Milli q synthesis a10 purification system

Stock solutions of LE (American Peptide, Sunnyvale, CA), ME (Sigma-Aldrich, St. Louis, MO), and yaGfl (GL Biochem, Shanghai, China) standards were prepared by dissolving the solid as received from the manufacturer in water, which was obtained from a Millipore Milli-Q Synthesis A10 purification system (Billerca, MA), to a concentration of 1 mM. Serial dilutions were then used to prepare 5 nM standards of each peptide. LE, ME, yaGfl standard mixtures were then further diluted to the desired concentrated (usually 100 pM) in either water or a modified Ringer’s solution consisting of 148 mM NaCl (EMD-Millipore, Darmstadt, Germany), 1.2 mM CaCl₂ (EMD-Millipore), 2.7 mM KCl (Sigma-Aldrich), and 0.85 mM MgCl₂ (Fisher Scientific, Fair Lawn, NJ) at pH 7.4. All standards were made fresh daily.

Uracil, acetanilide, methyl and ethyl esters of p-hydroxybenzoic acid (parabens), acetophenone, propiophenone, butyrophenone, benzophenone, valerophenone, hexanophenone, heptanophenone, and octanophenone were from Sigma-Aldrich (St. Louis, MO). Stock solutions, 25 mM, were made in acetonitrile (Chromsolv, LC-MS grad, Fisher Scientific, Fair Lawn NJ). Samples were diluted to concentrations from 5 to 20 µM with deionized water. Sample concentrations were selected to maximize concentration while factoring in column loadability and solubility. Water was from a Millipore Milli-Q Synthesis A10 purification system (Billerica, MA). Peptide samples were from an LC-MS grade BSA tryptic digest from Fisher. The lyophilized sample was diluted, as per reagent instructions, to 1 pmol/µL in 95:5 0.1% Formic acid/acetonitrile. Formic acid was from Sigma. The BSA sample was stored at −5 °C when not in use and used within 48 h of thawing/dilution.