Quantification of Folylpolyglutamate Substrates

FolGlu_n reaction mixtures were analyzed on a C18 Acquity UPLC HSS T3 2.1 × 100 mm column with 1.8 μm particles (Waters), guarded by a 0.22 μm pre-filter (Waters) and VanGuard pre-column (Waters), coupled to an Agilent 1200 Series UPLC instrument (Agilent Technologies, Santa Clara, CA, USA). Mobile phase A was 25 mM sodium phosphate buffer, pH 6.0 (Penta), supplemented with 0.02 % (w/v) sodium azide (Sigma Aldrich); mobile phase B was acetonitrile (VWR International, Radnor, PA, USA). Elution of individual FolGlu_1/2/3/4/5/6 molecules and their cleavage products was performed isocratically at 2.0 % / 1.5 % / 1.1 % / 0.4 % / 0.2 % / 0.0 % acetonitrile, respectively. The column temperature was set to 50.0 °C. The HPLC runs consisted of 1.8 min isocratic flow at 0.0 - 2.0 % B, 0.1 min transition to 10.0 % B, 1.1 min at 10.0 % B, 0.1 min transition back to 0.0 - 2.0 % B, and 7.4 min re-equilibration. Analytes were detected at 281 nm and 354 nm. Inhibition reactions with FolGlu₁ were analyzed in the same manner except that the percentage of acetonitrile was 2.1 % instead of 2.0 %. The substrate turnover was quantified as the ratio of the substrate and product peak areas. The sum of the areas of the substrate and the product served as an internal standard. The limit of quantification was at least 10 nM (when calculated as baseline height multiplied by 10).