Figure 1.

Bevacizumab quantitation using the Met-containing peptide STAYLQMNSLR. (A) Bevacizumab was digested using trypsin and analyzed by data-dependent acquisition (DDA) to identify proteotypic peptide candidates that can be used for an MRM-based assay. The Met-containing peptide STAYLQMNSLR showed the highest response during LC-MS. (B) STAYLQMNSLR either treated with (blue) or without H₂O₂ (orange), analyzed on a Sciex TripleTOF 6600 (AB Sciex). Extracted ion chromatograms of the oxidized (∼7.5 min) and nonoxidized (∼9.7 min) forms are shown, indicating the presence of both variants in the nontreated sample, while the nonoxidized variant was completely absent upon treatment with H₂O₂. (C) Bevacizumab was spiked at different concentrations into human plasma, followed by proteolytic digestion, addition of SIS peptide as normalizer, and analysis by LC-MRM (left: no oxidation, right: oxidation with H₂O₂). (D) Non-Met-containing control peptides are not significantly affected by H₂O₂ treatment. Light (NAT) peptides representing various mAbs were spiked into plasma and treated with (blue) or without H₂O₂ (orange), followed by addition of SIS peptides and LC-MRM analysis. n = 3. H₂O₂ oxidation did not considerably alter precision, nor did it considerably change the peak area ratios of corresponding NAT/SIS pairs. These ratios are indicated above the respective NAT peptide peak area bars. Error bars represent standard deviations. * = peak area × 10.