CRIMP2 Parameters

A full list of processing parameters, including advanced parameters.
  • “Cross-link Search Parameters”: General parameters for the cross-link search.
    • “Aggregation Level”: The level at which data aggregation should occur. The options follow the structure of your CRIMP project on how you group biological and/or technical replicates:
      • “Dataset”: All data from all runs across all groups and states will be aggregated together.
      • “State Group”: Data from each state group will be aggregated separately.
      • “State+Run Group”: Data from each state group and run group will be aggregated separately.
      • “Run Group”: Data from each run group is aggregated separately, but the aggregation will works across different state groups.
      • “Run”: Data from each run is aggregated individually.
    • “Normalize Retention Times” [Advanced]: Uses common identifications across runs to normalize RTs for all peptides across all runs.
    • “Extract XICs & Areas” [Advanced]: Whether to measure XIC areas during processing. This will add additional processing time, depending on the number of peptides identified (~10-20ms per peptide).
    • “Slices per Run” [Advanced]: The number of slices to divide each run into. This helps lower memory usage during processing and is recommended for really long gradients or really dense high-resolution MS2 data.
    • “Database Sharing Level”: Database sharing is meant to recover from stochastic differences between DDA runs by sharing linear peptide results across runs. The levels are the same as the point above (Aggregation Levels) where you can chose which runs or groups of runs should share information. For the best performance, it's best to allow propagation between technical replicates or the runs which are most similar. If you select the "Run" level, then there will be no database sharing and runs are searched in isolation.
    • “Resolve Spectrum Conflicts” [Advanced]: Whether to resolve conflicts between peptides of any form that claim the same MS2 data. The conflict resolution will compare fragmentation patterns between conflicting patterns to give more weight to peptides with more unique high-scoring fragments. If the differences are large, only the best candidate(s) are accepted and weaker scoring candidates are rejected.
  • “Fast-Tracked Export”: If you wish to go from processing directly to exporting results (skipping results validation), you can set paths and Q-value cutoffs for each desired aggregation target. This will generate .csv files at the end of the search. Note: The final results will still show up in the validation view, but you can access the .csv exports before it finishes writing and visualizing the results.
  • “Database Reducer”: Settings for the initial linear peptide search prior to crosslink pairing.
    • “% E-Threshold (% Library Size)” [Advanced]: The percent E-value threshold (E-beta - linear score during reduction) for the beta crosslinking partner at each RT. A value of 100% means any peptide at a given RT can be paired with. We recommend leaving this value high and only reduce it if you face performance problems to only narrow in on the best candidates.
    • “Minimum Fragment Count” [Advanced]: The minimum number of linear, tagged or complement fragments to consider a peptide a valid crosslink partner at any given RT.
    • “Number of Proteins per Chunk” [Advanced]: The number of proteins that are processed together as a batch during reduciton. The larger the chunk, the more memory is used. If you face high memory usage issues, we recommend lowering the value,
    • “Fragment Charge States”: The charge states for free fragments to be searched for each linear peptide during reduction (before forming cross-links).
    • “Include Complement Fragments”[Advanced]: Whether to check for complement fragments in the linear peptide search. This option allows poorly fragmenting crosslinked peptides to be boosted via an open-modification-style search. This is useful for cases where a peptide doesn't generate enough linear fragments on its own and must rely on crosslinked fragments for initial identification prior to pairing.
    • “Include Neutral Loss Fragments”[Advanced]: Whether to check for neutral loss during the linear search (-H2O, -NH3).
    • “Skip Y1 ions”[Advanced]: Whether to ignore Y1 ions during the linear search. This is useful for very specific digestions such as Trypsin where the terminus is shared by most peptides and will exist in almost every MS2 spectrum, providing very little identification value.
    • “Digestion”: The enzyme used for digestion.
    • “Minimum Peptide Length”: The minimum length in amino-acids of each linear peptide.
    • “Maximum Peptide Length”: The maximum length in amino-acids of each linear peptide.
    • “De-isotoped MS2”[Advanced]: Enable this if the MS2 data is de-isotoped. This mode will not validate charges, as everything will be assumed to be collapsed to a single peak (1+).
    • “Include Decoys”[Advanced]: Whether to automatically generate decoys. This is required to calculate False Discovery Rate and assign Q-valus. Results where no decoys are included are not reliable without error estimation.
    • “Squash Ambiguous Peptides”[Advanced]: Ambiguous peptides are treated as the same sequence and only searched once for all occurences of this sequence in the library. Results which share these ambiguous sequences are grouped togther as alternative sequences during export. Disabling this will make the search slower.
    • “Feature Driven Denoising”[Advanced]: Attempts to denoise MS2 by only keeping clear and distinct isotopic profiles and eliminating noise and single peaks at high-mass.
