Re: [spctools-discuss] combining multiple iprophet results / Run NSP in iprophet if processing through protein prophet afterwards?
I've uploaded the files (and some notes for interpretation) to Dropbox and hopefully you will have received a link? (I used a contact email from an old paper that is hopefully still valid!). Please let me know if you're unable to access the files / if you need any further files/info Thanks Pete On Thursday, 21 June 2018 11:11:58 UTC-7, David Shteynberg wrote: > > Hi Pete, > > Yes it would be helpful if I could take a look and you prot.xml and > pep.xml files. Can you put them on a shared drive and email me the link > directly? > > Thanks, > -David > > On Thu, Jun 21, 2018 at 11:05 AM, > > wrote: > >> Hi David, >> >> The 1% FDR I used is decoy-based, which works out as using a protein >> prophet cutoff of ~0.8 (in both cases). >> >> The dataset is large (~ 420,000 total spectra reported in the prot.xml) - >> perhaps this has an effect? >> >> Would it be useful for me to send across my prot.xml files (or pep.xmls?) >> >> Thanks >> Pete >> >> >> On Thursday, 21 June 2018 10:55:11 UTC-7, David Shteynberg wrote: >>> >>> Hi Pete, >>> >>> Thank you for the summary. I had a question about the 1% FDR. Is this >>> decoy-based or model-based? I am wondering what protein counts you will >>> observe when you compare at the 1% decoy-based FDR between running NSP in >>> both iProphet and ProteinProphet, running NSP only in iProphet and running >>> NSP only in ProteinProphet? >>> >>> Thank you, >>> -David >>> >>> >>> On Thu, Jun 21, 2018 at 10:39 AM, wrote: >>> Hi David, Thanks a lot for your reply. Your first answer makes a big difference - I had been combining iprophet results for a second round which had previously combined multiple search engines. By combining the results from different search engines only in the second iProphet run, my numbers are now more consistent irrespective of order of combination. Regarding the second point, switching NSP off in protein prophet (after running NSP in iProphet) makes quite a big difference to my final protein numbers (2632 entries versus 2336 at 1% FDR). Taking a look at the additional matches, they are all single peptide sequence hits - however, from manual inspection of several of these though, there are often multiple matches with high peptide prophet scores across several different biological replicates, and the spectra look good. Some of them do look like single matches though (albeit with good looking spectra). Given that both are 1% FDR, it's difficult to choose the most appropriate method to choose. From what I've said, I think the risk of false negatives is greater (running both NSP models) than the risk of false positives (running iprophet NSP only) - my thoughts are that it will be better to use NSP 'off' in protein prophet, but to consider protein IDs from single peptide sequence hits as being less confident. Thanks again for your help, Pete On Wednesday, 20 June 2018 13:29:06 UTC-7, David Shteynberg wrote: > > Hello Pete, > > I think the answer to your first question is it depends on the > specifics of your analysis. > > You can pass iProphet files through iProphet again, since it will just > use the PeptideProphet probabilities which are not modified (only > reported) > by iProphet. If the iProphet is from a single search engine this should > be > just fine. However, if the iProphet file contains results from multiple > search engines then you probably don't want to combine it with iProphet > again as in this case each spectrum search result comes from the highest > scoring search engine, so not all the information will be available for > iProphet in the second analysis. Also, for your large analysis that is > currently failing in Petunia you might consider running the tool on the > commandline. > > For your second question, the NSP model can be disabled on the > commandline using the NONSP flag. The ProteinProphet NSP model is > implemented differently in iProphet and in ProteinProphet. Although, in > theory applying the models both times could be problematic due to their > similarity, in practice the models are different enought that during > testing I have not observed deleterious effects from using both the > iProphet and the ProteinProphet NSP models. You can try running the tools > in different ways and comparing the performance. Using both NSP models > is > the current default and you would have to explicitly disable the models > when you run each tool. > > I hope this helps. > > -David > > > > > On Tue, Jun 19, 2018 at 5:29 PM, wrote: > >> Hi All, >> >> I have 2 questions I'd be grateful if people could help answer: >>
[spctools-discuss] combining multiple iprophet results / Run NSP in iprophet if processing through protein prophet afterwards?
