Fig. 2

Sampling elution peaks and co-isolating precursor ions during LC-MS/MS, each labeled peptide elutes from the chromatographic column as an elution peak over a time period typically ranging from 10 to 40 s (5–20 s at mid-height) while its ions are isolated (sampled) for MS2 analysis over much shorter intervals, typically ranging from 5 to 80 ms. If the elution peak is sampled too early (left panel) or too late (right panel), the fraction of the peptide ions used for quantification and sequence identification is smaller compared to sampling the apex (middle panel). To increase the fraction of sampled ions per peptide, we used DO-MS to increase the probability of sampling the apex (Fig. 3c), decreased the elution peak width (see the “Methods” section), and increased the MS2 fill time to 300 ms. SCoPE2 quantifies peptides sequentially, one peptide at a time. For each analyzed peptide, the MS instrument aims to isolate only ions from the peptide by applying a narrow mass filter (m/z isolation window) denoted by a red rectangle in the sketch above. Yet, ions from other peptides might also fall within that window and thus become coisolated, as shown with the blue and orange peptides in the third panel. Since coisolated peptides contribute to the measured reporter ions (RI), coisolation reduces the accuracy of quantification. To minimize coisolation, we reduced the isolation windows to 0.7 Th and improved apex targeting as described above. The success of these optimizations was evaluated by the precursor ion fraction (PIF), a benchmark computed by MaxQuant as an estimate for the purity of the ions isolated for fragmentation and MS2 analysis, Fig. 3d