OK. My short programming excursion is over and I went back to do my wet-lab experiments. It is tough work (see text) and very different to computer science :) Mainly there are three main differences which I have in my mind:

1. You prepare your sample, and you need a lot of patience to process it, e.g. the slicing of my gel took me about 2 days, but destaining, digestion & drying will take even longer, probably 3 or 4 days for my 4 plates (2 parallel). Simple tasks in general, but you have to be very precise and one is not allowed to shiver or to be unfocused! On the other hand, my preparations and experiments are very short, compared to some other experiments which last for months … The incubation times are also not to neglect, so you have 2-3 hours of “free” time for paper reading; I haven’t written a program yet, which compiles for 3 hours though – smile!

2. What about if the experiment doesn’t work? This is actually a every day experience in the wet-lab. But you can’t start debugging your sample, well at least not in a way you do it with source code :) Not working programms or algorithms are still a pain, but after days hunting the bug, you’ll hopefully find it! Some experimentalists told me, they had experiments which weren’t working even after a year …

3. It is different in another way too: you prepared your sample, and after weeks you ruin it by doing the wrong thing or you accidentally hit something/someone with your samples in your hand – no way to think of it. The whole work probably would be for nothing. Therefore, all sorts of in-silico work is more “save”. You can’t screw up your sample by accident (unless you perform a $> rm -rf /* and/or don’t make backups). Imagine, after years of programming your fancy application, you wrote a class which doesn’t work (of course it has never happened before – smile) or you misconfigured your config files – but your previous work is not gone, you can simply fix it. Nice, isn’t it!

I feel more “save” in front of a computer, but I love my experience I currently gain and I also need it to understand the problems, once I have to analyse the data. All tasks in the lab seem to be easy – in theory – but practice is different. Good to know and good that even Bioinformaticians have to do at least one wet-lab rotation here during our PhD time!

Best regards!

Apples PowerBooks!

Today there was the overdue update of the PowerBook series. Fine for Dani, and we think we will go for OS-X now. Basically every user is very pleased with this operating system and also with the build quality of the hardware. This thing is doing it’s job and runs a BSD underneath. Perfect, except for the price. But even that changed a bit; it dropped by about 250Eur. Apples update was fair, we didn’t expect a CPU upgrade, as this seems to be impossible, but the 15″ screen now offers 1440×960 and is 50% brighter, the battery last a hour longer and it comes with a DVD burner and the old 1.67GHz G4 (which is OK). Sadly there is no update for the crappy keyboard, which doesn’t fit into the nicely built machine … just my 2 cents!

All this doesn’t matter too much anyway, as Ecky was pointing out in his comment, we can do some coding on any machine ;-)

A different day: programming!

Hagen, a Masters student who just started a PhD in Spain is visiting our group for a week. He carried out his quite interesting final year project here at Team 17 in cooperation with Richard Durbins group. Now some data has to be verified and recalculated and I have the chance to get into it and continue what was started …

For me it was a good introduction into his work today, basically very important, as it could be highly relevant for a possible PhD project. Furthermore I wasn’t in the lab, rather we were programming. I just felt much more secure and confident, compared to wet-lab work – but no wonder, that is what I learned at University ;-)

We started implementing some scripts in Perl, but as this thing turned out as a memory hungry dinosaur (we used 3GB ram in the end) we ported it to Java, which was better for me anyway, as I am a very “fluent” Java speaker + it was 10x cleaner and OO. It was fun, sitting in front of computer again …

Yes, check out the new pictures of last week!

BUT to be honest, this entry is more about (K)ubuntu & Linux in general. Currently I am a bit tired of all that stuff. Well, sort of – I know I need a proper UNIX under the hood, as most bioinformatics tools run on this platform and I got used to it within the last couple of years. I don’t want to miss it and basically I can’t use Windows, too much things are missing.

OK so far, but what about this BUT? Well, I spent a lot of time struggling with some “simple” Linux issues (e.g. external monitor, Bluetooth, synchronisation of my mobile, Hibernation, webcam support, etc.) in the past. I have learned a lot, but now as I time is short, I don’t want to spend too much time any more on these issues. A few hours free time within a week isn’t much, and it is even less if there is something like Linux in the house.

Therefore I claim: A OS-X could be a nice solution, although I don’t like all the Apple hype. But it is a proper BSD, called “darwin” which is even free, and a working environment, where one doesn’t have to spend too much time working ON the machine, rather working WITH the computer. Furthermore all my loved Unix tools can be used … sounds promising. However, I don’t know if this is true – this is what most Apple users claim. I find out soon … probably!