Hi All, I have 2 questions I'd be grateful if people could help answer: 1) Is it valid to combine multiple iprophet.pep.xml files by passing through iprophet for a second time? - alternatively, is it valid to combine a single iprophet.pep.xml file with interact.pep.xml files in iprophet? I am trying to combine a lot of different experiments / search engines results etc, and have been combining in iprophet - but I appear to have maxed out the number interact.pep.xml files to pass into iprophet. Beyond a certain number of files (doesn't appear to be file-specific), iProphet fails. As a workaround, I wondered if I could simply run half of the files through iProphet, then combine the resulting file with the remaining files to be run, by running iprophet again prior to running protein prophet. - Would this be valid? 2) I attended a TPP course last year in which the course notes stated that NSP should be switched off in iProphet, if NSP model is to be used in protein prophet. I am using petunia (running protein prophet on the iprophet results), and I cannot see a NSP option in the protein prophet parameters. Does this mean that NSP is not being used when I run protein prophet? ... (i.e. am I ok to leave NSP on in iProphet?) Thanks Pete -- 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.
Re: [spctools-discuss] Re: No prot-MODELS
Hi Luis, I suspect that my problem may be due to multiple installations of TPP (from this post): https://groups.google.com/forum/#!msg/spctools-discuss/Tq-i-7SVoJE/4XZcq4EGCgAJ;context-place=forum/spctools-discuss I had originally installed 5.1 in a preferred location, but parts of TPP didn't run - so I uninstalled and installed at default path. Unknowingly I've done all my analysis in the location of what was the old installation. The temporary fix in the above thread didn't work for me when I tried to repeat it (substituting in the appropriate path). Please could you tell me is there a fix that you'd recommend? or will have I reinstall? (I'd prefer not to if possible - there's several days worth of searches etc in there and I'm concerned I'll mess up file paths). - would it help if I sent over an example interact.prot.xml file? Thanks Pete On Tuesday, 12 June 2018 15:47:47 UTC-7, pbell@gmail.com wrote: > > Hi Luis, > > I've got the same problem with my dataset (also a large dataset, combining > multiple experiments). - No models html file is generated. > > Please can you tell me if this issue was resolved? > > Thanks > Pete > > > On Monday, 7 May 2018 19:01:28 UTC-7, Luis wrote: >> >> Hi Heeyoun, >> >> Does it seem that the file is complete when you open it in the >> ProtXMLViewer? (can you see the last protein group, for example?) If so, >> maybe there is an issue with the script that generates the models html >> page. Are you able to share the interact.prot.xml file so I can test it >> locally? If so, feel free to email it to me directly. >> >> Cheers, >> --Luis >> >> >> On Mon, May 7, 2018 at 5:44 PM, Heeyoun Hwang wrote: >> >>> Hi, Luis >>> >>> Thank you for your kindness. >>> >>> I opened the ProtXMLViewer with interact.prot.xml file, but >>> interact.prot-MODEL.html file was not automatically generated. >>> >>> The MODEL.html file is not exist, so I cannot checked that is corrupt or >>> broken and I cannot delete it. >>> >>> In other words, PepXMLViewer can generate interact.pep-MODEL.html file. >>> PepXMLViewer is fine. >>> >>> This interact.prot.xml has 47k protein entries. Is this too many number >>> to generate the model? >>> >>> Best, >>> >>> Heeyoun >>> >>> >>> >>> >>> 2018년 5월 8일 화요일 오전 7시 24분 29초 UTC+9, Luis 님의 말: Hello, The MODELS.html file should be auto-generated when you open the ProtXMLViewer (if one does not exist). If for some reason the models file already exists but is somehow not displaying or is corrupt/broken, the easiest thing to do is to delete that models file and re-open the ProtXMLViewer. Let us know if that still fails, and of any errors that you may notice in this case. Hope this helps, --Luis On Fri, May 4, 2018 at 2:55 AM, Heeyoun Hwang wrote: > Hi, all again, > > Is there anybody to help me, please? > > I want to get prot-MODELS.html file which was not automatically > generated. > > Thank you. > > Heeyoun Hwang > > 2018년 5월 1일 화요일 오후 5시 12분 52초 UTC+9, Heeyoun Hwang 님의 말: > >> Hi, >> >> I have to use TPP for large DATA sets including 24 RAW files. >> >> Most search process have been worked well, but prot-MODELS.html file >> is not generated. >> >> Surely, interact.prot.xml and its index is good. >> >> How can I get the MODELS.html file? >> >> Regards, >> >> Heeyoun Hwang >> >> >> >> -- > 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-discu...@googlegroups.com. > To post to this group, send email to spctools...@googlegroups.com. > Visit this group at https://groups.google.com/group/spctools-discuss. > For more options, visit https://groups.google.com/d/optout. > -- >>> 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-discu...@googlegroups.com. >>> To post to this group, send email to spctools...@googlegroups.com. >>> Visit this group at https://groups.google.com/group/spctools-discuss. >>> For more options, visit https://groups.google.com/d/optout. >>> >> >> -- 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.
