Figure 2.

Reproducibility of the 2D-LC setup for four consecutive experiments: (A) extracted mass chromatograms of T49/50 peptide from BSA (DAIPENLPPLTADFAEDKDV(C)K, [MH₃]³⁺ = 820.06) which eluted only in Fraction 3 of 5 step elutions (with 15.4% ACN); (B) extracted mass chromatograms of T43 peptide from ENL (VNQIGTLSESIK, [MH₂]²⁺ = 644.86) eluted only in Fraction 4 out of 5 (using 18.6% ACN). All mass chromatograms were generated using an extraction mass window of 0.1 Da around the corresponding monoisotopic peaks. Second dimension chromatography runs were performed at 12 µL/min using a 30 min gradient (7–35% ACN, 0.1% FA). The amounts of digests loaded on column were 20 fmoles of ENL and 100 fmoles of BSA.