Thank you David, everything is clear. I will write to the PEAKS support team tomorrow.
Best, Sergio -------------------------- Sergio Ciordia Higuera Proteomics Facility National Center for Biotechnology C\Darwin, 3 Universidad Autónoma de Madrid Cantoblanco 28049 Madrid (Spain) Phone: +34 91 585 4540 / 4695 Fax: +34 91 585 4506 El dom, 13 oct 2024 a las 5:52, David Shteynberg (< [email protected]>) escribió: > Hello Sergio, > > My answers are in bold: > > *- Should the file collect all the PSMs or only the peptides?.* > > * If we are trying to replicate how TPP processes other search engines > then, yes collect all PSMs without filtering and let the TPP process the > pepXML.* > > *- Should the file contain the decoys? In this way, it would not be > necessary to use the strategy of the ‘unknown decoys’ or yes?* > > *The results should contains the ‘unknown decoys’, any ‘known decoys’ that > are exposed to the search engine are no longer reliable as independent > indicators of false positive results and should be excluded (ignored) from > further FDR estimates.* > > *- They have to correct the calculated neutral masses and mass > differences bug* > > *Yes!* > > *- Should the pepXML file be filtered by FDR?. This last point seems > important to me because the file I gave you seems to me to be filtered by > 1% FDR at the PSM level (you can't change it unless you repeat the search > in PEAKS and set a less strict value). In case the pepXML file has to be > exported with an FDR value, what should it be for the TPP analysis?* > > *No. You can have all results exported from PEAKS (maximum FDR without > filtering) and let TPP establish the thresholds for FDR. You can then > apply TPP based FDR thresholds without rerunning the analysis (assuming the > minimum probability for reported PSMs is set to zero.)* > > Best, > -David > > > On Oct 12, 2024, at 12:41 AM, Sergio Ciordia <[email protected]> wrote: > > Thank you very much David for such a thorough analysis, for the effort and > your time. I agree with you that there seems to be a bug in the pepXML > export. I'm going to write to support to see if they can fix it (and fast). > Since I'm going to write to them, what exactly would we need to do the > analysis with TPP? For example: > > - Should the file collect all the PSMs or only the peptides?. > - Should the file contain the decoys? In this way, it would not be > necessary to use the strategy of the ‘unknown decoys’ or yes?. > - They have to correct the calculated neutral masses and mass > differences bug. > - Should the pepXML file be filtered by FDR?. This last point seems > important to me because the file I gave you seems to me to *be filtered > by 1% FDR at the PSM level *(you can't change it unless you repeat the > search in PEAKS and set a less strict value). In case the pepXML file has > to be exported with an FDR value, what should it be for the TPP analysis? > > Let's see if we can get it to work. Thanks again for all your help in > trying to implement PEAKS in TPP and all the comparative analysis with > Comet. > > Best regards, > Sergio > > -------------------------- > Sergio Ciordia Higuera > Proteomics Facility > National Center for Biotechnology > C\Darwin, 3 > Universidad Autónoma de Madrid > Cantoblanco > 28049 Madrid (Spain) > Phone: +34 91 585 4540 / 4695 > Fax: +34 91 585 4506 > > > El sáb, 12 oct 2024 a las 2:18, David Shteynberg (< > [email protected]>) escribió: > >> Hello Sergio, >> >> After having a much closer look at this data I am having some doubts >> regarding the data as it is reported in the PEAKS pepXML output. First of >> all I noticed that the mass differences and the calculated neutral masses >> were off in the pepXML export, for example: >> >> <PastedGraphic-1.png> >> >> So I modified my tools to allow recomputing the calculated neutral masses >> and mass differences. Here is this entry after I run my tool: >> >> <PastedGraphic-2.png> >> >> Although the calculated neutral mass is now correct, the mass difference >> for this PSM is quite large, and possibly represents another modification >> in this peptide that is not annotated by PEAKS for this PSM. The mass of >> 16 Da matches oxidation and I see many of these types of peptides, with a >> massdiff of 16 and containing a Methionine. >> >> >> This leads me to suspect a bug in the pepXML export of these PSMs from >> PEAKS. >> >> I was able to get quite good results running your mzML file through >> comet. These are the comet modifications I used in my search: >> >> <PastedGraphic-3.png> >> >> *<PastedGraphic-4.png>* >> >> >> Although I am uncertain that this will fix all of the issues I am seeing >> in this result, is it possible to rerun PEAKS using a similar set of >> modifications, I used here for comet? >> >> Another concern was that out of over 26,000 PSMs in the pepXML file only >> 48 are hits to the DECOY portion of the database. This seems a bit low to >> me, and I am not sure why that is. >> >> >> Here is the summary from the comet + TPP analysis of this run: >> <image.png> >> >> Comet + PeptideProphet is finding about 24000 correct PSMs at an >> error-rate less than 1%. >> >> Running iProphet on the data boost the PSM number higher, especially at >> the lower error-rates: >> >> <PastedGraphic-5.png> >> >> >> To summarize, comet+PeptideProphet+iProphet is finding about 24000 >> correct PSMs, mapping to almost 21000 peptide sequences in this file: >> >> <PastedGraphic-6.png> >> >> >> Perhaps if the PEAKS issues can be solved, a similar number can be found >> using that software, but for now the results coming out of PEAKS are not >> close. >> >> Best, >> -David >> >> >> >> >> >> >> On Oct 10, 2024, at 2:43 PM, Sergio Ciordia <[email protected]> wrote: >> >> Hi David, >> >> I have anticipated your answer and I think this is what you asked for. I >> have used TPP to generate the DECOY database using these settings: >> >> <image.png> >> >> I have searched PEAKS again and uploaded the new file ‘ >> PEAKS_target_decoy.pep.xml’ and the decoy database to the shared folder. >> >> Link: PEAKS Dataset >> <https://drive.google.com/drive/folders/1Q3LF1wRTM62KfS20dfXmToQMY9ji9Dug> >> >> See what you think of the output. >> >> Best, >> Sergio >> >> -------------------------- >> Sergio Ciordia Higuera >> Proteomics Facility >> National Center for Biotechnology >> C\Darwin, 3 >> Universidad Autónoma de Madrid >> Cantoblanco >> 28049 Madrid (Spain) >> Phone: +34 91 585 4540 / 4695 >> Fax: +34 91 585 4506 >> >> >> El jue, 10 oct 2024 a las 22:47, Sergio Ciordia (<[email protected]>) >> escribió: >> >>> OK, I think I understand you now. I think what you are asking me to do >>> is to create a database containing the target and decoy entries and repeat >>> the search. Then in the output that PEAKS gives us, we could locate the >>> ‘unknown’ decoy. Is this what you need?. >>> >>> Sergio >>> >>> -------------------------- >>> Sergio Ciordia Higuera >>> Proteomics Facility >>> National Center for Biotechnology >>> C\Darwin, 3 >>> Universidad