Glycolic acid

Five hundred milligrams of total protein lysate were reduced with 5 mM DTT (Sigma) for 30 min at 60°C and alkylated with 10 mM of iodoacetamide (Sigma) in the dark at room temperature for 30 min. Trypsin (Promega) was added at ratio of 1:50 ratio for 18 h at 37°C. Samples were adjusted to 1% (v/v) trifluoroacetic acid (TFA) (Sigma), 80 mg/ml glycolic acid (Sigma), and the precipitated deoxycholate was removed by centrifugation. Five milligrams of TiO₂ beads (Titanisphere, 10 μm) were washed once in 0.1% (v/v) TFA, 70% (v/v) acetonitrile (ACN), and 80 mg/ml glycolic acid, added directly to the sample and incubated with shaking for 1 h. A C8 stage-tip was prepared and washed with methanol (Sigma), then 0.1% (v/v) TFA, 70% (v/v) ACN, and 80 mg/ml glycolic acid (40 μl). TiO₂ beads were added to the C8 stage-tip and tips were centrifuged 1000 × g until all liquid was dispensed. Beads were washed on tip with 300 μl of 0.1% TFA, 70% ACN, 80 mg/ml glycolic acid (300 μl) twice then thrice with 0.1% (v/v) TFA and 70% (v/v) ACN. Phosphopeptides were eluted from TiO₂ tip with consecutive 100 μl additions of 1% (v/v) ammonia (Sigma) with 0, 30, and 50% (v/v) ACN. Samples were immediately dried and resuspended in 1% (v/v) TFA and 5% (v/v) ACN for LC-MS/MS.

Calibration of the high-performance liquid chromatography (HPLC) system for carboxylic acid quantification was carried out with formic acid (98–100%) from Chem-Lab (Zedelgem, Belgium) and acetic acid, glycolic acid and sodium lactate (all >99%) from Sigma-Aldrich (St. Louis, MO, USA). The acid model solution for the ED experiment was prepared with formic acid (98–100%) from Chem-Lab, L-lactic acid (90%) from Roth (Karlsruhe, Germany) and glycolic acid (99%) and acetic acid (99.8%) from Sigma-Aldrich. Sodium hydroxide pellets (98%) for pH adjustment were purchased from Thermo-Fisher Scientific (Waltham, MA, USA).
The pretreated kraft BL was supplied by a local pulp mill. Twelve different wood species were processed by the mill, amongst which were beech, birch, acacia and oak. The liquor composition and sample characteristics are summarized in Table 1. Besides the components listed in Table 1, BL also contains inorganic pulping chemicals and salts such as sodium, sulphur compounds and carbonate as well as organic compounds such as other carboxylic acids, hemicelluloses and extractives that were not quantified.

The following materials and chemicals were purchased from Merck, Johannesburg, South Africa: standards (98%) of glycolic acid, α-ketoglutaric acid, succinic acid, lactic acid, malonic acid, succinylacetone, glutaconic acid, adipic acid, methylmalonic acid, fumaric acid, ethylmalonic acid, phenyllactic acid, vanillylmandelic acid, 4-hydroxyphenylpyruvic acid, sebacic acid, 4-phenylbutyric acid, stearic acid, 3-methylglutaconic acid, phenylacetic acid, glutaric acid, citric acid, pyruvic acid, 3-phenylbutyric acid, and sorbitol, hexane, diethyl ether, and butanol, derivatization reagents, namely N-bis (trimethylsilyl) trifluoroacetamide (BSTFA) with trimethylchlorosilane (TMCS), and pyridine. Spectrometry-grade solvents, including methanol, acetonitrile, water, acetone, isopropyl alcohol, and ethyl acetate, were obtained from Honeywell Burdick & Jackson, Anatech, Johannesburg, South Africa. Sodium and magnesium sulphate were obtained from Merck, South Africa. Synthetic urine (SurineTM Lot 72110) was purchased from Dyna-tek industries, Johannesburg, South Africa. For validation experiments, ERNDIM External Quality Assurance Samples (EQAS) 2016 Quantitative Organic Acids samples were utilized.