head comparisons among the various SPE workups. And comparing each of them to the same spiked amount in plain solvent (and to the same spiked amount in the un-cleaned-up protein mixture) gave a reading of how well they did compared to the maximum possible recovery, and how much better they did than what I had before.

Permalink to Comment

POST A COMMENT




Remember Me?



EMAIL THIS ENTRY TO A FRIEND

Email this entry to:

Your email address:

Message (optional):




RELATED ENTRIES
Conference in Basel
Messed-Up Clinical Studies: A First-Hand Report
Pharma and Ebola
Lilly Steps In for AstraZeneca's Secretase Inhibitor
Update on Alnylam (And the Direction of Things to Come)
There Must Have Been Multiple Chances to Catch This
Weirdly, Tramadol Is Not a Natural Product After All
Thiola, Retrophin, Martin Shkrell, Reddit, and More