The Tag to Subsequence Search node first performs a tag search and then performs a sequence tag search on the output of the tag search.

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 Tag to Subsequence Search node parameters

Parameter

Description

1. Input Data

Input Database

Select the search database.

2a. Subsequence Tag

Minimum Tag Score

Minimum probability that the probable tag is not found by chance.

Amino Acid Gap

Largest number allowed of sequential amino acids not having a fragment between them.

Max Tags

Greatest number of probable tags considered for each search per target.

Minimum Fragments Used

Minimum number of fragments required for each sequence tag.

Remove Water Losses

If set to True, water loss is not considered when searching sequence tags.

Consider Isobaric Dipeptide Gaps

If set to True, for example, both GA and Q are considered for gaps of 128.0 Da.

Compiler Tolerance

The permissible error, measured in ppm between two fragment ion masses that are still considered as matching an amino acid.

2b. Subsequence (Biomarker) Search

Subsequence Precursor Mass Tolerance

Specifies the tolerance when comparing subsequence mass to observed precursors. A large tolerance setting might 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.

Maximum Mass to Include PTMs

Maximum proteoform mass to search with PTMs.

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.

N-Term Modifications to Include

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

Delta M Mode

Determines whether the application conducts the search in Delta M (Δm) Mode. For more information on this mode, see Delta M Mode searches.

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.

Random Seed Value

User settable advanced parameter that ensures that the FDR calculation is reproducible in repeat runs.

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