OpenNuXL

Annotate NA to peptide crosslinks in MS/MS spectra.

1. Input files: centroided mzML file or Thermo raw file
2. Data preprocessing:
   • (optional, precursor_correction) precursor mass correction
   • (optional, autotune) step determines precursor and fragment mass accuracy
     • performs a peptide database search for non-XL peptides
     • tries to improve results using percoltator
     • based on the best identifications (1% FDR)
       • (optional, IDFilter) spectra are marked to be generated by a confident non-XLed peptide
       • new fragment mass tolerance = 4.0 * 68th percentile of median absolute fragment error (identipy)
       • (optional, pcrecalibration) correct global fragment error by subtracting the median

1. Configuring XL search
   • user configuration
   • presets for different protocols (UV, DEB, NM, etc.)
2. Generation of Precursor/MS1 adduct to nucleotides to fragment adduct rules
3. Fragment spectra preprocessing
   • Remove zero intensities
   • Remove isotopic peaks and determine charge (unknown charge set to z=1)
   • Remove interfering peaks from cofragmentation
   • Scale intensity by taking the root to reduce impact of high-intensity peaks
   • Normalize max intensity of a spectrum to 1.0
   • Keep 20 highest-intensity peaks in 100 m/z windows
   • Keep max. 400 peaks per spectrum to highly charged fragments in the low m/z region
   • Calculate and store TIC of filtered spectrum
4. Experimental feature precalculation from spectra
   • Precalculate nucleotide tags
   • Calculate intensity ranks
   • Calculate amino acid tags
5. Generate decoy sequences
   • Uses maximum number of attempts to shuffle in order to minimize sequence similarity
   • Sequence similarity target vs. decoy is calculated by overlap prefix with prefix and prefix to suffix (e.g, reversing is also discouraged if possible)
6. Search: For all proteins:
   • Diggest current protein
   • For all peptides of current digest
     • Skip peptide if already searched
     • Determine potential immonium ions of peptide
     • Apply fixed modification(s) to peptide
     • Apply variable modifications(s) to peptide to get modification peptidoforms
     • For all modified peptide sequences (of current peptide)
       • For peptides with methionine try to also score plain precursors - CH4S (NM and DEB related methionie cross-link)

The command line parameters of this tool are:

INI file documentation of this tool: