This page last modified: Feb 28 2007
title:OMSSA Hints and Howto keywords:omssa,ms,search,peptide,how,to,documentation, description:How to choose and determine options for OMSSA and omssacl Table of contents ----------------- Introduction How to convert average mass to OMSSA mods Choosing values for "te", "to", "tez", "mm", etc. Other factors that may effect mass tolerance Fixed and variable modifications Introduction ------------ Choosing the best values for OMSSA options can be a challenge. With the help of several people from various institutions, I've put together some notes. Many, many thanks to all who contributed. How to convert average mass to OMSSA mods ----------------------------------------- The problem: your MS/MS lab gives you a data set and says "We did this run with a static modification on Cys of 57.0215." This translates to an average mass of Cysteine of 57.0215. You can enter this mass value into a couple of web sites and determine the likely mod is Carbamidomethyl C. This you can look up in the OMSSA mods list. average mass >= 57.0215 http://www.unimod.org/modifications_list.php (Registration not necessary. Click "login as guest".) http://www.abrf.org/index.cfm/dm.home?AvgMass=all It provides all average masses, and some monoisotopic masses when you click on the highlighted modifications. Choosing values for "te", "to", "tez", "mm", etc. ------------------------------------------------- Many of the parameters are chosen based on the instrument and data type, although there don't seem to be hard and fast rules. For instance, a no-enzyme search which contains many potential modifications and sites requires a larger "mm" setting than a tryptic search with a limited number of possible mods. The mass tolerances change based on the type of instrument. Some labs have spent a lot of time coming up with sets of parameters, and have multiple sets depending upon data type. A precursor mass tolerance "te" of 0.75 (this is +/-) is very high (a wide range) when searching high resolution ms data. I've talked to labs that normally stay between the values of 0.01 and 0.06 for this parameter. On the other hand, a value of 0.75 is very low when are searching low resolution data. For low resolution ms data, a typical value is in the range of 1.0 to 1.5. Another recommendation is to set the "tez" parameter to 0 (zero). This disables the charge-dependent changes to the precursor mass tolerance value. I found (by trial and error) that a product mass tolerance "to" of 0.15 worked for a set of spectra (where "worked" means 0.15 yielded more peptides than other values for this setting). However, I'm told that this product mass tolerance could be too narrow. A typical value is somewhere in the range 0.30 to 0.50. I'm not clear why setting the value of "to" to a larger number (seemingly more relaxed) gives fewer peptides. An experienced person has told me that a bigger precursor mass tolerance "te" might give more significant hits since more potential matches will be available. The concern would be that some of these hits were false positives. Oddly, my personal experience is that values outside some optimal range yield fewer hits. Other factors that may effect mass tolerance -------------------------------------------- A friend tells me that -te and -to are very much dependent on which mass spectrometer you use. I don't mean to cast aspersions on the MS/MS scientists, but other important factors in mass tolerance relate to the MS/MS calibration: 1) How well the MS/MS was calibrated 2) How long ago the MS/MS was calibrated 3) How much the temperature in the room has changed since the MS/ME was calibrated 4) Other unspecified factors This is one area of possible improvement for the current generation of search tools. These tools could sample the spectra, and from that sample determine settings, especially mass accuracy. This would be a good idea because the mass accuracy values can be highly variable. In the near term, I may implement a crude work around by implementing an automated software pipeline that runs a search (such as OMSSA) many times with a range of settings. The intent is to find the "best" settings. Here are some rough suggestions for well-calibrated instruments: Ion trap -to 3.0 -te 0.4 Linear ion trap -to 1.5 -te 1.0 Q-TOF -to 0.2 -te 0.2 Fixed and variable modifications -------------------------------- Fixed modifications are better than variable modifications when possible because it limits the range of possible matches to be searched. In other words, the search will be faster and there may be fewer incorrect matches (bad hits, false positives). The speed difference can be significant. The computational overhead is less of an issue for many of my searches because I have the luxury of a cluster with 30 dual-cpu nodes and I have a high-throughput pipeline that will automataically split, run in parallel, and join results (this pipeline is open source, by the way). I have not done a direct comparison with the same modification fixed versus variable to confirm that search results are the same.