Glutathionyl-hemoglobin (HbSSG) is used as a human biomarker to pinpoint systemic oxidative stress caused by various pathological conditions, noxious lifestyles, and exposure to drugs and environmental or workplace toxicants. Measurement by MALDI mass spectrometry is most frequently used, however, the method suffers from excessive uncontrolled variability. This article describes the improvement of a MALDI-ToF mass spectrometry method for HbSSG measurement through enhanced precision, based on strict control of sample preparation steps and spreadsheet-based data analysis. This improved method displays enhanced precision in the analysis of several hundred samples deriving from studies in different classes of healthy and diseased human subjects. Levels span from 0.5% (lower limit of detection) up to 30%, measured with a precision (as SE%) < 0.5%. We optimized this global procedure to improve data quality and to enable the Operator to work with a reduced physical and psychological strain. Application of this method, for which full instruction and the data analysis spreadsheet are supplied, can encourage the exploitation of HbSSG to study human oxidative stress in a variety of pathological and living conditions and to rationally test the efficacy of antioxidant measures and treatments in the frame of health promotion.
Keywords: MALDI-ToF; glutathione; hemoglobin; method optimization; oxidative stress; red blood cells; spreadsheet.