David, This is Henry Lam, the original developer of SpectraST. I am very sorry for missing your post -- I have not been checking the spctools-discuss group frequently and I was referred to your post by another user who met a similar problem. Please do write to me directly in the future.
Your problem should be easy to solve. You have to include the "-M spectrast.usermods" file in the Search command also. The modifications defined by spectrast.usermods are not "permanent" -- they last as long as that command is finished. Even if those modifications have been included some library spectra, they are not internalized into SpectraST's list of recognized modifications. When the library is searched without the spectrast.usermods specified, SpectraST will not understand the new modifications. To cope, it will ignore all the spectra with those modifications. That is why you could not find them matched to anything. The design is actually intentional. Libraries are supposed to be shared. Once SpectraST allows users to define their own modifications, people will possibly define conflicting modifications (e.g. the same mod token meaning different things). By forcing the user to always supply the spectrast.usermods that supposedly works with a particular library when it is searched, there will be no ambiguity. Henry On Wednesday, December 16, 2015 at 3:22:01 AM UTC+8, David Zhao wrote: > > Hi Sarah, > > Thank you for sharing your experience with SpectraST, as regards to the > library building, did you use the "lib2html" tool in TPP to display the > imported hit in the created library, and check if the hit you wanted are > actually in the library? > And is there a SpectraST folk here in the group can help us out with > user-defined modification library creation and search? > Thanks in advance! > > David > > On Tuesday, December 15, 2015 at 2:05:53 AM UTC-8, Sarah P. wrote: >> >> Hi David, >> >> I'm not able to solve your problem, but I can comfort you, you are not >> the only one with these kind of problems. Some months ago, I had similar >> problems and until today, I could not really solve them. But in the >> meantime, I tried a lot of different things. First, I tried to figure out >> where exactly my problem is located. And in some points, it seems to be >> similar to yours. So, this is what I did: >> >> I'm also working with a user defined modification. Searches with comet >> are successful, just as in your case. In my case, however, the next step >> was kind of a quality control as to the respective modification. So I >> performed the TPP workflow to get an interact.ipro.pep.xml. Afterwards, I >> used the PepXML Viewer to check on the spectral quality of the modified >> peptides and to check whether the TPP assigns the same spectrum to the same >> modified sequence than other search engines (comparison of original spectra >> out of raw files). So far, it works. >> >> Just like you, I tried to build the spectral library afterwards by >> implementing the modification with the user defined modification file you >> described. My file spectrast.usermods looks just like yours, only with a >> different mass and a different amino acid residue. When I build the >> spectral library with the command line of my computer, I also get a >> confirmation of a successful building process. But then, I have problems to >> work with the library. Normally, I filter the spectral library for >> probability and then review the consensus spectra of the modified peptides >> in my spectral library. But in this case, when I build a consensus library >> with only high probability spectra, there are no modified peptides anymore, >> so I cannot perform any searches of data against the library. So I removed >> the probability filter and build up a library. But when I try to evaluate >> this spectral library to check on the quality of the modified spectra, to >> look at the spectra itself or to check whether the modification is set up >> correctly, I have some problems: >> >> · The same spectra I could look at on the level of ipro.pep.xml >> (high probability, correct sequence, correct modification) cannot be found >> in the splib anymore. >> >> · In other cases, I find the same spectra now with very low >> probabilities. >> >> · In a third case, the same spectrum is now displayed as a >> spectrum containing another phosphorylation or another sequence (comparison >> of scan numbers). >> >> · Most of my reference spectra will be eliminated as soon as I >> filter the spectral library for high probability. >> >> · Just in case I ignore the differences between PepXML Viewer >> and Lorikeet Spectrum Viewer and I look at the peptides displayed in the >> table of the splib.html, there are some differences between the table and >> the spectrum I see when I click at the peptide in the table. For example, >> in your case, in the table, I see the sequence DLK[324]DYFTK. When I click >> on the spectrum, it will be displayed with the sequence DLK and without the >> modification. >> >> · To be more precise, the corresponding fragment ion series >> can’t be matched or assigned as they don’t belong to the sequence >> displayed either in the table or next to the spectrum of the Lorikeet >> Spectrum Viewer. The precursor mass of the spectrum, however, belongs to >> the sequence of the splib.html-table. The fragment masses, interestingly, >> are sometimes even higher than the corresponding precursor masses. >> >> · My next step will be to find out to which sequence the >> displayed fragment ion series could belong, but even if I can figure this >> out, I don’t know how this could help. >> >> · Additionally, when I compare just the raw spectrum (the >> spectrum with the same scan number than the spectrum the library took for >> the display. I extracted the necessary information directly out of the xml >> files.) with the results of other programs, the other softwares will find >> another sequence belonging to this raw spectrum. I did not work with a >> consensus library when comparing different softwares of course. >> >> · To complete the view, I just searched for any modified >> peptides of the interact.ipro.pep.xml in the spectra of the splib.html, but >> I found none. I clicked at every single Lib-ID that corresponds to a >> modified peptide and the problem is always the same. >> >> I repeated these tests with different data sets, different versions of >> the TPP and of spectrast, different formats of spectrast.usermods, >> different locations of the TPP (server based and local) and also different >> user defined modifications. The problem is always the same. >> >> >> >> So, I’m sorry that I cannot really help you, but maybe I can guess that >> there is no problem with your data set or the way you perform your search >> against the library, but only with the user defined modification in the >> spectral library. >> >> >> >> Is there anyone else out there having similar problems or maybe already a >> solution for it? >> >> >> >> Thanks in advance! >> ... > > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To unsubscribe from this group and stop receiving emails from it, send an email to spctools-discuss+unsubscr...@googlegroups.com. To post to this group, send email to spctools-discuss@googlegroups.com. Visit this group at https://groups.google.com/group/spctools-discuss. For more options, visit https://groups.google.com/d/optout.
