Reporter Ions Quantifier node - Proteome Discoverer Software - Reporter Ions Quantifier node - Proteome Discoverer Software - Proteome Discoverer Software

General Quantification Settings

Peptides to Use

Specifies the peptides to use for protein quantification.

• Unique: Uses only peptides that are not shared among different proteins or protein groups for protein quantification.
• (Default) Unique + Razor: Uses all quantification results that are not shared among different peptide groups for protein quantification. All shared quantification results are used for one peptide group only.
• All: Uses all peptides for protein quantification, whether or not they are unique.

This parameter adds the # Razor Peptides column to the Proteins page of the result report when you use razor peptides for quantification.

Consider Protein Groups for Peptide Uniqueness

Defines peptide uniqueness on the basis of protein groups rather than individual proteins.

• (Default) True: Considers a peptide unique if it is included in only one protein group.
• False: Considers a peptide unique if it is included in only one protein.

For best results, use the default. When you use larger protein (FASTA) databases, peptides commonly appear in multiple homologous proteins. If you set this parameter to False, the results might not contain many quantification results.

Use Shared Quan Results

If set to True, the shared quan results are used for quantification; otherwise, they are not.

A quan result is a Reporter Quan Spectrum in reporter quantification, and it is a consensus feature in precursor quantification. Quan results are shared if they are associated with multiple peptides.

Reject Quan Results with Missing Channels

Determines whether to set all quantification values from all channels to zero if one or more of the quantification channels have a detected intensity of zero.

• True: Does not use a PSM for calculating peptide group and protein quantification values if one or more of the quantification channels has a detected intensity of zero.
• (Default) False: Calculates and displays the quantification values for those channels that the application found and ignores the missing channels.

Reporter Quantification

Reporter Abundance Based On

Specifies whether to use S/N values or intensity values as quantification channel values instead of ion reporter peak values in reporter ion quantification results.

• (Default) Automatic: Uses S/N values instead of reporter ion quantification peak values when all spectrum files have S/N values; otherwise, uses intensity values.
• S/N: Uses S/N values instead of reporter ion quantification peak values.
• Intensity: Uses intensity values instead of reporter ion quantification peak values.

The workflow fails when the spectra do not contain S/N values, as can happen with ion trap spectra.

Apply Quan Value Corrections

Determines whether to apply a correction (for example, for isotopic or labeling impurities) to the raw quantification values.

• (Default) True: Applies a correction to the raw quantification values.
• False: Does not apply a correction to the raw quantification values.

Co-Isolation Threshold

Specifies a threshold for the amount of interference allowed by coisolation. Interference is the amount of ion current within the isolation window not originating from the identified peptide. Peptides with percentages higher than this threshold should not be used for quantification. The default value of 100% means that no peptide is excluded. This parameter is only used for reporter ion quantification. For more information on coisolation, see Excluding PSMs with high levels of coisolation.

Range: 0–100; default: 50

Average Reporter S/N Threshold

Specifies the minimum average reporter S/N threshold value that determines which PSMs the application excludes from quantification. The application excludes all PSMs that have an average S/N value smaller than this threshold value.

This parameter adds the Average Reporter S/N column, which displays average reporter S/N values, to the PSMs and Quan Spectra pages. The application calculates these values as the sum of S/N values found, divided by the number of defined mass tags. For detailed information about the calculation, see Filtering quantification data with average reporter ion signal-to-noise values.

For the PSMs that this parameter excludes from quantification, the Quan Info column displays the notation Excluded by Method, and the Quan Usage columns displays the notation Not Used.

Range: 0–no maximum; default: 0

CHIMERYS Coefficient Threshold

Only spectra with a CHIMERYS Coefficient above this threshold are considered for the quantification. The CHIMERYS Coefficient represents the contribution of the PSM to the MS2 spectrum.

SPS Mass Matches [%] Threshold

This parameter specifies the percentage of all selected SPS masses that have to match a peptide fragment for considering this PSM in the quantification. That is, when there are ten SPS masses, then at least seven have to match a peptide fragment, otherwise the PSM is discarded from quantification.

Default: 65

Minimum Channel Occupancy [%] Threshold

Only peptides with at least this percentage of non-zero channel values are considered for quantification.

Range: 0 – 100; default: 0

Normalization and Scaling

Normalization Mode

Specifies how to perform normalization to correct for experimental bias.

• (Default) None: Does not apply any normalization.
• Total Peptide Amount: After aggregating all abundance values per quantification channel, the node normalizes the abundance values of each channel with a constant channel-specific factor so that all channels have the same total peptide abundance at the end.
• Specific Protein Amount: Calculates the normalization factor from the abundances of selected proteins in the specified FASTA file. You can specify a FASTA file (use the Proteins for Normalization parameter to specify the name of the FASTA file) that can contain one or multiple proteins. The Reporter Ions Quantifier node uses all proteins in the FASTA file that are contained in the result file and that have any protein abundance. It calculates the maximum sum for each file. The normalization factor is the factor of the sum of the sample and the maximum sum in the same file.
• When normalization is based on specific proteins, the node sums the abundance values of all proteins in the result file that are contained in the selected FASTA file. It compares the protein sequences in the FASTA file to the sequences in the result file, not to the title lines. You can therefore use the same FASTA file when the title line is slightly different.
• The node sums up the abundances for each sample. Then it calculates the normalization factor on the basis of these abundance sums.
• If you set the Normalization Mode parameter to Specific Protein Amount, but do not select a FASTA file, or if the result file contains no proteins that appear in the FASTA file, you cannot perform normalization.
• For more information on the Normalization Mode parameter, see Normalizing peptide groups and protein abundances.

