StPeter error - Could not read spectra

[Jason Winget]

I am trying to process my data using StPeter and am getting the following error:

Extracting peak intensities from 1 spectral files:
  1 of 1: C:\TPP\data\Supernatant\PG_SS_RAW\SS1_Slot2-4_1_789.mzML    0%ERROR: Could not read spectra.

The data was collected on a Bruker timsTOF and converted using the msconvert options "combine ion mobility scans" as well as vendor peak picking and scan summing.

I searched the mzML with Comet, then processed those results with PeptideProphet, iProphet, and ProteinProphet. I am using TPP v6.3.3 Arcus, Build 202308242222-9003 (Windows_NT-x86_64). Before it crashes, StPeter appears to be working:

****** BEGIN StPeter ANALYSIS ******
 Time at start of analysis: Thu Aug 31 15:39:02 2023

 Parameters:
  degenerate peptides = yes
  fdr = 0.01
  minimum probability = 0
  sample load = 0
  tolerance = 0.4

 Reading protXML: .\ss1_slot2-4_1_789.iproph.prot.xml
  Probability at 0.01 FDR: 0.965
  Number of proteins above FDR cutoff: 418
  Number of quantifiable proteins above FDR cutoff: 418
  Number of peptides for proteins: 822
  Extracting true protein lengths for zero length proteins. Database: uniref_taxonomy_allcombined_DECOY.fasta
  Number of proteins fitting criteria for StPeter analysis: 418****** BEGIN StPeter ANALYSIS ******

 Time at start of analysis: Thu Aug 31 15:39:02 2023

 Parameters:
  degenerate peptides = yes
  fdr = 0.01
  minimum probability = 0
  sample load = 0
  tolerance = 0.4

 Reading protXML: .\ss1_slot2-4_1_789.iproph.prot.xml
  Probability at 0.01 FDR: 0.965
  Number of proteins above FDR cutoff: 418
  Number of quantifiable proteins above FDR cutoff: 418
  Number of peptides for proteins: 822
  Extracting true protein lengths for zero length proteins. Database: uniref_taxonomy_allcombined_DECOY.fasta
  Number of proteins fitting criteria for StPeter analysis: 418

The mzML files are in the same directory as the protXML file, and the head of the mzML appears normal:

<?xml version="1.0" encoding="utf-8"?>
<indexedmzML xmlns="http://psi.hupo.org/ms/mzml" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://psi.hupo.org/ms/mzml http://psidev.info/files/ms/mzML/xsd/mzML1.1.2_idx.xsd">
  <mzML xmlns="http://psi.hupo.org/ms/mzml" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://psi.hupo.org/ms/mzml http://psidev.info/files/ms/mzML/xsd/mzML1.1.0.xsd" id="SS1_Slot2-4_1_789" version="1.1.0">
    <cvList count="2">
      <cv id="MS" fullName="Proteomics Standards Initiative Mass Spectrometry Ontology" version="4.1.56" URI="https://raw.githubusercontent.com/HUPO-PSI/psi-ms-CV/master/psi-ms.obo"/>
      <cv id="UO" fullName="Unit Ontology" version="09:04:2014" URI="https://raw.githubusercontent.com/bio-ontology-research-group/unit-ontology/master/unit.obo"/>
    </cvList>
    <fileDescription>

      <fileContent>
        <cvParam cvRef="MS" accession="MS:1000579" name="MS1 spectrum" value=""/>

I also checked within the mzML file to confirm that it does contain MS2 scan information.

Any advice?

Jason

[David Shteynberg]

Hey Jason,

If you are able to compress the dataset directory and share it I would be happy to troubleshoot this particular issue.

Cheers!

-David