Figure 3.

Optimization of tryptic digestion using MDA‐MB 231 lysate spiked with 2.5 fmol of both SIS and dSIS peptide. A) PTEN (¹⁴⁸AQEALDFYGEVR¹⁵⁹) peptide recoveries at different incubation times (0.5–4 h) and at protein:trypsin (P:T) ratios of 1:10 (dark red) and 2:1 (light red). (*) indicates a significant difference (p < 0.01) between two protein:trypsin ratios at the same incubation time. N = 4 per tested incubation time. B) p110α (⁵⁰³EAGFSYSHAGLSNR⁵¹⁶) recoveries at different incubation times (0.5–4 h) at protein:trypsin ratios of 1:10 (light blue) and 2:1 (dark blue). (*) indicates significant difference (p < 0.01) between two protein:trypsin ratios at the same incubation time. N = 4 per tested incubation time. C,D) Overlaid mass spectra of enriched PTEN and p110α peptides after 1 h digest using C) 10:1 and D) 1:2 protein:trypsin, recorded in the linear mode. Spectra show similar background for both tested protein:trypsin ratios and no peaks are interfering with the target peptide peaks.