Identification of Endogenous Site-specific Covalent Binding of Catechol Estrogens to Serum Proteins in Human Blood.
Protein adducts covalently modified by catechol estrogens (CEs), referred to as estrogenized proteins, are potential biomarkers for estrogen homeostasis or exposure to environmental toxicants. However, serum proteins endogenously modified by CEs and the modification sites remain elusive. In this study, liquid chromatography-mass spectrometry (LC-MS)-based shotgun proteomics is applied to identify site-specific protein estrogenization in human blood via a systematic approach and stringent validation. We showed CEs, namely 2- and 4-hydroxyl estrogens which are regarded as biomarkers for estrogen homeostasis, form covalent bonds with proteins, mainly via side chain Cys, Lys, or His residue. Estrogenization of purified human serum albumin (HSA) and immunoglobulin G (IgG) at specific sites was achieved by co-incubation and used as the standards to confirm the identified estrogenization in serum proteins. Based on a database search, estrogenized peptides derived from serum proteins in patient blood were identified; endogenous estrogenization of HSA and IgG-1 at multiple sites were confirmed as compared to the standards. Based on a test using Ellman's reagent, estrogenization produced stable products and irreversibly abolished the reactivity of Cys34-HSA, which is the most important antioxidant and nitric oxide carrier in blood. Given the importance of estrogen metabolism in environmental toxicology, further exploration of estrogenized proteins is warranted for biomarker discovery and/or new mechanisms in disease process.
*Department of Chemistry and.
Chieh-Ming
CM
Fang
*Department of Chemistry and.
Ming-Chun
MC
Ku
*Department of Chemistry and.
Che-Kai
CK
Chang
*Department of Chemistry and.
Huei-Chen
HC
Liang
*Department of Chemistry and.
Tzu-Fan
TF
Wang
Department of Family Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
Chih-Hsing
CH
Wu
*Department of Chemistry and shchen@mail.ncku.edu.tw.
Shu-Hui
SH
Chen
D001665:Binding Sites; D002853:Chromatography, Liquid; D030562:Databases, Protein; D004958:Estradiol / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry; D002393:Estrogens, Catechol / Q000097:blood* / Q000737:chemistry; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D008875:Middle Aged; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D040901:Proteomics / Q000379:methods; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D000075462:Serum Albumin, Human; D053719:Tandem Mass Spectrometry
C422455:ALB protein, human; D002393:Estrogens, Catechol; D007074:Immunoglobulin G; D012709:Serum Albumin; D004958:Estradiol; C001390:2-hydroxyestradiol; C014036:4-hydroxyestradiol; D000075462:Serum Albumin, Human
catechol estrogen; estrogen homeostasis; estrogenization; protein adducts; shotgun proteomics