Humans are continually exposed to various reactive oxygen species (ROS) due to endogenous aerobic metabolism as well as from environmental pollution. Excessive production of ROS plays an important role in disease onset and progression, including cancer. Post-translational oxidative modifications of hemoglobin have been used as surrogate biomarker for monitoring the oxidative stress in vivo. In this study, Fenton reaction was used as a model to generate ROS and to react with human hemoglobin in vitro. After trypsin digestion, the sites and types of modification were identified by nanoflow liquid chromatography nanospray ionization coupled with high-resolution mass spectrometry. In addition to oxidation at certain sites of Met, His, Tyr, and Lys, conversion of His to Asp and hydroxyl-Asp was also identified. Furthermore, advanced oxidation of His to hydroxyl Asp was identified at two sites. Because elevated oxidative stress is tightly associated with oral cancer, we measured the relative extent of modification at the identified sites were quantified in hemoglobin from oral cancer patients and compared them with those from healthy subjects. Our results showed that the extents of oxidation at α-His-45, α-Lys-99, and β-His-77 in globin were significantly higher in oral cancer patients than in healthy subjects, while the extents of conversion of histidine residues at α-20, α-50, and β-2 to aspartate were significantly lower. Moreover, the advanced oxidation of His to hydroxyl Asp at α-His-50 and β-His-2 are also significantly higher in oral cancer patients than in healthy subjects (<i>p</i> &lt; 0.0005). To our knowledge, this is the first report on advanced oxidation of His to hydroxyl Asp in human hemoglobin. This advanced oxidative modification in hemoglobin might serve as potential biomarker to assess oxidative stress in oral cancer patients.

Higher advanced oxidative modifications in hemoglobin of oral cancer patients as measured by nanoflow liquid chromatography tandem mass spectrometry

2019 Fall National Meeting

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