Multiple oxidative and advanced oxidative modifications of hemoglobin in gastric cancer patients measured by nanoflow LC-MS/MS.

Overproduction of the reactive oxygen, nitrogen, and chlorine species by the immune systems during chronic infection and inflammation can cause structural and functional changes of cellular proteins. The high abundance of hemoglobin in blood makes hemoglobin adducts suitable biomarkers for assessing the damage of these reactive species in the body. In this study, a total of 23 types and sites of modification in human hemoglobin were simultaneously analyzed, including monooxygenation of histidine, tyrosine, methionine, and aspartate, conversion of histidine to aspartate and hydroxyaspartate, as well as chlorination and nitration of tyrosine residues. Hemoglobin was isolated from the blood of the study subjects, digested into peptides, and the extents of these modifications were quantified relative to their parent peptides using nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry under selected reaction monitoring (nanoLC - NSI-MS/MS-SRM). The extents of monooxygenation at β-His-77 and β-Tyr-130, chlorination at α-Tyr-24 and β-Tyr-130, and nitrosylation at α-Tyr-24 were elevated in gastric cancer patients. Conversely, conversion of histidine to aspartate at α-His-20, α-His-50, β-His-2, β-His-143, and monooxygenation at β-His-143 were decreased in gastric cancer patients. The areas under the receiver operating characteristics (AUC of ROC) curve of these ten types and sites of hemoglobin modifications were between 0.7644 and 0.9644. The ratio of conversion of histidine to hydroxyaspartate versus conversion of histidine to aspartate was significantly higher in gastric cancer patients at α-His-20, α-His-50, and β-His-143 (p < 0.05) with AUC of ROC ranging between 0.7689 and 0.9178. To our knowledge, this is the first report of simultaneous measurement of multiple types of oxidative and advanced oxidative hemoglobin modifications in gastric cancer patients. The results revealed elevated levels of oxidative stress-induced protein damage in gastric cancer patients and the potential of using these modifications of hemoglobin as biomarkers for evaluation of oxidative stress in one drop of blood.