Glutathionylated γG and γA subunits of hemoglobin F: a novel post-translational modification found in extremely premature infants by LC-MS and nanoLC-MS/MS.

Oxidative stress plays an important role in the development of various disease processes and is a putative mechanism in the development of bronchopulmonary dysplasia, the most common complication of extreme preterm birth. Glutathione, a major endogenous antioxidant and redox buffer, also mediates cellular functions through protein thiolation. We sought to determine if post-translational thiol modification of hemoglobin F occurs in neonates by examining erythrocyte samples obtained during the first month of life from premature infants, born at 23 0/7 - 28 6/7 weeks gestational age, who were enrolled at our center in the Prematurity and Respiratory Outcomes Program (PROP). Using liquid chromatography-mass spectrometry (LC-MS), we report the novel finding of in vivo and in vitro glutathionylation of gamma G and gamma A subunits of Hgb F. Through tandem mass spectrometry (nanoLC-MS/MS), we confirmed the adduction site as the Cys-gamma 94 residue and through high-resolution mass spectrometry determined hat the modification occurs in both. subunits. We also identified glutathionylation of the beta subunit of Hgb A in our patient samples; we did not find modified a subunits of Hgb A or F. In conclusion, we are the first to report that glutathionylation of gamma G and gamma A of Hgb F occurs in premature infants. Additional studies of this post-translational modification are needed to determine its physiologic impact on Hgb F function and if sG-Hgb is a biomarker for clinical morbidities associated with oxidative stress in premature infants. Copyright (C) 2014 John Wiley & Sons, Ltd.