Enhanced-Precision Measurement of Glutathionyl Hemoglobin by MALDI-ToF MS

Abstract

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.

Glutathionyl Hemoglobin and Its Emerging Role as a Clinical Biomarker of Chronic Oxidative Stress

“…In this case, the mass difference due to GSH binding can be verified by direct electrospray ionization (ESI) measurements in intact proteins [62]. As an alternative to HPLC-ESI-MS analyses, a novel technique based on linear mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed for the quantitative determination of HbSSG in blood samples [63,64]. There are also qualitative methods to detect S-glutathionylation, such as the use of specific antibodies [65], or specific chemical labeling like the use of biotin [66].…”

“…The second one is mainly used to quantify the levels of hemoglobin species, like glycated hemoglobin and HbSSG, in blood samples, thus obtaining a quantitative analysis of a potential biomarker of oxidative stress in several pathologies. Moreover, it is characterized by high sensitivity and resolution, and allows for low-cost analysis, enabling the evaluation of a large number of samples in a short time [63,64]. Lastly, hydrogen/deuterium exchange based MS represents a potent technique able to explore the solution-phase conformational dynamics, and hence correlate functional aberrations with structural modifications of the HbSSG [51].…”