Figure 1.

Theoretical consideration of peptide co-elution. (A) Frequency count (with bin width equals to five Dalton (Da)) of tryptically-digested peptides, allowing up to two miscleavages per singly-charged peptide, of all the 9393 Saccharomyces Cerevisiae proteins present in the NCBI's non-redundant protein database (04/23/2007). This results in a total of 771,753 unique peptides with molecular weights less than 6300 Da. (B) Theoretical retention times calculated for all of the 771,753 peptides [24]. The scale color code represents the number of peptides per grid but averaged over 25 grids (five mass bins × five retention time bins). Each grid has mass width of one Da and retention time width of 6 seconds. Since the retention time variation of a given peptide is typically much larger than 6 seconds, we may view the number encoded by color as the expected minimal number of co-eluted peptides whose mass differences are within one Da. This is due to the fact that a given peptide may appear in the mixture with charge states other than that is singly charged. Panels (C) and (D) result from zooming in on different regions of panel (B) to illustrate the minimal number of co-eluted peptides occurring within a small time window and with very similar masses. It is important to note that the complexity of the pictures above will significantly increase if one were to take into account post-translational modifications, incorrect cleavage sites, and multiple charge states.