Proteins for Normalization

Specifies the name of the FASTA database containing the proteins that the normalization should be based on. The database can contain a single protein sequence or multiple protein sequences.

This parameter is relevant only when you set the Normalization Mode parameter to Specific Protein Amount.

Scaling Mode

Determines how Precursor Ions Quantifier node scales abundances.

After it aggregates all the abundance values or optionally normalized abundance values per quantification channel, the node scales the abundance values of each channel as follows:

• On All Average: For every protein and peptide in a file, the average of all samples is 100.
• On Controls Average: For every protein and peptide in a file, the average of all control samples is 100. The node proportionally scales up or down all other channels by using the same factor. When you use multiplexed files, it processes samples from one file separately.
• (Default) None: Does not scale abundances.

Exclude Peptides from Protein Quantification

For Normalization

Specifies how the normalization based on the summed abundance of all peptides and the RT-dependent normalization are done:

• Use All Peptides: all peptides are use (the default value)
• Exclude Modified: peptides having one of the specified modifications are excluded
• Use Only Modified: only peptides having one of the specified modifications are used

For Protein Roll-Up

Specifies how the roll-up of peptide abundances to protein level is done:

• Use All Peptides (the default value)
• Exclude Modified
• Use Only Modified

For Pairwise Ratios

Specifies which peptides to use to calculate protein ratios based on the pairwise peptide ratios mode:

• Use All Peptides
• Exclude Modified (the default value)
• Use Only Modified

Considered Peptide Modification

Peptides with the specified modification will be considered for inclusion or exclusion in protein ratios based on the other settings in this section.

N-Terminal Considered Peptide Modification

Does not use peptides with the specified modification to calculate the normalization factors. Only effective when you set the Normalization Mode parameter to Total Peptide Amount.

C-Terminal Considered Peptide Modification

Does not use peptides with the specified modification to calculate the normalization factors. Only effective when you set the Normalization Mode parameter to Total Peptide Amount.

N-Terminal Considered Protein Modification

Does not use peptides with the specified modification to calculate the normalization factors. Only effective when you set the Normalization Mode parameter to Total Peptide Amount.

C-Terminal Considered Protein Modification

Does not use peptides with the specified modification to calculate the normalization factors. Only effective when you set the Normalization Mode parameter to Total Peptide Amount.

Quan Rollup and Hypothesis Testing

Protein Ratio Calculation

Specifies how protein ratios are calculated.

• Protein Abundance Based: Protein ratios are directly calculated from the grouped protein abundances.
• Pairwise Ratio Based: Protein rations are calculated as the median of all possible pairwise peptide ratios calculated between replicates of all connected peptides.

Maximum Allowed Fold Change

Specifies the maximum fold change that can be reasonably expected for the observed ratios.

Reports the quantification ratios based on the maximum values. Values greater than the value selected by this option are replaced by the maximum or minimum value.

Range: 1–100 000; default: 100

With the default setting, ratios above 100 are set to 100, and ratios below 0.01 are set to 0.01.

Imputation Mode

Specifies how missing values should be treated.

• None: Does not impute any missing values.
• Replicated Based Resampling: Imputes low abundance values by using the already available method random sampling from the distribution of the lower fifth percentile of abundance values.
• Low Abundance Resampling: Replaces missing values with random values sampled between the minimum and the lower 5% of all detected values.
• (Default) k-Nearest Neighbors: Replaces missing values with the k-nearest neighbors detected among all samples.

For more information on the Imputation Mode parameter, see Impute missing values.

Hypothesis Test

Specifies how p-values are calculated for the reported quan ratios.

• t-test (Background Based): t-test based on background population of proteins or peptides.
• ANOVA (Individual Proteins): ANOVA based on the abundances of individual proteins or peptides. For this you need to specify that ratios are calculated based on protein abundances. In addition, you need at least three (biological) replicates in each condition of the ratio.
• None: No hypothesis test is performed.

Quan Ratio Distributions

1st Fold Change Threshold

Specifies the threshold for ratio changes to be highlighted as upregulated or downregulated. Ratios higher than the specified value or lower than 1/value are highlighted in the first highlight color. A ratio of 1.0 indicates no change.

Range: 1.0–1000.0; default: 2

2nd Fold Change Threshold

Specifies the threshold for ratio changes to be highlighted as upregulated or downregulated. Ratios higher than the specified value or lower than 1/value are highlighted in the second highlight color. A ratio of 1.0 means no change.

Range: 0.0–1000.0; default: 4

3rd Fold Change Threshold

Specifies the threshold for ratio changes to be highlighted as upregulated or downregulated. Ratios higher than the specified value or lower than 1/value are highlighted in the third highlight color. A ratio of 1.0 means no change.

Range: 0.0–1000.0; default: 6

4th Fold Change Threshold

Specifies the threshold for ratio changes to be highlighted as upregulated or downregulated. Ratios higher than the specified value or lower than 1/value are highlighted in the fourth highlight color.

Range: 0.0–1000.0; default: 8

5th Fold Change Threshold

Specifies the threshold for ratio changes to be highlighted as upregulated or downregulated. Ratios higher than the specified value or lower than 1/value are highlighted in the fifth highlight color.

Range: 0.0–1000.0; default: 10