Hi Thanks for the reply, No, there was no crash, I'll try the different with the different mass. This iTRAQ data set had no replicates, but since then I've received a data set from a similar experiment, but with replicates and labelled with TMT6plex. I used the TPP workflow consting of X!tandem, peptide- and protein prophet and Libra and everything ran smoothly I'm looking at the quantitation.tsv file now :)
Which brings me to my next question, There were two groups, each with three replicates. But each replicate had a different mass tag, so group one consited of replicate1:TMT126 rep2:TMT127 and rep3:TMT128 group two consisted of rep1:TMT129 rep2:TMT130 and rep3:TMT131 I would like to compare the level of expression of a given protein in Group1 vs Group2 In the quantitation.tsv file I have 6 intensity values per peptide. Can I take the average intesity value of the 3 replicates for a given group and then normalise in a similar fashion as outlined by the Libra documentaion? or normalise first and then take an average? how should I proceed? Thank you in advance, Armin On Wednesday, 5 December 2012 01:58:50 UTC+2, Luis wrote: > > Hello, > By "unable to get it to work using Libra", so you mean that you get > bad results? Or does the software crash? > I think that you are using the iTRAQ-8 reagent kit, which carry a mass > of 304.205360 (see the Unimod entry for more detail); the mass you are > using (144.1) applies to the 4-channel version of the reagent. > > You can set the "normalization channel" in Libra as the channel that > you want to use as a reference (i.e. assign abundance ratio of 1, > adjust the other channels accordingly). > > Hope this helps, > --Luis > > > > On Fri, Nov 9, 2012 at 6:18 AM, Armin <[email protected] <javascript:>> > wrote: > > Hi all > > > > I’m performing the analysis of an iTRAQ data for the first time, I have > > files in .mzML format. The petides were labelled with two iTRAQ reagents > 117 > > and 119. > > > > I am able to run the data through the pipeline (X!Tandem, peptide > prophet , > > protein prophet ) without quantitation, however I am unable to get it to > > work using Libra. > > > > Firstly, I’ve added these lines to the “modifications” section of the > tandem > > parameters file: > > > > <note type="input" label="residue, modification > > mass">144.102063@[,144.102063@K</note> > > > > <note type="input" label="residue, potential modification > > mass">144.102063@Y</note> > > > > Is this correct? > > > > Secondly, for the Libra condition file I selected the commonly used > values > > for an iTRAQ 8-channel and deselected the labels that are not in my > dataset? > > > > this is the Libra condition file that I used: > > > > <?xml version="1.0" encoding="UTF-8"?> > > > > <SUMmOnCondition> > > > > <fragmentMasses> > > > > <reagent mz="117.1" /> > > > > <reagent mz="119.1" /> > > > > </fragmentMasses> > > > > <isotopicContributions> > > > > <contributingMz value="1"> > > > > <affected mz="2" correction="0" /> > > > > </contributingMz> > > > > <contributingMz value="2"> > > > > <affected mz="1" correction="0.0014" /> > > > > </contributingMz> > > > > </isotopicContributions> > > > > <massTolerance value="0.2" /> > > > > <centroiding type="2" iterations="1" /> > > > > <normalization type="-1" /> > > > > <targetMs level="2" /> > > > > <output type="1" /> > > > > <quantitationFile name="quantitation.tsv" /> > > > > <minimumThreshhold value="20" /> > > > > </SUMmOnCondition> > > > > > > Also, to what do I set the "Normalization channel" > > > > > > Any help and advice would be appreciated > > > > > > > > Thank you > > > > -- > > You received this message because you are subscribed to the Google > Groups > > "spctools-discuss" group. > > To view this discussion on the web visit > > https://groups.google.com/d/msg/spctools-discuss/-/2kwPGA4LyVsJ. > > To post to this group, send email to > > [email protected]<javascript:>. > > > To unsubscribe from this group, send email to > > [email protected] <javascript:>. > > For more options, visit this group at > > http://groups.google.com/group/spctools-discuss?hl=en. > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To view this discussion on the web visit https://groups.google.com/d/msg/spctools-discuss/-/IgfVjq8iM6cJ. 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.