  • “Crosslinked Peptide Generator”: Settings for generating crosslinked peptides and validating precursors (MS1).
    • “Alpha % E-Threshold (% Library Size)” [Advanced]: The percent E-value threshold (E-alpha - linear score during reduction) for the alpha crosslinking partner at each RT. We recommend constraining this between 0.01 (for dense MS2 data) to 25 (for most cases). The lower the value, the more selective the selection process for quality alpha peptides.
    • “Top N” [Advanced]: The number of similar-scoring groups of peptides (sorted by E score) that will undergo pairing after E-alpha and E-beta selection. We recommend a value less than 20 to avoid entrapment with too many poor-quality peptides. On the flip side, a really small value (less than 5) will severly impact sensitivity.
    • “Minimum m/z”: The minimum m/z for candidates (crosslinked peptide pairs or single peptidews).
    • “Maximum m/z”: The maximum m/z for candidates (crosslinked peptide pairs or single peptides).
    • “Minimum Charge”: The maximum z for candidates (crosslinked peptide pairs or single peptides).
    • “Maximum Charge”: The maximum z for candidates (crosslinked peptide pairs or single peptides).
    • “Search product ions”[Advanced]: Whether to allow for in-source fragmentation of a labile crosslink. When enabled, this option will create peptides where the linker is cleaved off in MS1 at either binding site.
    • “Generate Source -H2O Precursors”[Advanced]: Whether search for neutral losses (-H2O) as a result of in source fragmentation (MS1).
    • “Generate Source -NH3 Precursors”[Advanced]: Whether search for neutral losses (-NH3) as a result of in source fragmentation (MS1).
    • “Use reported charge”[Advanced]: Whether to restrict charge checks to those reported by the instrument for each MS2 Spectrum. Disabling this option will search for other possible charges of candidates that can produce the same MS2 spectrum. Disabling this option will also increase search time.
    • “Minimum Raw Peak Intensity”[Advanced]: The minimum absolute intensity for a precursor (MS1).
    • “Precursor Peak % Intensity Threshold (theoretical peaks to check)”[Advanced]: The minimum relative (%) intensity for checking precursor peaks (MS1).
  • “Crosslinked Fragment Search”: Settings for searching and scoring MS2 spectra after pairing.
    • “Allowed Fragmentation Events”[Advanced]: The number of fragmentation events to consider. A value of 2+ will also generate internal ions.
    • “Search Neutral Loss Ions”[Advanced]: Whether to include neutral loss fragments (-H2O, -NH3).
    • “Skip Y1 ions”[Advanced]: Whether to ignore Y1 ions during the main scoring (different step than linear search). This is useful for very specific digestions such as Trypsin where the terminus is shared by most peptides and will exist in almost every MS2 spectrum, providing very little identification value.
    • “Relative Intensity Threshold”[Advanced]: The minimum relative (%) intensity for checking fragment peaks.
    • “Fragment Count Threshold”[Advanced]: The minimum number of fragments to accept a candidate.
    • “Feature Driven Denoising”[Advanced]: Attempts to denoise MS2 by only keeping clear and distinct isotopic profiles and eliminating noise and single peaks at high-mass. This applies during scoring of crosslinked candidates (different than the same setting during the linear search -- reduction).
  • “CSM Flagging Controller”: Post-search flagging for manual validation.
    • “Minimum Contiguous Fragments”[Advanced]: The number of contiguous or sequential fragments. Any results below this value will be flagged.
  • “MS1 Mass Accuracy”: The mass tolerance for finding peaks for MS1 spectra. This will depend on instrument resolution and is one-sided.
  • “MS2 Mass Accuracy”: The mass tolerance for finding peaks for MS2 spectra. This will depend on instrument resolution and is one-sided.
  • “Peptide Elution Time”: The average length of time a peptide spends in the LC column (the average width of an XIC peak).
  • “XIC Extraction”: The m/z window used in extract the XIC for a given peptide. This setting is similar to the “MS Mass Accuracy” where the PPM value is one-sided. This mass range will be applied around the best peak or the monoisotopic peak for XIC extraction, depending on the “Always use monoisotopic m/z (lock)”.
  • “XIC Peak Picking”: The settings for finding peaks in the XIC data.
  • “MS1 Peak Picking”: The settings for finding peaks in the aggregated MS data.
  • “MS2 Peak Picking”: The settings for finding peaks in each MS2 spectrum. If you have data which is acquired in centroid mode, we recommend a really low value for the "Peak to Background Ratio" because the intrument already performed a first-pass peak selection from the underlying noise.
  • “XIC Smoothing”: The type of smoothing for the XIC.
  • “MS1 Smoothing”: The type of smoothing for the aggregated MS1 data.
  • “MS2 Smoothing”: The type of smoothing for each MS2 spectrum.