Autónoma de Madrid >>> Cantoblanco >>> 28049 Madrid (Spain) >>> Phone: +34 91 585 4540 / 4695 >>> Fax: +34 91 585 4506 >>> >>> >>> El jue, 10 oct 2024 a las 22:33, 'David Shteynberg' via spctools-discuss >>> (<[email protected]>) escribió: >>> >>>> I think we have a bit of misunderstanding here. I am not looking for >>>> decoys that are "known" to PEAKS, I want to include entrapment decoys that >>>> are "unknown" to PEAKS and known to us as true negatives. Then we can >>>> utilized the true negatives to estimate error rates. The true negative >>>> entrapment decoys should not be revealed to the search algorithm, just like >>>> the false positives among the target sequences are not known to the >>>> algorithm, but represent the error we are trying to control. >>>> >>>> I hope this makes sense. >>>> >>>> -David >>>> >>>> On Thu, Oct 10, 2024 at 10:48 AM Sergio Ciordia <[email protected]> >>>> wrote: >>>> >>>>> Hi David, >>>>> >>>>> There are several commercial softwares such as PEAKS or Proteome >>>>> Discoverer that you upload the target database directly and they >>>>> internally >>>>> generate the Target-Decoy database. The problem is that when you launch >>>>> the >>>>> search and get the output, the software usually removes the decoy entries >>>>> from the final results and that's why they don't appear in the list. >>>>> >>>>> I understand then that we would need the same ‘pep.xml’ file but >>>>> containing all the target and decoy entries. Is that what you need?. >>>>> >>>>> Best, >>>>> Sergio >>>>> >>>>> -------------------------- >>>>> Sergio Ciordia Higuera >>>>> Proteomics Facility >>>>> National Center for Biotechnology >>>>> C\Darwin, 3 >>>>> Universidad Autónoma de Madrid >>>>> Cantoblanco >>>>> 28049 Madrid (Spain) >>>>> Phone: +34 91 585 4540 / 4695 >>>>> Fax: +34 91 585 4506 >>>>> >>>>> >>>>> El jue, 10 oct 2024 a las 19:40, 'David Shteynberg' via >>>>> spctools-discuss (<[email protected]>) escribió: >>>>> >>>>>> Hello Sergio, >>>>>> >>>>>> Thanks for sending this. After taking a look I have another request. >>>>>> The database you searched against seems to not contain any entrapment >>>>>> DECOYS to help independently validate any computed scores or >>>>>> probabilities. Are you able to search this data against a database >>>>>> containing some decoys? You can use the TPP decoy generator to create >>>>>> decoys (we have been using deBruijn randomized sequences), or should I >>>>>> create it and give you the database to search? >>>>>> >>>>>> Best, >>>>>> -David >>>>>> >>>>>> On Thu, Oct 10, 2024 at 3:52 AM Sergio Ciordia <[email protected]> >>>>>> wrote: >>>>>> >>>>>>> Thank you David. You are right, I attach in this new link the 3 >>>>>>> files you need: mzML, fasta and pep.xml. >>>>>>> >>>>>>> Link: PEAKS Dataset >>>>>>> <https://drive.google.com/drive/folders/1Q3LF1wRTM62KfS20dfXmToQMY9ji9Dug> >>>>>>> >>>>>>> I hope you can do something. Thanks anyway for your concern. >>>>>>> >>>>>>> Best regards, >>>>>>> Sergio >>>>>>> >>>>>>> -------------------------- >>>>>>> Sergio Ciordia Higuera >>>>>>> Proteomics Facility >>>>>>> National Center for Biotechnology >>>>>>> C\Darwin, 3 >>>>>>> Universidad Autónoma de Madrid >>>>>>> Cantoblanco >>>>>>> 28049 Madrid (Spain) >>>>>>> Phone: +34 91 585 4540 / 4695 >>>>>>> Fax: +34 91 585 4506 >>>>>>> >>>>>>> >>>>>>> El jue, 10 oct 2024 a las 1:34, David Shteynberg (< >>>>>>> [email protected]>) escribió: >>>>>>> >>>>>>>> Thank you Sergio! Would you mind also sending the mzML data and >>>>>>>> the sequence