You wonder what I did the whole last week ? OK, I try to explain a bit …

First of all, all these technologies are quite new for me. Well, I know the basics of Proteomics, but I rarely touched a wet-lab. Anyway – I learned how to prepare a cell culture to extract the proteins, I learned running a protein gel (1D SDS-PAGE), how to rinse and stain the gel, how to cut it and finally how to destain it, digest & dry the proteins. First I looked over the shoulder of Mercedes (Thanks for all your patience!), then I started my own experiment, which will take about 2 months including data analysis. Sure, I’ll have to ask quite often for help :)

I will examine proteins from nuclear and cytoplasmic samples (don’t want to tell you more here!) by LC/MS/MS, to see which proteins are abundant and which aren’t. This result can be incorporated into a bigger running experiment and will help to judge their results. But that’s not all. I will interpret the spectra from our machines and also compare the data acquired. We have the most recent machines in our lab, a LTQ-FT and a Q-TOF, and it will be interesting how they compete on the same samples (currently I prepare everything for more equal runs on these machines). Time per run, resolution, mass accuracy and sensitivity are important parameters. Anyway, I don’t want to go into details… Another issue I will cover with my experiments is a comparison between a RP HPLC and a more sophisticated technology, which is supposed to have the same resolution, but with a higher throughput. We’ll see …

Besides practical work, I read over a dozen papers, and there are still soooo many which are important to get the big picture! Within the next couple of weeks, I’ll have to go through the most important literature about my research, otherwise I won’t be able to write my first report in 2 month.

For me, the whole thing is a great opportunity. I learn the basic wet-lab techniques, get into operating the machines and also learn about the spectra & data. Considering a PhD in this group it is extremely helpful, as this would mean to implement computer aided analysis software & algorithms besides research about new approaches to mine the data. But that’s future, possibly starting in about 6-7 month ;-)

Tomorrow there is an UNIX course, which is compulsory – will be fun for me I guess! At least I hope I can support the biologists tomorrow …

Currently I am quite nervous, because tomorrow is a big day – the “real” start of my PhD ;-)

I am going to work in Team17, as I am very interested in the automated interpretation of MS data, which is a challenging undertaking. To get a feeling for this subject, I’ll do my wet-lab in MS. The project description:

Identification & characterisation of a multiprotein complex by proteomic mass spectrometry

Proteins often operate in concert with one another, organising into multisubunit complexes. The interactions between proteins modulate to a large extent several aspects of their function, including subcellular localisation, activity, specificity and regulation. Understanding the relationships between complex subunits and among complexes themselves therefore provides vital clues of molecular function and protein organisation. With recent improvements in technology and availability of genome sequences, mass spectrometry is ideally suited for the identification and characterision of proteins from complex biological samples.

The central theme of our research is to develop and apply procedures to describe native in vivo protein interactions, based on systematic targeted isolation of multiprotein complexes, followed by identification of the associated components by mass spectrometry. Our efforts are directed towards regulators of gene expression that are important in stem cell differentiation and early embryonic development.

The objective of the rotation project will be to investigate and characterise the multiprotein assembly(ies) associated to one of our candidate gene expression regulator proteins. A cell line has been constructed where the target protein is modified with a sequence tag that allows its isolation by two-step affinity purification. The tagged gene is under the control of its own promoter and therefore tagged protein levels are kept very close to physiological. The project will involve the purification of the bait protein, together with its associated proteins, from tagged cells. The isolated proteins will be processed and analysed by liquid chromatography / tandem mass spectrometry (LC/MS/MS). In instances where there is indication of post-translational modifications in the bait or interacting proteins, further mass spectrometry analysis will be carried out to confirm such features. The project will use the most modern MS technology and an array of pioneering biochemical techniques, which might include relative quantification by SILAC or ICAT.

Tomorrow I will now more about what I will learn, what I’ll have to do and which books/papers are waiting for me. Good night for now!

Hi All! Well, the first week is over and it wasn’t very exhausting at all. We had an introduction at the Sanger Institute on Monday, security training at the University of Cambridge on Tuesday & Wednesday, a journal club session on Thursday and another security training on Friday ;-) Next week proper work is waiting, as we all start our first rotation and I am looking forward to learn more about mass spectrometry!

Flo (yes, yet another friend from my old “Weihenstephan” semester) has finally arrived here as well. We met on Wednesday and he stayed here for two days, as he was looking for an accommodation. He was quite successful – I thought it’s more complicated around this season.

Ah, by the way, yesterday we all came together for a game evening: Bene, Flo, Georg, Micha, Dani & me (all originally from FH-Weihenstephan!) and Ernst & Tessa. Check out the picutres ;-)

Parts of my free-time I used to look at the scripting language Ruby! As far as I understood the concept, it seems to me as a very good scripting language, esp if you have proper OO background ;-) I have not much experience in Perl nor in Python, however I need some proper scripting language for rapid prototyping, everyday scripts and tasks (besides satisfying my own interest). I won’t use any Java for the moment, even I have to get fluent with the new 1.5 features. But that can wait a bit, as my guess is, that I will use it in my studies anyway. A bit more about Ruby soon!

Hi all! Just a rough outline of the day: indoor climbing with Dani, Bene & Pete. Was quite nice to be back on a “wall”. I need that more often I think ;-) Afterwards we all went to Coffee Nero to meet Micha and Jerven. And finally “The Eagle” pub in the evening to meet all Sanger PhD 1st years – it was very nice to meet all of them before the official start.

Now I need sleep, it’s going to be a very interesting day tomorrow ;-)