Figure 1.

An illustration of the on-the-fly OAO strategy. (A) Total ion chromatogram (TIC) of the 8c2 spiked-in brain extract followed by digestion and nano-LC/SRM-MS analysis. SRM events were grouped by retention time windows. In total 675 SRM transitions, corresponding to 15 peptide candidates for 8c2, were monitored with a 125-min gradient. The full list of candidate peptides and SRM transitions is presented in Supplementary Table S-1. (B) Chromatogram of the scheduled retention time window of 60-65 min, within which several peptides were optimized. The peak represents A₆₇TIITDTSSNK₇₇. Each data point on the profile marks one independent optimization cycle. (C) The orthogonal array design (L₂₅) for SRM optimization of three key factors, where each was investigated at five levels. (D) 25 SRM trials were performed in sequence within a short period (<1s total per cycle, and >20 cycles within the peak elution ). The extracted ion current (XIC) peak was obtained to evaluate the S/N for each trial (c.f. Supplementary Figure S-2).