database that goes along with these search results from >>>>>>>> PEAKS? >>>>>>>> >>>>>>>> Best, >>>>>>>> -David >>>>>>>> >>>>>>>> On Oct 9, 2024, at 3:58 PM, Sergio Ciordia <[email protected]> >>>>>>>> wrote: >>>>>>>> >>>>>>>> Hi David, >>>>>>>> >>>>>>>> Thank you for your quick and honest response. I am sorry to hear >>>>>>>> that you have no funds to continue TPP implementation. I hope this >>>>>>>> problem >>>>>>>> will be solved. >>>>>>>> Nevertheless, I appreciate your consideration of evaluating a >>>>>>>> sample file. This file contains what the PEAKS team calls ‘Peptide >>>>>>>> output >>>>>>>> in pepxml’. I hope it is adequate but in any case I don't think there >>>>>>>> will >>>>>>>> be a problem in getting a suitable data output for TPP if necessary. >>>>>>>> The >>>>>>>> other format that can be obtained is mzidentML in case it is of >>>>>>>> interest. >>>>>>>> >>>>>>>> LINK: pepXML PEAKS sample >>>>>>>> <https://drive.google.com/file/d/185t5m7vJPLQAj8vVpcviX_p58DHZfyAB/view?usp=sharing> >>>>>>>> >>>>>>>> Thanks again for your reply. >>>>>>>> >>>>>>>> Best regards, >>>>>>>> Sergio >>>>>>>> >>>>>>>> -------------------------- >>>>>>>> Sergio Ciordia Higuera >>>>>>>> Proteomics Facility >>>>>>>> National Center for Biotechnology >>>>>>>> C\Darwin, 3 >>>>>>>> Universidad Autónoma de Madrid >>>>>>>> Cantoblanco >>>>>>>> 28049 Madrid (Spain) >>>>>>>> Phone: +34 91 585 4540 / 4695 >>>>>>>> Fax: +34 91 585 4506 >>>>>>>> >>>>>>>> >>>>>>>> El jue, 10 oct 2024 a las 0:46, David Shteynberg (< >>>>>>>> [email protected]>) escribió: >>>>>>>> >>>>>>>>> Hello Sergio, >>>>>>>>> >>>>>>>>> Thank you for your email. As you know PEAKS is not a search >>>>>>>>> engine that we have integrated in the TPP, mainly because we have not >>>>>>>>> had >>>>>>>>> any requests for this feature before your email. It is something >>>>>>>>> that can >>>>>>>>> be done with a bit of work and testing, but unfortunately there is >>>>>>>>> currently no funding for us to continue this work. I wish I had a >>>>>>>>> more >>>>>>>>> satisfying answer to give you, but perhaps, if you can forward your >>>>>>>>> sample >>>>>>>>> pep.xml file, we can do this if more funding becomes available. >>>>>>>>> >>>>>>>>> Best, >>>>>>>>> -David >>>>>>>>> >>>>>>>>> On Oct 9, 2024, at 3:35 PM, Sergio Ciordia <[email protected]> >>>>>>>>> wrote: >>>>>>>>> >>>>>>>>> Hi, >>>>>>>>> >>>>>>>>> I have been using TPP for some time now mainly to validate with >>>>>>>>> PeptideProphet the results I get with various search engines and to >>>>>>>>> generate a spectral library that I use in various programs. The thing >>>>>>>>> is >>>>>>>>> that in my lab we are now using PEAKS (v12) and the output is really >>>>>>>>> good. >>>>>>>>> I was wondering if it would be possible to include in TPP an analysis >>>>>>>>> pipeline of the PEAKS data from the pep.xml file generated by the >>>>>>>>> program. >>>>>>>>> I know it is a commercial software but the output is very good and >>>>>>>>> I would be very grateful if you could consider including it like >>>>>>>>> Sequest or >>>>>>>>> Mascot. I think it would not be complicated since they already have >>>>>>>>> the >>>>>>>>> pep.xml output, it would only have to be compatible with XInteract to >>>>>>>>> be >>>>>>>>> able to validate peptide-spectrum matches. >>>>>>>>> >>>>>>>>> If necessary I can provide a sample pep.xml file. >>>>>>>>> >>>>>>>>> Thank you very much. >>>>>>>>> >>>>>>>>> Best regards, >>>>>>>>> Sergio >>>>>>>>> >>>>>>>>> -- >>>>>>>>> 