Figure 2.

Quantitative LC/MS/MS analysis of hemoglobin Providence. (A) Total Ion Chromatogram (TIC) collected during a 60-min mobile phase gradient separation of tryptically digested Hb Providence peptides (after treatment with 10× H₂O₂), using C18 reverse phase chromatography. (B) LC/MS/MS fragmentation spectra of the βK82D containing +3 peptide KKVLGAFSDGLAHLDNLDGTFATLSELHCDK (1039.872 m/z), which eluted at 36.11 min. Step 1: Database searches from LC/MS/MS analysis were utilized to identify all oxidized and unoxidized C93 containing βK82D peptides. The y an b ion series from this spectrum provided (representing the βK82D peptide with unoxidized C93) sequence information that was used to confirm the peptide identify prior to quantification. (C) Extracted ion Chromatogram (XIC) of the same peptide identified from MS/MS spectra represented in panel 2. Step 2: After database searches were used to identify all βK82D peptides containing oxidized and unoxidized C93, extracted ion chromatograms (XICs) from full MS scans were used to quantify the relative abundance of oxidized βK82D.