Hi Mike Alright, that made it much more clear :)
So for a given successfully identified MS2 spectrum, *i* is the sum of intensities of all b and y ions that contributed to the identification. Therefore one would need to have access to the MS2 intensities when computing the SI, and not just the MS1, if I am correct. In the Griffin paper <http://peak height of the MS/MS fragments are summed for SIN, whereas for the AUC, the precursor ions are integrated. Overlapping peptides in the precursor/ MS scan increases the chance for error with the integration process.>, it is mentioned Using the fragment ion intensity, specifically only those intensities that > match the peptide of interest, may facilitate more accurate measurements as > there is less chance of including the signal of co-eluting precursors or > background noise. Peak height of the MS/MS fragments are summed for SIN, whereas for the AUC, > the precursor ions are integrated. Overlapping peptides in the precursor/MS > scan increases the chance for error with the integration process. I guess these are some of the advantages of using the MS2 intensities over MS1. Please correct me if I am wrong. Thanks for your help! Cheers Antonio lørdag den 2. juni 2018 kl. 23.43.04 UTC+2 skrev Michael Hoopmann: > > Hi Antonio, > Sure, I can see where there may be some confusion. Peptide k is not the > precursor ion, it is merely a sequence identification. That sequence is > quantified based on the MS/MS spectra that match to it. > > For example, suppose after doing peptide validation and protein > inference, you identify albumin with peptides SEIAHR and DLGEEHFK. > Thus, for albumin, pn=2, where k[1]=SEIAHR and k[2]=DLGEEHFK. > > Now for k[1]=SEIAHR, there may be three MS/MS spectra acquired that > produced that peptide sequence identification. Therefore sc=3. If you go > into each of those spectra, you can identify the b-ion peaks and y-ion > peaks that were evidence for the peptide identification. If you sum the > intensity of those b- and y-ions, then you have the spectral index for that > spectrum. > > Summing together all the spectral indexes for each of the spectra used as > evidence for a protein produces the spectral index of the protein. > > I hope that clarifies everything. > > Cheers, > Mike > > On Saturday, June 2, 2018 at 8:58:42 AM UTC-7, Antonio Ortega wrote: >> >> Hi Mike >> >> Thanks for your fast answer! I am sorry I couldn't get back to you >> earlier. >> I understand protein inference must be carried out prior to running >> StPeter. However, I still don't understand the Spectral Index (SI) >> definition from the original Griffin paper >> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805705/ . I would >> appreciate if you could shed some light. >> >> The spectral index (SI) for a protein is the cumulative fragment ion >>> intensity for each spectrum of each peptide giving rise to a protein, which >>> can be represented using the equation from Griffin et al. where *i* is >>> the fragment ion intensity of peptide *k*, *j* is the *j*th spectral >>> count of *sc* total spectral counts for peptide *k* and *pn* is the >>> number of peptides identified for that protein >> >> >> >> <https://lh3.googleusercontent.com/-fxXf4Vlx0m4/WxJHqr8AafI/AAAAAAAANwQ/uaPdP9JDycE4hCuE0amGXvJv_hYcLpUVACLcBGAs/s1600/SI.png> >> >> >> I don't understand what's the fragment ion intensity of peptide *k. *Please >> correct me if I am wrong here. Peptide *k* is a precursor ion and it has >> a single intensity (measured in MS1). After going through MS1, it fragments >> generating multiple fragment ions. So according to my understanding, >> there's a single precursor ion intensity and several fragment ion >> intensities for peptide *k*. Therefore, I don't comprehend what's *i* in >> the equation above. >> >> >> I understand that *i* is summed over for all *sc *spectra identified as >> coming from peptide *k,* and then for all *pn* peptides coming from >> protein *K.* This total sum whould make protein's K SI. >> >> >> Thanks for your help and time!! :) >> >> >> Best >> >> >> Antonio >> >> >> onsdag den 23. maj 2018 kl. 01.05.15 UTC+2 skrev Michael Hoopmann: >>> >>> Hi Antonio, >>> To use StPeter, you must perform peptide validation and protein >>> inference first. These are done, minimally, with the PeptideProphet and >>> ProteinProphet tools, respectively. They are needed because StPeter >>> quantifies proteins, not peptides, and thus you need to first perform >>> protein inference. >>> >>> We do have a tutorial for StPeter, sorry if our links don't line up >>> precisely from the launch location you started at. Updating the >>> documentation site is a work in progress. Try here: >>> http://tools.proteomecenter.org/wiki/index.php?title=Tutorial:StPeter1 >>> <http://www.google.com/url?q=http%3A%2F%2Ftools.proteomecenter.org%2Fwiki%2Findex.php%3Ftitle%3DTutorial%3AStPeter1&sa=D&sntz=1&usg=AFQjCNEogKp8slamhq2lozPqyziMcnFZyA> >>> >>> Unfortunately, it does assume using the GUI. StPeter (and the other TPP >>> tools) should all have command line usage statements if you simply type >>> them without parameters. But it is easiest to try solving your Apache2 >>> problems. >>> >>> And regarding your last question, no, the precursor intensity is never >>> used as part of StPeter. The quantification is done entirely with spectral >>> indexes which utilize the intensity of fragment ions, not precursor ions. >>> >>> Cheers, >>> Mike >>> >>> On Saturday, May 19, 2018 at 5:03:25 PM UTC-7, Antonio Ortega wrote: >>>> >>>> Hi all! My name is Antonio Ortega, and I am a Master student in >>>> Bioinfomatics at the University of Copenhagen, currently doing my Master >>>> Thesis on proteomics and mass spectrometry. >>>> >>>> I was wondering how can I use StPeter as a quantification tool if I >>>> pass as input the output of search engines like MSGF or Comet. I can see >>>> in >>>> the tutorial that Stpeter receives as input protXML files, however Comet >>>> produces pepXML output and MSGF produces mzid files that apparently can be >>>> converted to pepXML with idconvert >>>> >>>> https://groups.google.com/forum/#!topic/spctools-discuss/tbkcovbFxEc >>>> >>>> Why is the intermediate proXML file produced by PeptideProphet, >>>> iProphet and ProteinProphet needed? Is there any step by step tutorial on >>>> how to use these tools from the command line? The tutorial here >>>> http://tools.proteomecenter.org/wiki/index.php?title=TPP_Tutorial >>>> applies for people using the GUI Petunia, however, Apache2 gives me a >>>> forbidden access error when I try to access the TPP port., so I cannot >>>> follow the instructions there. >>>> >>>> Regarding the method, the fragment ion intensity of the j spectrum of >>>> peptide k corresponds to the intensity of the precursor that generated >>>> spectrum j right? And this number can be found in .mgf spectrum files as >>>> the second number in the PEPMASS field right? >>>> PEPMASS=XXXX YYY >>>> being YYY equal to the intensity. >>>> >>>> Thank you very much for your help in advance and keep up the good work! >>>> >>>> Best regards >>>> >>>> >>>> Antonio >>>> >>>> >>> -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. 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