Re: [spctools-discuss] Valid combination of multiple samples, fractions and digestions with different enzymes, using prophets
Please note - I've discovered a similar problem (no models html file generated) in the following thread: https://groups.google.com/forum/#!searchin/spctools-discuss/interact$20prot%7Csort:date/spctools-discuss/Yrg2yEO4otg/8u0bNKzpAAAJ Cheers Pete On Monday, 11 June 2018 14:58:53 UTC-7, pbell@gmail.com wrote: > > Hi David, > > Thanks for your advice - I've had success using the experiment flag in > Petunia, and have found that using an experiment label including fraction, > replicate and also enzyme (used for digest) works well when processed > through iProphet/protein prophet. > > However, my problem now is that the 'models' tab when I open the resulting > interact.ipro.prot.xml file is not visible - so I don't know where to set > the cutoff for 1% FDR > > I notice in my .params folder that there is no interact.ipro.prot html > file - but no errors appear while protein prophet is running. > > Please could you shed any light on how I can make the models visible? > Perhaps I've deleted some file in the .params folder that's required to > generate the html? > > Thanks > Pete > > > > On Wednesday, 6 June 2018 00:34:47 UTC-7, David Shteynberg wrote: >> >> Hello Pete, >> >> iProphet has a sibling experiments model and uses the replicate spectra >> model for replicate PSMs that are in the same experiment.This is enabled by >> running InteractParser with -X flag, which labels the >> spectra in the pepXML file. If you are using xinteract or Petunia web >> interface the option is -E. You have to make sure that >> for each search engine analysis you assign the same label to the same >> data. The experiment label is flexible and allows you to separate the data >> into "experiments" as defined by you. It makes sense in your case to make >> the experiment labels either the "fraction_name" or the >> "fraction_name"+"replicate". Other than that I think you are on the right >> path. >> >> Cheers, >> -David >> >> On Tue, Jun 5, 2018 at 3:13 PM, wrote: >> >>> Hi, >>> >>> I'd really appreciate advice regarding the most valid way to combine my >>> searches with peptide / i / protein prophet. >>> >>> I have 3 samples, 3 fractions per sample, and each fraction was digested >>> with multiple enzymes. Each of these digests were injected twice. >>> >>> The resulting data were then searched with different search engines; all >>> in an attempt to increase number of protein IDs. >>> >>> My idea of the workflow was as follows: >>> >>>1. combine results of 1 search engine for duplicate injections of a >>>single fraction using peptide prophet >>>2. combine results of multiple search engines using iprophet >>>3. combine iprophet results from different enzymatic digestions of a >>>single fraction of a single sample using protein prophet (to group >>> sibling >>>peptides) >>> >>> I'm unclear whether/when it is valid for me to combine: >>> a) different fractions (note - fractions are expected to have some >>> overlap in peptide and protein IDs) >>> b) different samples (note- samples are biological replicates, and are >>> expected to contain the same peptides / proteins) >>> >>> The reason I would like to combine them all together, is so that I can >>> have a single protein FDR for the whole experiment. >>> >>> Thanks! >>> Pete >>> >>> >>> >>> -- >>> 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-discu...@googlegroups.com. >>> To post to this group, send email to spctools...@googlegroups.com. >>> Visit this group at https://groups.google.com/group/spctools-discuss. >>> For more options, visit https://groups.google.com/d/optout. >>> >> >> -- 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.
