Ben, There are two separate searches, one with light mods static, one with heavy mods static. We use a front-end to the TPP which does all the steps, including translating unimod PTM specifications into search engine mods, so I've just run a test and looked up exactly what Tandem is using. These searches also use fixed Carbamidomethyl (C) and variable Oxidation (M).
Light Search <note type="input" label="residue, modification mass">28.0...@k, 28.0...@[,57.0...@c,</note> <note type="input" label="residue, potential modification mass">15.9...@m,</note> Heavy Search <note type="input" label="residue, modification mass">32.0...@k, 32.0...@[,57.0...@c,</note> <note type="input" label="residue, potential modification mass">15.9...@m,</note> The output from the searches is combined using xinteract. The ASAPRatio command lines are: /wwwdata/pipeline/live/extern/tpp_4.3.1/bin/ASAPRatioPeptideParser 'interact.pep.xml.gz' -lnK -S -r0.02 /wwwdata/pipeline/live/extern/tpp_4.3.1/bin/ ASAPRatioProteinRatioParser 'interact.prot.xml.gz' /wwwdata/pipeline/live/extern/tpp_4.3.1/bin/ASAPRatioPvalueParser 'interact.prot.xml.gz' Cheers, -- Dr. David Trudgian Bioinformatician in Proteomics University of Oxford Mon-Thu: CCMP, Roosevelt Drive Tel: (+44) (01865 2)87784 Friday : Dunn School of Pathology, S. Parks Rd. Tel: (+44) (01865 2)75557 On 5 May, 16:35, Ben Collins <[email protected]> wrote: > Hi DT, > > I'm trying to use dimethylation labelled samples with TPP but I think > I might not be specifying the modifications correctly. Is there any > chance that you could post the relevant sections from your search > params file (preferably X!Tandem if you're using this)? > > Many thanks, > Ben > > On Mar 19, 11:10 am, dctrud <[email protected]> wrote: > > > > > Bernard, > > > Sorry for the slow reply. ASAPRatio will know which label is heaviest, > > it shouldn't matter which way around the searches are. > > > With regard to the questions r.e.dimethylfalse positives, the > > procedure we are using this for involves labeling for quantitation > > where all lysines should be dimethylated / heavy dimethylated. I > > probably wrongly assumed you were doing this labeling, whereas I guess > > you are not forcing complete dimethylation of lysines? What exactly is > > your experiment? What biological changes are you trying to measure? > > > DT > > > On 11 Mar, 17:59, Bernt <[email protected]> wrote: > > > > Hi DT, > > > > Really grateful for your advice. I get what you mean, I have 2 more > > > concerns: can we change which form to be the numerator and which form > > > to be the denominator? When we specify -A-lnK-S, would the ratio be > > > Search1 / Search 2 or Search 2 / Search 1? Do you get Peptide Prophet > > > probabilities when you place a static mod on Lys? > > > > Another interesting point for discussion in methylation: would you be > > > able to distinguishdimethylLys from Arginine? Would placing static > > > mod ofdimethylon Lys give you many false positives which might be > > > just Arginines instead? How do you manage them? > > > > Bernard > > > > P.S: Has anybody come across methylated tryptophan W? > > > > On Mar 11, 10:00 pm, dctrud <[email protected]> wrote: > > > > > Bernard, > > > > > We've successfully used ASAPRatio for heavydimethyllabeled > > > > quantitation using the following workflow, which might help you get to > > > > where you want: > > > > > * Run *two* searches, with the light and heavy methyl n-term and > > > > lysine mods specified as *static* modifications, i.e. > > > > > Search 1:Dimethyl(K) +Dimethyl(n-term) as static mods. > > > > Search 2:Dimethyl:2H4 (K) +Dimethyl:2H4 (n-term) as static mods. > > > > > * Run xinteract on the resulting pair of search results using the > > > > static mods option for ASAPRatio, i.e. > > > > > -A-lnK-S-r0.05 > > > > > There's no need to run XPRESS if you are only interested in ASAPRatio > > > > results. > > > > > DT > > > > > On 11 Mar, 07:05, Bernt <[email protected]> wrote: > > > > > > Dear all, > > > > > > I have a question pertaining to ASAPRatio. > > > > > > Can I actually get ASAPRatio to give me a ratio of Kmethylated / > > > > > Kheavy > > > > > +methylated (i.e. K142/K148)? If it can be done, how do I specify on > > > > > the commandline different modified forms of Lys as the numerator and > > > > > denominator of the ratio? Both modifications have been specified under > > > > > variable mods in Xtandem. Does Xpress need to be specified everytime > > > > > ASAPRatio is run, or can it be omitted? Can the following work? > > > > > > xinteract -NinteractHeavyMethyl2.pep.xml -p0 -l6 -OA -A- > > > > > lR170.11676K142.11061-F-r0.1-mK148.13061R180.12476 > > > > > "*HeavyMethyl.tandem.pep.xml" > > > > > > I also tried specifying static mod on Lys (i.e. 1...@k), and variable > > > > > mod of 2...@k to represent the Heavy+Methyl form in Xtandem first, > > > > > then : > > > > > > xinteract -Ninteractstatic.pep.xml -p0 -l6 -OA -X-nR,10K,6 -A-lRK-F- > > > > > r0.1-mK148.13061R180.12476 "*static.tandem.pep.xml" > > > > > > But in this case, PeptideProphet's probabilities were all 0, > > > > > presumably because there were very few significant peptide assignments > > > > > due to the static mod specified on all Lys. Does anybody have any idea > > > > > how I can get around this problem with ASAPRatio? > > > > > > I look forward to any suggestions. Thanks in advance. > > > > > > Cheers, > > > > > Bernard > > -- > You received this message because you are subscribed to the Google Groups > "spctools-discuss" group. > To post to this group, send email to [email protected]. > To unsubscribe from this group, send email to > [email protected]. > For more options, visit this group > athttp://groups.google.com/group/spctools-discuss?hl=en. -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To post to this group, send email to [email protected]. To unsubscribe from this group, send email to [email protected]. For more options, visit this group at http://groups.google.com/group/spctools-discuss?hl=en.
