title: 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.
abstract: 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 γG and γA subunits of Hgb F. Through tandem mass spectrometry (nanoLC-MS/MS), we confirmed the adduction site as the Cys-γ94 residue and through high-resolution mass spectrometry determined that the modification occurs in both γ subunits. We also identified glutathionylation of the β subunit of Hgb A in our patient samples; we did not find modified α subunits of Hgb A or F. In conclusion, we are the first to report that glutathionylation of γG and γA 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.
authors.affiliation: Division of Neonatology, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Ehrmann
authors.forename: Kristie
authors.initials: K
authors.lastname: Rose
authors.forename: M Wade
authors.initials: MW
authors.lastname: Calcutt
authors.forename: Amy B
authors.initials: AB
authors.lastname: Beller
authors.forename: Salisha
authors.initials: S
authors.lastname: Hill
authors.forename: Theresa J
authors.initials: TJ
authors.lastname: Rogers
authors.forename: Steven D
authors.initials: SD
authors.lastname: Steele
authors.forename: David L
authors.initials: DL
authors.lastname: Hachey
authors.forename: Judy L
authors.initials: JL
authors.lastname: Aschner
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D005319:Fetal Hemoglobin / Q000032:analysis* / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry*; D006801:Humans; D062071:Infant, Extremely Premature / Q000097:blood*; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005319:Fetal Hemoglobin; D005978:Glutathione
keywords: LC-MS; glutathionylation; hemoglobin A; hemoglobin F; high-resolution tandem MS; nanoLC-MS/MS; oxidative stress; prematurity