Re: [spctools-discuss] Re: No prot-MODELS
Hi Luis, I've got the same problem with my dataset (also a large dataset, combining multiple experiments). - No models html file is generated. Please can you tell me if this issue was resolved? Thanks Pete On Monday, 7 May 2018 19:01:28 UTC-7, Luis wrote: > > Hi Heeyoun, > > Does it seem that the file is complete when you open it in the > ProtXMLViewer? (can you see the last protein group, for example?) If so, > maybe there is an issue with the script that generates the models html > page. Are you able to share the interact.prot.xml file so I can test it > locally? If so, feel free to email it to me directly. > > Cheers, > --Luis > > > On Mon, May 7, 2018 at 5:44 PM, Heeyoun Hwang > wrote: > >> Hi, Luis >> >> Thank you for your kindness. >> >> I opened the ProtXMLViewer with interact.prot.xml file, but >> interact.prot-MODEL.html file was not automatically generated. >> >> The MODEL.html file is not exist, so I cannot checked that is corrupt or >> broken and I cannot delete it. >> >> In other words, PepXMLViewer can generate interact.pep-MODEL.html file. >> PepXMLViewer is fine. >> >> This interact.prot.xml has 47k protein entries. Is this too many number >> to generate the model? >> >> Best, >> >> Heeyoun >> >> >> >> >> 2018년 5월 8일 화요일 오전 7시 24분 29초 UTC+9, Luis 님의 말: >>> >>> Hello, >>> >>> The MODELS.html file should be auto-generated when you open the >>> ProtXMLViewer (if one does not exist). If for some reason the models file >>> already exists but is somehow not displaying or is corrupt/broken, the >>> easiest thing to do is to delete that models file and re-open the >>> ProtXMLViewer. >>> >>> Let us know if that still fails, and of any errors that you may notice >>> in this case. >>> >>> Hope this helps, >>> --Luis >>> >>> >>> On Fri, May 4, 2018 at 2:55 AM, Heeyoun Hwang >>> wrote: >>> Hi, all again, Is there anybody to help me, please? I want to get prot-MODELS.html file which was not automatically generated. Thank you. Heeyoun Hwang 2018년 5월 1일 화요일 오후 5시 12분 52초 UTC+9, Heeyoun Hwang 님의 말: > Hi, > > I have to use TPP for large DATA sets including 24 RAW files. > > Most search process have been worked well, but prot-MODELS.html file > is not generated. > > Surely, interact.prot.xml and its index is good. > > How can I get the MODELS.html file? > > Regards, > > Heeyoun Hwang > > > > -- 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-discu...@googlegroups.com. To post to this group, send email to spctools...@googlegroups.com. Visit this group at https://groups.google.com/group/spctools-discuss. For more options, visit https://groups.google.com/d/optout. >>> >>> -- >> 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-discu...@googlegroups.com . >> To post to this group, send email to spctools...@googlegroups.com >> . >> Visit this group at https://groups.google.com/group/spctools-discuss. >> For more options, visit https://groups.google.com/d/optout. >> > > -- 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.
