Fish lipid protocols

I spent most of today cobbling together a method for extracting lipids from yeast cells for analysis by mass spectrometry. A reasonably straight-forward task you might think, but let me explain why you'd be wrong.

First of all, most of the time when I do journal searches I'm on a computer physically at UNSW, where I have a staff number, and probably a password that allows me to access their database remotely, but I have no idea what it is. No problem, I also have a UWS student number that gives me access to their database - except that for some inexplicable reason my access has been revoked, and they don't seem to want to tell me why. Still, I also have a UTS student number which gives me access to their database, and it actually works! I also have temporary access to Cambridge's resources, but unfortunately the combined UTS/Cambridge resources are still less than those available at UNSW. Neither, for example, has access to the journals Methods in Enzymology or the Journal of Lipids Research, which are both fairly handy when you're trying to put together a method for extracting lipids.

Things were further complicated by the fact that just about everyone, whether they're dealing with bacteria, yeast, plants, rats or humans, uses a technique devised by two individuals by the names of Bligh and Dyer in 1959 for analysing lipids from fish plasma. They published this method in the Canadian Journal of Biochemistry (or somesuch stupid journal - I'm typing this from memory and I refuse to look the bloody thing up again). Despite the fact that just about everyone uses it, no one really says anything other than: "lipids were extracted using the method of Bligh and Dyer", which isn't particularly useful, because I defy anyone to get hold a paper from 1959 that was published in the Canadian Journal of Biochemistry. I did manage to find a number of things online whinged about the fact that everyone cited the Bligh and Dyer paper without actually reading it, which still isn't particularly handy. So I tried a search on "lipid extraction yeast", and turned up a book chapter co-authored by Gunther Daum, who is pretty much the guru in yeast lipid biology. So things were looking up, except for the fact that neither Cambridge nor UTS had electronic access. UTS did have a hard copy, so if push came to shove I could see whether my sister was willing to go and photocopy the relevant pages, but that still left the question of how she would get them to me. My next step was to ask Pnar if she had a copy, but unfortunately she didn't. To cut a long story short, it turned out that both Steve and Juan had copies, and that for the 30-odd minutes that I'd been trying to get hold of it, a copy was literally 3 metres away in Juan's desk drawer. Shit.

However, the method outlined was adapted from a paper published even earlier than Bligh and Dyer's method, and was not going to be compatible with MS. Fuck. Somewhat exasperated by this stage, I decided to try searching some other databases, such as Google (that is an in-joke, people), and eventually turned up enough bits of information to sort it out. I knew it required chloroform and methanol in a 1:2 mixture, but it was the nuts and bolts that were eluding me. The methods part of my thesis will be easy to write though:

Lipids were extracted by the method of Bligh and Dyer (1959).

As a postscript, the good news is that I'll be able to measure both steryl esters and free ergosterol using direct injection ESI MS/MS, as well as get a shit-ton of data from a bunch of NMR experiments. All while working with Jules Griffin who is pretty much The Dude when it comes to metabolomics (his paper is the first hit when you type "metabolomics NMR" into Pubmed). So, um yeah. The phrase that springs to mind is a NoFX lyric: "Some people have all the luck, no matter how much we fucking suck".

Some of the above may have been exaggerated for comic purposes.