DNA repair is essential for maintaining genomic stability and plays an important role in preventing disease processes. Recent years, DNA repair proteins have emerged as predictive, prognostic, and therapeutic targets in cancer. In particular, inhibiting specific enzymes in the base-excision-repair (BER) pathway may be a useful anticancer strategy. DNA polymerase β (Polβ) an essential component of BER pathway involved in the repair of oxidative DNA damage. Among the DNA glycosylases involved in the first step of BER, human endonuclease 8-like 1 (NEIL1) protein is a bifunctional enzyme with an additional β,δ-elimination activity. In order to clearly demonstrate the role of these proteins in cancer development and treatment, absolute quantification of proteins in biological samples is required. This study shows the simultaneous measurement of NEIL1 and Polβ proteins by liquid chromatography-high resolution mass spectrometry (HR-LC-MS) with targeted proteomics approach. 15N-labeled analogues of NEIL1 (15N-NEIL1) and Polβ (15N-Polβ) as internal standard, were used to develop an accurate method for the measurement of NEIL1 and Polβ. Both 15N-NEIL1 (1.6 µg/µL) and 15N-Polβ (1.8 µg/µL) were hydrolyzed with trypsin. High resolution LC-MS analyses were performed using a Thermo Scientific™ Dionex™ UltiMate™ 3000 HPLC coupled to an orbitrap mass spectrometer (Thermo Scientific™ Exactive Plus™ Orbitrap™ MS) equipped with a heated electrospray-ionization (HESI) ion source in the positive ionization mode. The XCalibur Software (Thermo Scientific™) was used for data analyses. The monoisotopic masses of the doubly-charged molecular ions [(M+2H)2+] were used to analyze mixtures of 15N-NEIL1 and 15N-Polβ on the basis of precursor ions. Fifteen tryptic peptides from 15N-NEIL1 and nine tryptic peptides from 15N-Polβ were identified, which matched to a subset of the theoretically predicted tryptic peptides of proteins. These peptides provided a statistically significant protein score that would unequivocally identify both proteins. The results obtained showed that the HR-LC-MS measurement method is highly suitable for the multiple analysis of NEIL1 and Polβ proteins in biological samples. Thus, by absolute quantification of these DNA repair proteins, the nature of DNA repair can be explained in more detail. ORCID NO: 0000-0002-7311-4020
Anahtar Kelimeler: High resolution mass spectrometry, Human DNA repair protein, NEIL1, Polβ
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