Re: [spctools-discuss] Issues with processing .d (Bruker) files run in DDA-LFQ mode

[David Shteynberg]

Hello Shagun, I was able to run comet and the TPP on these files from the latest TPP version 7.1.0.  After adding two independent deBruijn randomized decoys to the database using the Petunia Decoy Database tool, my pipeline found approximately 80 thousand to almost 90 thousand PSMs at 1% spectrum error rate, per mzML file.  These map to about 68 thousand unique peptides at 1% peptide error rate, after iProphet combining all the files.  ProteinProphet mapped all of these to just over 7000 proteins at 1% protein error rate (or lower.) The biggest issue I found in your comet params file was the was n-terminal acetylation was specified using MSFragger notation ‘[^’ for the amino acid (should be ’n’ instead for comet.)  Unfortunately this confused the pipeline and exposed downstream assumptions that broke the analysis.  I am attaching the comet params file I used to process this data.   Cheers! -David P.S.  Please update your TPP to the latest 7.1.0 to get the latest features and bug-fixes! -- 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 view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/78BF9834-8CE6-40E6-8D4B-5EC6D221DE1C%40systemsbiology.org.

ShagunGupta.comet.params
Description: Binary data

On Aug 13, 2024, at 2:15 PM, Shagun Gupta <sg2...@cornell.edu> wrote: Hi David Apologies for the hassle! I have uploaded the .d files in an unzipped format under TPP_diagnosis/TPP_diagnosis. Let me know if this works instead? Best Shagun On Tuesday, August 13, 2024 at 3:09:37 PM UTC-4 David Shteynberg wrote: Hello Shagun, I downloaded the larger zip file twice and tried to decompress, but each time it told me there was a corruption in the zip file.  The smaller files downloaded fine.  Can you check your file and upload again? Thanks! -David On Tue, Aug 13, 2024 at 9:47 AM Shagun Gupta <sg2...@cornell.edu> wrote: Hi David I have attached a link to the following files  - one replicate per condition .d file (file starting with "C_" had IDs but no values that could be extracted for quantification) - comet parameter file I used - fasta file I ran the search with. Let me know if I can share anything else! Box_link_diagnosis Best Shagun On Tuesday, August 13, 2024 at 11:05:42 AM UTC-4 David Shteynberg wrote: Hello Shagun, Thank you for the detailed report.  If you are able, please first compress (into a zip or similar) and then share some of the problem .d files so I can try to replicate this issue on my computer before I offer any suggestions.   Cheers! -David On Tue, Aug 13, 2024 at 7:50 AM Shagun Gupta <sg2...@cornell.edu> wrote: Hi all I have been experiencing issues processing .d files obtained from a Bruker timsTOF HT in DDA-LFQ mode, more specifically getting quantification - precursor intensity - per spectra. I am using TPP V6.3.3 Arcus on a windows computer.  Details - There are 12 .d files (4 repeats of 3 conditions) composed of a human with yeast proteome spike in at different ratios. - Converted to .mzXML using msconvert - Searched with COMET and a search database taken from UniProt for Homo sapiens+Yeast - Processed with PeptideProphet (filtered at probability associated with 1% FDR), XPRESS, ProteinProphet. Ran with -PREC flag (PeptideProphet), -i flag (XPRESS) - Want to do hypothesis testing (comparing the three conditions pairwise) using MSstats. So require raw precursor intensity values to make a file that can be used as input to MSstats. Unfortunately after trying the above, and a few more things, while I get a large number of PSMs passing FDR (~30k), a large proportion of them do not have any precursor intensity value (<1k have some "light area" values). Using the "light area" values also does not give expected results (its a benchmarking dataset and processing with MSFragger gave excellent results that align with expected ratios etc.). Could you suggest things I could be doing differently to get the right results? (I imagine it might have something to do with the initial conversion to mzXML itself?)  Happy to share any other details needed! Best Shagun -- 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 view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/cf666974-5c1f-4de0-80ad-d9b1df2173dan%40googlegroups.com. -- 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 view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/21faed8c-1c4d-45b8-a281-f3f572864ed1n%40googlegroups.com. -- 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 view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/1e530c4b-d879-4117-9708-ae204d2198e5n%40googlegroups.com. -- 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 view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/78BF9834-8CE6-40E6-8D4B-5EC6D221DE1C%40systemsbiology.org.