Specifying the quantification channels - Proteome Discoverer Software - Specifying the quantification channels - Proteome Discoverer Software - Proteome Discoverer Software

The first step in setting up the quantification is to specify the quantification channels to use. The process of specifying label modifications is similar for precursor ion quantification and reporter ion quantification, but it also has some differences:

• For precursor ion quantification, you specify the label modifications for each quantification channel. For precursor ion quantification, specifying the label modifications for quantification channels other than the unlabeled channel is mandatory.
• For reporter ion quantification, you set the label modification that produces all reporter ion quantification channels. For reporter ion quantification, specifying the label modifications is optional because the information about the modification of the peptides is not necessary for processing the data. It is only used to verify the peptides when the application loads the reports.
• When you specify at least one of the label modifications in the quantification method, the application verifies that each identified peptide has at least one of the specified modifications during processing.
• When the application identifies a peptide with none of the specified label modifications, this peptide cannot be the source of reporter peaks in the MS/MS spectra. As a result, the application marks the peptide “No Quan Labels” in the result file. It does not use these peptides when it calculates the protein quantification values.
• When the application finds a peptide that does not have an iTRAQ or TMT label as a modification, even though reporter ions were present, it leaves the Ratio columns in the result file blank.

When you install the application, the default methods for TMT and iTRAQ do not include the correct label modification. When you set up a workflow, ensure that these modifications are selected as either fixed or variable. The application does not automatically update already existing reporter methods; you must manually specify the label modifications.

When you open old result files that contain reporter quantification data, the label modifications of the quantification method of the result file appear as None on the Quan Channels page of the Quantification Method Editor dialog box. You can manually specify the label modification, which then triggers the validation of the peptides, and save the change in the quantification method in the result file.

When you do not set the label modifications on the Quan Channels page, the application does not perform the validation.