Free base l arginine

All electrolyte solutions contained Optima™ LC/MS Grade water (Thermo Scientific PN W6500) and or Optima™ LC/MS Grade ACN (Fisher Chemical PN A955) solvents. Anolyte was an aqueous solution of 1% formic acid (ACROS Organics™ PN 27048). Catholyte was an aqueous solution of 1% diethylamine >99.5% Reagent Grade (Sigma Aldrich PN 47216) and the mobilizer was a 1% formic acid, 50% ACN, and 49% water.
A 500 mM cathodic spacer solution containing Free Base l‐Arginine ≥ 99.5% (Arg) (Sigma Aldrich PN 11009–100G‐F) was prepared by dissolving 0.870 mg of Arg powder into 10 mL of Optima™ LC/MS Grade water. A 200 mM anodic spacer solution containing Free Acid Iminodiacetic Acid 98% (IDA) (Sigma Aldrich PN 220000–500G) was prepared by dissolving 0.270 mg of IDA powder into 10 mL of Optima™ LC/MS Grade water. Spacer solutions were stored at room temperature.
Peptide amino acid sequences initially reported by Shimura were synthesized and individually dissolved in LC/MS grade water at 5 mg/mL to act as internal pI markers 19.
Prior to desalting, lyophilized mAb material was reconstituted with MS grade water. All mAb samples were desalted with a 0.5 mL Zeba™ 7K MWCO spin desalting column (Thermo Fisher Scientific PN 89882).

RAW264.7 cells and A549 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in either arginine-free DMEM (Thermo Fisher Scientific Life Sciences, Waltham, MA, USA) or standard DMEM (containing 400 µM l-arginine; Sigma-Aldrich, St. Louis, MO, USA), both supplemented with fetal calf serum (10%; LabTech, Uckfield, United Kingdom). Confluent cultures were treated with LPS (RAW264.7 cells; 1 μg/ml; Sigma-Aldrich) or IL-1β (A549 cells; 10 ng/ml; R&D Systems, Minneapolis, MN, USA) for 24 h to induce iNOS and COX-2, in some cases in the presence of the NOS inhibitors, l-NAME (300 μM; Sigma-Aldrich) or ADMA (1 mM; Sigma-Aldrich). Increasing concentrations of ibuprofen arginate (Zambon Pharma), ibuprofen sodium (Sigma-Aldrich), l-arginine free base (Sigma-Aldrich), or the combination of ibuprofen sodium and l-arginine were also added such that the molar concentration of l-arginine present in each preparation was the same, or for ibuprofen sodium, the molar concentration of ibuprofen. After 24 h, media were collected for measurement of nitrite accumulation using the Griess reaction (Sigma-Aldrich) or PGE₂ using immunoassay (Cisbio, Codolet, France). Cell viability was assessed using the alamarBlue metabolic activity assay (Thermo Fisher Scientific).