Re: [spctools-discuss] Valid combination of multiple samples, fractions and digestions with different enzymes, using prophets
Hi David, Thanks for your advice - I've had success using the experiment flag in Petunia, and have found that using an experiment label including fraction, replicate and also enzyme (used for digest) works well when processed through iProphet/protein prophet. However, my problem now is that the 'models' tab when I open the resulting interact.ipro.prot.xml file is not visible - so I don't know where to set the cutoff for 1% FDR I notice in my .params folder that there is no interact.ipro.prot html file - but no errors appear while protein prophet is running. Please could you shed any light on how I can make the models visible? Perhaps I've deleted some file in the .params folder that's required to generate the html? Thanks Pete On Wednesday, 6 June 2018 00:34:47 UTC-7, David Shteynberg wrote: > > Hello Pete, > > iProphet has a sibling experiments model and uses the replicate spectra > model for replicate PSMs that are in the same experiment.This is enabled by > running InteractParser with -X flag, which labels the > spectra in the pepXML file. If you are using xinteract or Petunia web > interface the option is -E. You have to make sure that > for each search engine analysis you assign the same label to the same > data. The experiment label is flexible and allows you to separate the data > into "experiments" as defined by you. It makes sense in your case to make > the experiment labels either the "fraction_name" or the > "fraction_name"+"replicate". Other than that I think you are on the right > path. > > Cheers, > -David > > On Tue, Jun 5, 2018 at 3:13 PM, > wrote: > >> Hi, >> >> I'd really appreciate advice regarding the most valid way to combine my >> searches with peptide / i / protein prophet. >> >> I have 3 samples, 3 fractions per sample, and each fraction was digested >> with multiple enzymes. Each of these digests were injected twice. >> >> The resulting data were then searched with different search engines; all >> in an attempt to increase number of protein IDs. >> >> My idea of the workflow was as follows: >> >>1. combine results of 1 search engine for duplicate injections of a >>single fraction using peptide prophet >>2. combine results of multiple search engines using iprophet >>3. combine iprophet results from different enzymatic digestions of a >>single fraction of a single sample using protein prophet (to group >> sibling >>peptides) >> >> I'm unclear whether/when it is valid for me to combine: >> a) different fractions (note - fractions are expected to have some >> overlap in peptide and protein IDs) >> b) different samples (note- samples are biological replicates, and are >> expected to contain the same peptides / proteins) >> >> The reason I would like to combine them all together, is so that I can >> have a single protein FDR for the whole experiment. >> >> Thanks! >> Pete >> >> >> >> -- >> 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-discu...@googlegroups.com . >> To post to this group, send email to spctools...@googlegroups.com >> . >> Visit this group at https://groups.google.com/group/spctools-discuss. >> For more options, visit https://groups.google.com/d/optout. >> > > -- 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.
Re: [spctools-discuss] Valid combination of multiple samples, fractions and digestions with different enzymes, using prophets
Thanks David, I wasn't aware of the experiment label - that sounds ideal. I'll give it a try On Wednesday, 6 June 2018 00:34:47 UTC-7, David Shteynberg wrote: > > Hello Pete, > > iProphet has a sibling experiments model and uses the replicate spectra > model for replicate PSMs that are in the same experiment.This is enabled by > running InteractParser with -X flag, which labels the > spectra in the pepXML file. If you are using xinteract or Petunia web > interface the option is -E. You have to make sure that > for each search engine analysis you assign the same label to the same > data. The experiment label is flexible and allows you to separate the data > into "experiments" as defined by you. It makes sense in your case to make > the experiment labels either the "fraction_name" or the > "fraction_name"+"replicate". Other than that I think you are on the right > path. > > Cheers, > -David > > On Tue, Jun 5, 2018 at 3:13 PM, > wrote: > >> Hi, >> >> I'd really appreciate advice regarding the most valid way to combine my >> searches with peptide / i / protein prophet. >> >> I have 3 samples, 3 fractions per sample, and each fraction was digested >> with multiple enzymes. Each of these digests were injected twice. >> >> The resulting data were then searched with different search engines; all >> in an attempt to increase number of protein IDs. >> >> My idea of the workflow was as follows: >> >>1. combine results of 1 search engine for duplicate injections of a >>single fraction using peptide prophet >>2. combine results of multiple search engines using iprophet >>3. combine iprophet results from different enzymatic digestions of a >>single fraction of a single sample using protein prophet (to group >> sibling >>peptides) >> >> I'm unclear whether/when it is valid for me to combine: >> a) different fractions (note - fractions are expected to have some >> overlap in peptide and protein IDs) >> b) different samples (note- samples are biological replicates, and are >> expected to contain the same peptides / proteins) >> >> The reason I would like to combine them all together, is so that I can >> have a single protein FDR for the whole experiment. >> >> Thanks! >> Pete >> >> >> >> -- >> 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-discu...@googlegroups.com . >> To post to this group, send email to spctools...@googlegroups.com >> . >> Visit this group at https://groups.google.com/group/spctools-discuss. >> For more options, visit https://groups.google.com/d/optout. >> > > -- 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.
