Seven different scenarios can occur when you derive protein quantification ratios from peptide quantification ratios. These cases show how the validity of using a given quantification result for the quantification of a certain protein depends on whether this particular quantification result is unique or shared among other peptides.
The peptide quantification ratios are taken from the associated quantification results. The term quantification result in this topic refers to MS/MS reporter intensities taken from the same scan as the identification (for example, ID-CID) or from a separate quantification scan (for example, Quan-HCD). The term also refers to intensities derived from the precursor scans in precursor ion quantification. A quantification result here is a general quantity associated with one or more peptides that are, in turn, associated with one or more proteins.
Case 1: Quantification result associated with one spectrum, one peptide, and one protein
Case 1 is the simplest case. The quantification result is associated with one identification spectrum—whether the quantification results come from the same identification spectrum, from a different quantification spectrum, or from the precursor ion—and one peptide that is contained in one protein. The quantification result is unique for this protein. The application can mark peptide A “Unique” in the Quan Info column of the PSMs page if the quantification result meets other criteria.
Case 2: Two quantification results associated with two spectra, one peptide, and one protein
Case 2 is a variant of case 1. Each of two different quantification results is associated with a different identification spectrum. Both identification spectra identify peptide A, which is a peptide with the same sequence. Peptide A is only contained in one protein. Each of the two different quantification results is unique for just one protein. The peptides are redundantly identified and quantified, and you can use both for the quantification of protein A.
Case 3: Quantification result associated with two Spectra, two peptides, and one protein
Case 3 is similar to case 2 but varies from it in a slight but important way. In case 3, the two identification spectra are associated with the same quantification result rather than with two different quantification results. For example, you might obtain these results if you trigger the same precursor two times for MS/MS. It does not matter whether peptide A and peptide B are the same peptides (redundantly identified) or different peptides that are accidentally contained in the same protein. It also does not matter whether they are identified by the same search engine or by two different search engines, for example, a CID spectrum and an ETD spectrum. The quantification result is still unique for just one protein. However, you cannot use the quantification ratio of both peptides A and B to calculate the quantification ratio of protein A, because it is the same quantification result, and you do not want to use the same quantification result multiple times for the same protein. In this case, the application marks peptide A—the peptide with the better identification—as “Unique” and the other peptide as “Redundant” for quantification (rather than redundant for identification).
Case 4: Quantification result associated with one spectrum, two peptides, and one protein
In case 4 the two peptides can be identified by the same search engine and have different ranks, or they can be identified by different search engines and both have rank 1. It does not matter whether peptide A and B have the same sequence with different PTM states or different sequences. The quantification result is unique for protein A. You can use it to calculate the protein ratio, but you must only use it once. The application marks the “better” peptide as “Unique” and the other as “Redundant” for quantification.
In case 5 the quantification result is associated with one identification spectrum and one peptide, but this peptide is contained in more than one protein. The quantification result is potentially shared between these proteins, and you do not know how to share it. If the quantification method specifies using only unique peptides for protein quantification, you would not use peptide A in this case. If the quantification method specifies using all peptides for protein quantification, the quantification result of peptide A would be divided equally between both proteins.
Case 5: Quantification result associated with one spectrum, one peptide, two proteins
Case 6: Quantification result associated with one spectrum, two peptides, and two proteins
In case 6 the quantification result is associated with one identification spectrum from which two different peptides are identified either by the same search engine as different ranks or by different search engines. The two different peptides are contained in two different proteins. The two different peptides are both unique to just one protein. Nevertheless, the associated quantification result is the same, and you do not want to use it to calculate the protein ratios if you specified in the quantification method to use only unique peptides. Only if you specify using all peptides can you use them for protein quantification. This case illustrates the discrepancy between the uniqueness of peptides and the uniqueness of the quantification results.
Case 7: Quantification result associated with two spectra, two peptides, and two proteins
Case 7 is a variant of case 6. Either the same search engine or different search engines identify different identification spectra, for example, CID and ETD spectra. As in case 6, the peptides are unique, but the quantification result is not. The result depends on whether you specified in the quantification method to use only unique peptides or all peptides.