The Subsequence Search node performs a subsequence search. Increasing the precursor tolerance results in longer run times.

To search for modified proteoforms, select the Include Modified Forms check box in the Predefined Search dialog box for biomarkers.

The following table lists the parameters for the 4.4 version of the node. For information about the 3.0 version, refer to the user interface.

Table ProSightPD 4.4 Subsequence Search parameters

Parameter

Description

1. Input Data

Input Database

Specifies the name of the database to search.

2. Subsequence (Biomarker) Search

Subsequence Precursor Mass Tolerance

Specifies the maximum mass difference between the subsequence mass and the observed proteoform mass for a proteoform to be included in the search. Setting this to a large tolerance can potentially increase search time with large databases.

Minimum value = 0.01 Da (10.1 ppm)

Maximum value = 1,000,000 Da (1000 ppm)

Fragment Mass Tolerance

Specifies the mass tolerance in Da or ppm used for matching fragment peaks.

Range: 0–2.0 Da; 1–50 ppm

Maximum PTMs per Isoform

Limits the number PTMs to add to any given proteoform. If more than this number of PTMs are present on an isoform, the proteoforms created are limited to the ones with combinations of PTMs having this many PTMS or fewer.

Maximum SNPs per Isoform

Limits the number SNPs to add to any given proteoform. If more than this number of SNPs are present on an isoform, the proteoforms created are limited to the ones with combinations of SNPs having this many SNPs or fewer.

Remove Disulfide Bonds

Thermo Fisher Scientific recommends setting this parameter to False. You can set this parameter to True if there are annotated disulfide bonds in the search database that you want to avoid searching, or when there is a known error in the disulfide bond annotation in the database. Errors in UniProt where disulfide bonds are placed on non-cysteine residues have been observed and can cause the search to crash.

Maximum Mass to Include PTMs

Maximum proteoform mass to search with PTMs.

N-Term Modifications to Include

Selected modifications are included on all intact proteoforms n-termini.

Calculate FDR

Specifies whether the FDR is calculated.

You might see fewer results passing the FDR threshold if you use Delta M Mode.

Decoy Reps

The number of times a decoy search is run. A higher number of repetitions produces more stable decoy results but increases search times.

Maximum PrSMs per Precursor

Maximum number of PrSMs matched per precursor limits the number of results to the highest scoring hits.

Minimum Matched Fragments

Return only hits with this many or more matched fragments.

Include Labile Modification Mass Shift

Target mass will be shifted to account for loss of labile modification mass.

Labile Modification Mass Shift

Comma separated list of Target mass shifts.

Number of Off by 1

Specifies the number of off-by-one errors to accommodate. For example, if set to 1, the search considers three candidate precursor masses.

  • deconvoluted precursor mass
  • deconvoluted precursor mass plus 1.003354838 Da
  • deconvoluted precursor mass minus 1.003354838 Da

Thermo Fisher Scientific recommends setting this parameter to 2.

3. Static (Fixed) Modifications

Static (Fixed) Modification

Specifies a static (also known as fixed) modification from a list of known modifications. Static modifications apply universally to every instance of the specified residues.

4. Multithreading Options

CPU Usage

The amount of CPU to direct to data processing:

  • High—The total number of CPU cores less 1
  • Medium—Half the number of available cores
  • Low—1 core

5. Fragmentation Method

Override Fragmentation Method

Manually set the fragmentation method.

Auto: allows the software to determine the correct fragmentation type. (Default)

6. UVPD Method

Default UVPD Method

Set the number of ion types to consider for the score of UVPD fragment ion spectra:

  • 4—recommended for maximizing IDs
  • 9—recommended for maximizing characterization