An LC/ESI-SRM/MS method to screen chemically modified hemoglobin: simultaneous analysis for oxidized, nitrated, lipidated, and glycated sites

Proteins are continuously exposed to various reactive chemical species (reactive oxygen/nitrogen species, endogenous/exogenous aldehydes/epoxides, etc.) due to physiological and chemical stresses, resulting in various chemical modifications such as oxidation, nitration, glycation/glycoxidation, lipidation/lipoxidation, and adduct formation with drugs/chemicals. Abundant proteins with a long half-life, such as hemoglobin (Hb, t_1/2 63 days, ∼150 mg/mL), are believed to be major targets of reactive chemical species that reflect biological events. Chemical modifications on Hb have been investigated mainly by mechanistic in vitro experiments or in vivo/clinical experiments focused on single target modifications. Here, we describe an optimized LC/ESI-SRM/MS method to screen oxidized, nitrated, lipidated, and glycated sites on Hb. In vivo preliminary results suggest that this method can detect simultaneously the presence of oxidation (+16 Da) of α-Met³², α-Met⁷⁶, β-Met⁵⁵, and β-Trp¹⁵ and adducts of malondialdehyde (+54 Da) and glycation (+162 Da) of β-Val¹ in a blood sample from a healthy volunteer. [Figure not available: see fulltext.]