Quantification of the Endogenous Adduction Level on Hemoglobin and Correlation with Albumin Adduction via Proteomics: Multiple Exposure Markers of Catechol Estrogen

Hung Hsiang Jen, Husam Kafeenah, Ting Yao Chang, Yu Min Lin, Yan Shen Shan, Chih Hsing Wu, Shu Hui Chen

Research output: Contribution to journalArticlepeer-review

Abstract

Catechol estrogens (CEs) are genotoxic metabolites whose detection is challenging due to their low concentrations and high variability in the blood. By intact protein and free CE measurement of the spiked hemolysate, endogenous CEs were revealed to mainly (>99%) exist as hemoglobin (Hb) adducts in red blood cells. In order to detect endogenous CE-Hb adducts, we developed a two-step method that involved protein precipitation and solid phase extraction to purify Hb from red blood cells, and the method was coupled with proteomics using liquid chromatography-tandem mass spectrometry. Using bottom-up proteomics and standard additions, we identified C93 and C112 of Hb-β as the main adduction sites of Hb, and this accounted for CE-induced oxidization of adducted peptides by sample preparation. The non-adducted, adducted, and oxidized tryptic peptides that covered the same Hb-β sequences were targeted by parallel reaction monitoring to determine the adduction level in red blood cells. A quantification limit (S/N < 8) below the endogenous CE-Hb adduction level with relative standard errors that ranged from 5 to 22% was achieved and applied to clinical samples. The human serum albumin (HSA) adduction levels from the same patient were also determined using a previously developed method (Anal. Chem. 2019, 91, 15922-15931). A positive correlation (R2 = 0.673) between the CE-HSA and CE-Hb adduction level was obtained from all clinical samples, and both levels were significantly (p < 0.005) higher for patients with breast cancer compared to healthy controls. However, double indexes derived from the red blood cell and the serum, respectively, provide higher precision and confidence in predicting cancer risk than the single index. This study reported an efficient sample preparation for proteomics-based Hb adducts and revealed the potential of using multiple blood proteins for developing more reliable and specific markers based on protein adductomics.

Original languageEnglish
Pages (from-to)4248-4257
Number of pages10
JournalJournal of Proteome Research
Volume20
Issue number9
DOIs
Publication statusPublished - 2021 Sep 3

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Chemistry(all)

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