Friday, April 19, 2013
Wednesday, April 17, 2013
Wow! What a busy couple of weeks. I've completely fallen behind, but you'll soon see that there were some really good reasons for it (I hope...)
I'm somewhat back now with something really cool. I didn't discover this on my own. I've been working at the AMAZING proteomics core facility at the University of Wisconsin School of Pharmacy this week and they brought this engine to my attention. Side note, if you are looking to work or study with a premier collection of just about every top end mass spectrometer, consider moving to beautiful (and cold!!!) Madison, WI. You name it and they have it here. If I told you what they had, almost everyone would be jealous.
Back on track: The little icon above is more than just a way to flirt with a lawsuit from Fox Studios. It is also a very nice and rapid no-frills search engine that you can get for nothing at all. My gun toting friend here was described in last month's JPR paper from Wenger and Coon.
It is downloadable, in about 6 seconds on a hotel wireless network. It installs almost as quickly, and it runs through your massive proteomics files in only slightly more time. Adding a checkmark in the right place gives you "FDR on the fly" with no real measurable increase in your processing time.
I need to do some more digging. Mostly because I am skeptical of anything that is free, and I am extremely skeptical of things that are free and seem great, but this is a quick look at the protein level of a single run of a nuclear extract processed against the Uniprot Human database with Morpheus and Sequest HT in PD.
Thursday, April 11, 2013
Just got the alert that registration is open for the Mascot User's meeting at/before ASMS.
The location is The Hyatt Regency Minneapolis, 8:30 am to 12:30 pm, Sunday June 9th 2013, (immediately preceding the 61st ASMS).
There is no charge for attending this workshop, but advance registration is required. For program and on-line registration, click here
Maybe even better is the fact that you can watch the user's meeting as a webcast. Good for those of us who can't go, or who have lecture-induced narcolepsy!
Sunday, April 7, 2013
I'll save the sample processing details for the manuscript, but I will say that this is actually not a very fair comparison for MSAmanda. This is because of FDR. On Sequest HT and Mascot, Percolator gave me a significant boost, giving me 20-40% more peptides than using a simple 1% FDR. For some reason I haven't investigated yet, searches of this data set on MSAmanda with Percolator do not complete. So this is MSAmanda with a 1% FDR and both of the other data sets boosted with Percolator cutoffs.
If you use 1% FDR on all three of them: MSAmanda wins by far, finding dramatically more peptides than either Mascot or Sequest. I am using the data above because I am more concerned with total peptides right now than dumping a lot of time into troubleshooting FDRs. I am a pathway-centric biologist, after all.
BTW, yes these numbers are low. When we see phosphoproteomics papers we like to see thousands of sites, there is a good reason for this, but I can't go into it without revealing what I'm working on.
Anyway! Download MSAmanda and instructions for installing it in Proteome Discoverer here.
UPDATE: 4/16/13: Scratch what I've said about MSAmanda and Percolator. It works just fine with Percolator. You simply need to make sure that you aren't ordering the same modification twice. I set Carbamidomethylation of cysteine as both a static and dynamic mod. That would error out just about everything.
Also, if you download MSAmanda now, it installs with no problems on Win 7 32-bit PCs. Again, it may have been my mistake. At the end of my incredibly long days I get to work on my own projects. Sometimes I'm really tired and sleepy by then....
Saturday, April 6, 2013
Now we're getting somewhere. My argument against most targeted techniques falls right here: There may be as many (or more) than 1,000,000 variants of proteins expressed in human cells. The most comprehensive studies to-date can only identify a small fraction of these. By targeting, I feel like we're often limiting ourselves to what has already been identified. This is one of my many arguments against the SWATHish techniques where we're looking at matching a MS1 mass found in a total MS range to some potential daughter ions that are found when everything within that same mass range is fragmented. Without a comprehensive library, there are far too many false positives.
In a step toward negating this objection, last month in Nature Methods a paper from Paola Picotti et al., announced a "nearly complete" mapping of the yeast proteome. If you have a complete map, these technologies become a whole lot more feasible in my eyes. You can read more about this paper here, and obtain the map at the peptide atlas here.
Update 4/7/13: You can download this database as a spectral library. Not only that, but you can download it as an Orbitrap/LTQ (High-low experiment) spectral library. This aspect of the database is going into use real soon.
Friday, April 5, 2013
Geez, talk about appropriate timing. At KHUPO it was pretty obvious that glycomics/glycoproteomics is just about to blow up. A staggering number of the posters/talks were glyco centric. I was thinking that perhaps this was a regional focus, but with 20+ articles on glycomics in this month's MCP, I think that we're looking at more of a global trend. If this is your field, I'm sure you're way ahead of me on this one. If this isn't your field, it still might be time to start paying attention because the collaborators/clients are going to start calling with glycomics/glycoproteomics requests. Fortunately, there are a lot of teams working on the biggest hurdle (in my humble opinion) to these analyses -- the software side of things. I know of some really cool stuff that is currently in the pipeline and I'm sure that there is a whole lot more that I haven't heard about yet.
Thursday, April 4, 2013
Something I say a lot is along the lines of: "One day I'm not going to learn something about mass spectrometry..." I neither hope for nor expect that day to come. I particularly like when old paradigms get knocked down. Holy cow. And this is one. And it's not new, it's just impossible to keep up (for me, anyway).
Anyway, every search engine forever has required that we set leucine and isoleucine as equal, right? And we always have this uncertainty in the backs of our minds that this could also be an artifact of a hydroxyproline conversion. And here comes this paper (and a couple others as well, but this is the one that I was given) that says "Hey! We don't have to guess. ETD can tell the difference."
The paper in question is from Kallol Gupta et. al., and was a study with contributions from researchers in 3 facilities in Bangalore, India. In it, they show how ETD fragmentation demonstrates differences between these three isomeric amino acids. I love this paper. I especially love that it is yet another example of the meme that I've been trying to popularize, one talk at a time.
In comparing the ETD to the great honeybadger, it's great because the ETD don't care about: high energy bonds, low energy, weird PTMs, the 5/18 cutoff rule or even about isomeric peptides. The ETD just does what it wants, busting peptide bonds and elucidating sequences.
Anyway, everyone should check out this paper. Whether you are using ETD or not, it is great to be reminded that this is a young field and that these rules that we're so used to may just be the next set to be broken by the next advance.
Monday, April 1, 2013
This month's Proteomics (Wiley) has another interesting paper on jumping phosphorylation site assignments. The article is from Li Ciu and Gavin Reid at Michigan State University. The focus is on using some detailing ion trap CID fragmentation pathways and how these pathways correlate with mistaken phospho-site assignments. I am so glad that there are chemists out there who do these types of studies. Every new insight into what these instruments are doing internally ultimately helps us understand the biology side of things.
Sadly, my tour of South Korea is nearly at an end. Don't worry, I'm not going to be done talking about it for a while. This country is an absolute powerhouse for proteomics ideas and we're going to be seeing/hearing a lot out of the talented researchers here. As an example, I spend today at the Yongsei Proteome Research center (proteomix.org). They are doing high level and innovative work that is fully grounded in solid fundamental techniques. Not to brag, or put anyone down, but my job is to help people get better proteomics data. Most of the time I can look through a tune or instrument method and find things that I would change that should help. These are extremely tough things to find at the YPRC. I walked away from this facility thoroughly impressed, not only with the skill of the mass spectrometrists, but with the extremely clever ideas that they are working on. Needless to say, I learned a lot and while it is obvious that I can't share what I saw, I think it is safe to say that we'll be hearing a lot from these researchers soon enough.