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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