Fig. 2

A small modification of the peptide identification approach drastically increases the number of peptides with 1–10% label that can be identified. Number of peptide spectral matches (PSMs) identified at different ¹³C label percentages using six different peptide identification strategies. Cultures of a B. subtilis and b E. coli were grown in Bacillus minimal medium or M9 minimal medium (E. coli) in which a percentage of the glucose was replaced with ¹³C_1-6 glucose for > 10 generations to achieve close to complete labeling. Three biological replicates were run for each label percentage. Peptides were identified using the SEQUEST HT Node in Proteome Discoverer (version 2.2) with six different strategies to account for the mass shifts caused by addition of heavy atoms. Standard search: no dynamic modifications to account for addition of label; open search: the precursor mass tolerance was set to 20 Da allowing for the potential addition of 20 neutrons (e.g., ¹³C atoms) in a peptide; dynamic modifications: allowing for up to three dynamic modifications each of two custom peptide modifications adding a 1 neutron mass shift and a 2 neutron mass shift (up to 9 neutrons in total per peptide); modifications on termini: six dynamic modifications were set up that were restricted to either the C or the N-terminus of the peptide. The modifications account for mass shifts of 1 to 6 neutrons and depending on the search strategy the low mass shifts (1, 2, and 3 neutrons) were set up as modifications on the C or the N-Terminus or low and high mass shift modifications were distributed between both termini. Each modification can only be added to a terminus once. This strategy allows for a total of 21 neutron additions to a peptide