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 [email protected]. >>>>>>>>> To view this discussion on the web visit >>>>>>>>> https://groups.google.com/d/msgid/spctools-discuss/621aa092-4079-4dc8-963f-92ca8eb7e6acn%40googlegroups.com >>>>>>>>> <https://groups.google.com/d/msgid/spctools-discuss/621aa092-4079-4dc8-963f-92ca8eb7e6acn%40googlegroups.com?utm_medium=email&utm_source=footer> >>>>>>>>> . >>>>>>>>> >>>>>>>>> >>>>>>>>> >>>>>>>>> -- >>>>>>>>> You received this message because you are subscribed to a topic in >>>>>>>>> the Google Groups "spctools-discuss" group. >>>>>>>>> To unsubscribe from this topic, visit >>>>>>>>> https://groups.google.com/d/topic/spctools-discuss/VptRGKWbkvM/unsubscribe >>>>>>>>> . >>>>>>>>> To unsubscribe from this group and all its topics, send an email to >>>>>>>>> [email protected]. >>>>>>>>> To view this discussion on the web visit >>>>>>>>> https://groups.google.com/d/msgid/spctools-discuss/26147492-1510-42FC-A601-FB34DCABBC69%40systemsbiology.org >>>>>>>>> <https://groups.google.com/d/msgid/spctools-discuss/26147492-1510-42FC-A601-FB34DCABBC69%40systemsbiology.org?utm_medium=email&utm_source=footer> >>>>>>>>> . >>>>>>>>> >>>>>>>> >>>>>>>> -- >>>>>>>> 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 [email protected]. >>>>>>>> To view this discussion on the web visit >>>>>>>> https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W5q3RRYqwOQ_5W3YQmfcYV%2BHNgbNgnMdJL_aV-eyeOH6g%40mail.gmail.com >>>>>>>> <https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W5q3RRYqwOQ_5W3YQmfcYV%2BHNgbNgnMdJL_aV-eyeOH6g%40mail.gmail.com?utm_medium=email&utm_source=footer> >>>>>>>> . >>>>>>>> >>>>>>>> >>>>>>>> >>>>>>>> -- >>>>>>>> You received this message because you are subscribed to a topic in >>>>>>>> the Google Groups "spctools-discuss" group. >>>>>>>> To unsubscribe from this topic, visit >>>>>>>> https://groups.google.com/d/topic/spctools-discuss/VptRGKWbkvM/unsubscribe >>>>>>>> . >>>>>>>> To unsubscribe from this group and all its topics, send an email to >>>>>>>> [email protected]. >>>>>>>> To view this discussion on the web visit >>>>>>>> https://groups.google.com/d/msgid/spctools-discuss/5E738AA1-A696-4510-8D4C-D509926D065C%40systemsbiology.org >>>>>>>> <https://groups.google.com/d/msgid/spctools-discuss/5E738AA1-A696-4510-8D4C-D509926D065C%40systemsbiology.org?utm_medium=email&utm_source=footer> >>>>>>>> . >>>>>>>> >>>>>>> >>>>>>> -- >>>>>>> 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 [email protected]. >>>>>>> To view this discussion on the web visit >>>>>>> https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W7%2BQqcKvPBMcMTLWR2oahP8Qz%2BQVpyop9dUusFZC4vL0A%40mail.gmail.com >>>>>>> <https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W7%2BQqcKvPBMcMTLWR2oahP8Qz%2BQVpyop9dUusFZC4vL0A%40mail.gmail.com?utm_medium=email&utm_source=footer> >>>>>>> . >>>>>>> >>>>>> >>>>>> -- >>>>>> You received this message because you are subscribed to a topic in >>>>>> the Google Groups "spctools-discuss" group. >>>>>> To unsubscribe from this topic, visit >>>>>> https://groups.google.com/d/topic/spctools-discuss/VptRGKWbkvM/unsubscribe >>>>>> . >>>>>> To unsubscribe from this group and all its topics, send an email to >>>>>> [email protected]. >>>>>> To view this discussion on the web visit >>>>>> https://groups.google.com/d/msgid/spctools-discuss/CAGJJY%3D9CSRn1sgnyOVDvj1n5at44wc16XFSz5bTchD2%3D2k-nPw%40mail.gmail.com >>>>>> <https://groups.google.com/d/msgid/spctools-discuss/CAGJJY%3D9CSRn1sgnyOVDvj1n5at44wc16XFSz5bTchD2%3D2k-nPw%40mail.gmail.com?utm_medium=email&utm_source=footer> >>>>>> . >>>>>> >>>>> >>>>> -- >>>>> 