Methanoic acid

The analysis of the aromatic intermediates and short chain carboxylic acids obtained during the MO degradation was carried out in a Shimadzu Prominence HPLC equipment. 3.0 mL of methanol (HPLC/PDA grade, Panreac) were added on 7.0 mL of sample collected from the catalytic test developed under optimal conditions of reaction, then filtered (ϕ = 0.45 μm, PVDF filters), and measured. The fractions were separated in a Premier C18 column (5.0 μm × 4.6 mm × 250 mm, Shimadzu) at 30°C. Injection volume was 30 μL and flow 0.8 mL/min, using a gradient method (A/B) with methanol (A) and water acidified with phosphoric acid at pH 2.3 (B) as mobile phases (0–5 min, 0/100 v/v; 10 min, 10/90 v/v; 15 min, 40/60 v/v; 20 min, 60/40 v/v; and 30 min 70/30 v/v). Following compounds were used to calculate response factors of targeted analytes: methyl orange (Sigma-Aldrich, 85%), 4-aminobenzenesulfonic acid (Sigma-Aldrich, 99%), Propanedioic acid (Sigma-Aldrich, 99%), maleic acid (Sigma-Aldrich, ≥99%), Butanedioic acid (Merck, 99.99%), ethanoic acid (Panreac, 99.8%), methanoic acid (Carlo Erba, 85%), oxalic acid (Alfa Aesar, 99%), benzenesulfonic acid (Sigma-Aldrich, 98%), benzene-1,4-diol (Panreac, 99%), Cyclohexa-2,5-diene-1,4-dione (Fluka, ≥99.5%), phenol (Sigma-Aldrich, 99%), p-nitrophenol (Alfa Aesar, 99%), and Benzene-1,2-diol (Alfa Aesar, 99%).

Briefly, the dried solutions were incubated with 50 mM Tris [2-carboxyethyl]phosphine (TCEP, Sigma) in 25 mM NH₄HCO₃ at 56°Cfor 1 hour to reductively cleave the disulfide bonds of proteins. The resulting sulfhydryl functional groups were alkylated with 100 mM iodoacetamide (IAA, Amersham) in 25 mM NH₄HCO₃ at room temperature for 0.5 hour in the dark. Subsequently, the proteins were digested with 20 ng/μl porcine trypsin (modified proteomics grade, Sigma) at 37°C overnight. The mixture of the resulting tryptic peptides was extracted twice from the gel pieces with 5% trifluoroacetic acid (TFA, Fluka) in a 50% ACN solution. The pooled extracts were evaporated in a vacuum centrifuge (Labconco, Kansas, MO), and resuspended in 0.1% methanoic acid (Sigma) prior to the LC separation and MS detection. In-house database was constructed with the FASTA protein sequences downloaded from uniprot mapping (http://www.uniprot.org) containing all proteins from mouse. For each experiment, MS/MS spectra were extracted from the raw data files by extractmsn program in the Proteome Discoverer 2.0 (ThermoFinnigan, San Jose, CA).