3no2 tyr

The analytical standards employed for the determinations were purchased from Sigma-Aldrich (St. Louis, MO, USA) for o-Tyr, Phe, 3NO₂-Tyr, 3Cl-Tyr, p-Tyr, 8OHdG and 2dG (>96% w/w purity). The isotopically labeled compounds used as internal standards were deuterated phenylalanine (Phe-D₅) from CDN Isotopes (Pointe-Claire, QC, Canada); 8-Oxo-2′-deoxyguanosine-¹³C¹⁵N₂ (8OHdG-¹³C¹⁵N₂) and 2′-deoxyguanosine-¹³C¹⁵N₂ (2dG-¹³C¹⁵N₂) from Toronto Research Chemicals (Toronto, ON, Canada); and deuterated para-tyrosine (p-Tyr-D₂) from Cambridge Isotope Laboratories (Tewksbury, MA, USA). The purity of all isotopic labeled compounds was > 98% w/w. Milli-Q^® grade water (>18.2 MΩ) was used from a Milli-Q^® Integral system (Darmstadt, Germany). Solvents used (Methanol (CH₃OH) and acetonitrile (CH₃CN)) (LC-MS grade) were obtained from JT Baker (Deventer, The Netherlands) and sodium hydroxide, phosphoric acid, and formic acid (HCOOH) were purchased from Panreac Química (Barcelona, Spain).

Standards of o-Tyr, p-Tyr, Phe, 3NO₂-Tyr, 3Cl-Tyr, 8OHdG and 2dG (>96% w/w purity) were from Sigma-Aldrich (St. Louis, MO, USA). Internal standards (ISs) p-Tyr-D₂, 2dG-¹³C¹⁵N₂, and 8OHdG-¹³C¹⁵N₂ were acquired from Cambridge Isotope Laboratories and Phe-D₅ from CDN Isotopes (Pointe-Claire, Canada).
Individual stock solutions of o-Tyr (2 mM), 3NO₂-Tyr (2 mM), 3Cl-Tyr (2 mM), 8OHdG (2 mM), 2dG (2 mM), 2dG-¹³C¹⁵N₂ (5 mM), 8OHdG-¹³C¹⁵N₂ (5 mM), Phe-D₅ (10 mM) and Phe (75 mM) were prepared and dissolved as previously described [21 (link)]. Once prepared, solutions were stored at −20 °C. Multi-component working solutions were prepared and kept at −20 °C. Standard solutions were prepared by serial dilution of the working solutions. The preparation of the standards and reagents that we used were described in detail by Cascant-Vilaplana and colleagues [21 (link)].

Acetonitrile (LC-MS grade), methanol (LC-MS grade) and formic acid (analytical grade) were purchased from Sigma Aldrich Quimica SA (Madrid, Spain). Water was Milli-Q grade (>18.2 MΩ) from a Millipore purification system. Standards of o-Tyr, m-Tyr, Phe, 3NO₂-Tyr, 3Cl-Tyr, p-Tyr, 8OHdG and 2dG (>96% w/w purity) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Deuterated phenylalanine (Phe-D₅) was purchased from CDN Isotopes (Pointe-Claire, Canada).