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 [email protected]. >>>>> To view this discussion on the web visit >>>>> https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W6CWMdM-8nURTkybHZ19%3DN-nR6asE-oQMqQ6ZuZzC9dMg%40mail.gmail.com >>>>> <https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W6CWMdM-8nURTkybHZ19%3DN-nR6asE-oQMqQ6ZuZzC9dMg%40mail.gmail.com?utm_medium=email&utm_source=footer> >>>>> . >>>>> >>>> >>>> -- >>>> You received this message because you are subscribed to a topic in the >>>> Google Groups "spctools-discuss" group. >>>> To unsubscribe from this topic, visit >>>> https://groups.google.com/d/topic/spctools-discuss/VptRGKWbkvM/unsubscribe >>>> . >>>> To unsubscribe from this group and all its topics, send an email to >>>> [email protected]. >>>> To view this discussion on the web visit >>>> https://groups.google.com/d/msgid/spctools-discuss/CAGJJY%3D-FRoThPZkMBERYLME8QfKvGXJcyJZLy_XV__c%3DDD-6yw%40mail.gmail.com >>>> <https://groups.google.com/d/msgid/spctools-discuss/CAGJJY%3D-FRoThPZkMBERYLME8QfKvGXJcyJZLy_XV__c%3DDD-6yw%40mail.gmail.com?utm_medium=email&utm_source=footer> >>>> . >>>> >>> >> -- >> 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 [email protected]. >> To view this discussion on the web visit >> https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W63F-Cp-Bk7QruzBSMF%2BWnMEKabNJeJ%3DspiFXVhyZB%3D8g%40mail.gmail.com >> <https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W63F-Cp-Bk7QruzBSMF%2BWnMEKabNJeJ%3DspiFXVhyZB%3D8g%40mail.gmail.com?utm_medium=email&utm_source=footer> >> . >> >> >> >> -- >> You received this message because you are subscribed to a topic in the >> Google Groups "spctools-discuss" group. >> To unsubscribe from this topic, visit >> https://groups.google.com/d/topic/spctools-discuss/VptRGKWbkvM/unsubscribe >> . >> To unsubscribe from this group and all its topics, send an email to >> [email protected]. >> To view this discussion on the web visit >> https://groups.google.com/d/msgid/spctools-discuss/47171376-CD67-493E-A762-D71469974F4C%40systemsbiology.org >> <https://groups.google.com/d/msgid/spctools-discuss/47171376-CD67-493E-A762-D71469974F4C%40systemsbiology.org?utm_medium=email&utm_source=footer> >> . >> > > -- > 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 [email protected]. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W6zA0L%3Dy9k5bkNbOEAA%2BVDEr8K9H6pU8dkMjseiEDmnQQ%40mail.gmail.com > <https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W6zA0L%3Dy9k5bkNbOEAA%2BVDEr8K9H6pU8dkMjseiEDmnQQ%40mail.gmail.com?utm_medium=email&utm_source=footer> > . > > > -- > You received this message because you are subscribed to a topic in the > Google Groups "spctools-discuss" group. > To unsubscribe from this topic, visit > https://groups.google.com/d/topic/spctools-discuss/VptRGKWbkvM/unsubscribe > . > To unsubscribe from this group and all its topics, send an email to > [email protected]. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/8C8534AE-3FA2-4345-BD7D-9DBDE481A945%40systemsbiology.org > <https://groups.google.com/d/msgid/spctools-discuss/8C8534AE-3FA2-4345-BD7D-9DBDE481A945%40systemsbiology.org?utm_medium=email&utm_source=footer> > . > -- 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 [email protected]. To view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/CAOgO5W5S8%3Dgb5wmZkyDYkbpFFjTc5E4%2BcMz0rMOVTTjjBvnqaw%40mail.gmail.com.
