The default parameters of the Annotated Proteoform search are designed to work in most situations. However, analyses can be customized to improve performance by optimizing the advanced parameters listed below. For a full list of advanced parameters, see Annotated Proteoform Search node.

The Advanced parameters for the Annotated Proteoform table is a list of advanced parameters and a brief description of how they impact the Annotated Proteoform search and results.

Table Advanced parameters for the Annotated Proteoform

Parameter

Description

Maximum PTMs per Isoform

Limits the number of PTMs that are expanded and searched during search time. For example, if an isoform has 10 PTMs in the database and the maximum is set to 4, then only proteoforms with up to 4 PTMs are searched. This improves the search time. However, in cases of highly modified proteoforms, you should increase this value.

Maximum SNPs per Isoform

Limits the number of amino acid mutations of single nucleotide polymorphisms (SNPs) in the same way as the Maximum PTMs per isoform parameter.

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

Limits the size of proteoforms that are considered with PTMs. It is unlikely to observe very large proteoforms, therefore it is unnecessary to consider all of the modified forms beyond the size expected to be observed in the experiment. Setting the limit to a reasonable size (100 kDa) allows the search to complete sooner.

N-Term Modifications to Include

N-terminal modifications are added at search time unless specifically added to the database. These modifications generally include acetylation or formylation, and the default is set to include N-terminal acetylation.

Delta M Mode

To identify unknown mass shifts, consider setting Delta M Mode to True. Searching with Delta M Mode will slow the search. Thermo Fisher Scientific recommends turning off FDR when utilizing Delta M Mode as the Delta M search affects the FDR threshold. Delta M Mode is suggested for data that has already been searched or in a target case.

Decoy Reps

Sets the number of times the decoy database is searched. The default setting is 1, which results in the fastest search. However, results can vary minimally run-to-run: 1-2 proteoform differences based on exactly where the FDR thresholds are set. Increasing the number of times the decoy database is searched to 3 results in slower searches but reduces the amount of variation between analyses.

Maximum PrSMs Per Precursor

Limits the number of spectral matches attributed to a single precursor. Any given precursor can have many associated spectral matches depending on search parameters and the data. In general, there are very few highly confident matches (based on P-Score or E-Value.) This parameter dictates the top N scoring PrSMs to return per precursor. The default value is 3, however not all 3 PrSMs can pass the FDR cutoff depending on their confidence. Due to the nature of the MS isolation window, the occurrence of off-by-one Dalton deconvolution errors and modifications such as deamination, which are ~1 Dalton, do not expect to see multiple high-scoring PrSMs for a single precursor (this is why the default setting is 3). If you expect multiple proteoforms to have the same precursor mass, (for example, positional isomers such as histones), you can increase this setting to 5 or 10.

Minimum Matched Fragments

Lets you discard spectra that deconvoluted with less than n fragment ions. The default value is 3 and you can increase it to 5 or 10 without major loss of IDs. For smaller proteoforms (< 5kDa), set the minimum to 5 or lower. By discarding sparse spectra, the search completes faster.

Include Labile Modification Mass Shift

Lets you apply a mass shift to all precursors to account for loss of a labile modification. To activate the setting, set Include Labile Modification Mass Shift to True and input a mass in the Labile Modification Mass Shift parameter.

Random Seed Value

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