title: Accurate, Direct, and High-Throughput Analyses of a Broad Spectrum of Endogenously Generated DNA Base Modifications with Isotope-Dilution Two-Dimensional Ultraperformance Liquid Chromatography with Tandem Mass Spectrometry: Possible Clinical Implication.
abstract: Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA. A set of ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can be also generated by TET enzymes. All of the aforementioned modifications seem to play some regulatory roles. We applied isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of the 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. Analyses of DNA extracted from matched human samples showed that the 5-(hydroxymethyl)-2'-deoxycytidine level was 5-fold lower in colorectal carcinoma tumor in comparison with the normal one from the tumor's margin; also 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine were lower in colorectal carcinoma tissue (ca. 2.5- and 3.5-fold, respectively). No such differences was found for 2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxyuridine. The presented methodology is suitable for fast, accurate, and complex evaluation of an array of endogenously generated DNA deoxynucleosides modifications. This novel technique could be used for monitoring of cancer and other diseases related to oxidative stress, aberrant metabolism, and environmental exposure. Furthermore, the fully automated two-dimensional separation is extremely useful for analysis of material containing a considerable amount of coeluting interferents with mass-spectrometry-based methods.
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, and ‡Department of Surgery, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń , 85-095 Bydgoszcz, Poland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gackowski
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, and ‡Department of Surgery, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń , 85-095 Bydgoszcz, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Starczak
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, and ‡Department of Surgery, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń , 85-095 Bydgoszcz, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Zarakowska
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, and ‡Department of Surgery, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń , 85-095 Bydgoszcz, Poland.
authors.forename: Martyna
authors.initials: M
authors.lastname: Modrzejewska
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, and ‡Department of Surgery, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń , 85-095 Bydgoszcz, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Szpila
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, and ‡Department of Surgery, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń , 85-095 Bydgoszcz, Poland.
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Banaszkiewicz
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, and ‡Department of Surgery, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń , 85-095 Bydgoszcz, Poland.
authors.forename: Ryszard
authors.identifier: 0000-0002-7259-1511
authors.initials: R
authors.lastname: Olinski
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009708:Nucleotidases / Q000032:analysis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013950:Thymus Gland / Q000378:metabolism
chemical_list: C528358:5-carboxydeoxycytidine; C402104:5-formyl-2'-deoxycytidine; D015415:Biomarkers; D003841:Deoxycytidine; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D004247:DNA; D006899:Mixed Function Oxygenases; D009708:Nucleotidases; C028331:deoxynucleotidase