Re: [spctools-discuss] Peptide prophet - use of decoy database for X! searches?
Hi David, Thanks for your reply, that's very helpful. I've now tried including an equal number of decoys (reversed) in my database, processing the same data through peptide prophet, iprophet and protein prophet. My number of identified proteins has gone down, but the PSMs are much more believable (judging from the spectra). As an aside - if I were to play with the proportion of decoys in the database, (reducing the number to say 25% of the total database, rather than 50%), in your experience would this reduction in the search space be likely to have much of an effect on PSM / protein IDs? Thanks again, Pete On Tuesday, 5 June 2018 16:26:37 UTC-7, David Shteynberg wrote: > > Hello Pete, > > Use of decoys in the X!Tandem workflow is not strictly required but it is > helpful. X!Tandem is one of a handful of search engines for which there > exists a parametric mixture model for PSM-level validation. In my > experience, using the parametric models may not always give you the best > results in terms of validation, and the lack of decoys precludes the > ability to compute decoy-based error rates (FDR). In this case you have to > only the FDR as estimated by the PeptideProphet model, and you cannot > compare this FDR to decoy-based estimates. The best results are usually > obtained by using a database that includes some proportion of decoys. You > can then validate the search results using PeptideProphet's semi-supervised > semi-parametric models, which tend to estimate more accurate error rates. > However you decide to search your data, I also suggest you also include > iProphet in your validation analysis for peptide-level validation. > > Cheers, > -David > > On Tue, Jun 5, 2018 at 12:47 PM, > > wrote: > >> Please can someone advise - is it recommended to include decoys in my >> database when using X! for searches then peptide/protein prophet in TPP? >> >> >> As I understand, X! recommends not including decoys in searches (when >> used standalone) – however when I analyse X! identified peptides using >> peptide/protein prophet, I assume that I will need decoy matches to help >> pin down the correct distribution? >> >> >> Thanks >> >> Pete >> >> -- >> 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-discu...@googlegroups.com . >> To post to this group, send email to spctools...@googlegroups.com >> . >> Visit this group at https://groups.google.com/group/spctools-discuss. >> For more options, visit https://groups.google.com/d/optout. >> > > -- 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.
[spctools-discuss] Valid combination of multiple samples, fractions and digestions with different enzymes, using prophets
Hi, I'd really appreciate advice regarding the most valid way to combine my searches with peptide / i / protein prophet. I have 3 samples, 3 fractions per sample, and each fraction was digested with multiple enzymes. Each of these digests were injected twice. The resulting data were then searched with different search engines; all in an attempt to increase number of protein IDs. My idea of the workflow was as follows: 1. combine results of 1 search engine for duplicate injections of a single fraction using peptide prophet 2. combine results of multiple search engines using iprophet 3. combine iprophet results from different enzymatic digestions of a single fraction of a single sample using protein prophet (to group sibling peptides) I'm unclear whether/when it is valid for me to combine: a) different fractions (note - fractions are expected to have some overlap in peptide and protein IDs) b) different samples (note- samples are biological replicates, and are expected to contain the same peptides / proteins) The reason I would like to combine them all together, is so that I can have a single protein FDR for the whole experiment. Thanks! Pete -- 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.
[spctools-discuss] Peptide prophet - use of decoy database for X! searches?
Please can someone advise - is it recommended to include decoys in my database when using X! for searches then peptide/protein prophet in TPP? As I understand, X! recommends not including decoys in searches (when used standalone) – however when I analyse X! identified peptides using peptide/protein prophet, I assume that I will need decoy matches to help pin down the correct distribution? Thanks Pete -- 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.
