Reversed phase hplc

Brain slices were incubated with GDF-15 or inhibitors in ACSF bubbled with 95% O₂–5% CO₂ for 1 h. The ACSF was then collected and frozen at −80 °C for later analyses. The slices were homogenized in ice lysis buffer containing protease inhibitor (Sigma, St Louis, Missouri, USA), rotated on ice for 40 min, and centrifuged at 13,800 × g for 20 min at 4 °C. The supernatants were collected to measure the protein concentrations. The samples were subjected to reversed-phase HPLC (ThermoFisher, Waltham, Massachusetts, USA) with fluorometric detection following pre-column derivatization with o-phthalaldehyde to analyze glutamate concentrations, as described previously62 (link). Chromatography was performed on a reversed-phase C-18 column using a pH sodium acetate methanol gradient. Methionine sulfone was added to each sample as an internal standard. External standards containing 40, 400, or 4000 pmol/20 ml glutamate were run at the beginning and end of every group. The peak heights of glutamate were initially normalized to the methionine sulfone peak and then quantified according to the linear relationship between peak height and the amounts of the corresponding standards. Glutamate release was normalized by the total protein in each single brain slice, and expressed as ng/mg protein63 (link).

For extraction of the product remaining inside the cells, the biomass pellets were resuspended in 30 ml TE buffer (100 mM TRIS, 10 mM EDTA, pH 7.4), with the exception of the reference sample, which was processed as the original cell suspension in the culture broth. The suspension was homogenized in a high-pressure homogenizer (Emulsiflex C-3; Avestin, Ottawa, Canada) in three passages at 1,000 bar, which are optimal parameters for extraction of soluble product from E. coli according to Pekarsky et al. (2019) (link). The homogenate was then centrifuged at 10,000 rcf (10 min, 4°C) to separate the cell debris from the soluble extract. SpA concentrations in the extracts from homogenization (intracellular, c_SpA,in) and from the PEF extract (extracellular, c_SpA,ex) were then quantified in triplicate via reversed phase HPLC (Thermo Fisher Scientific, Waltham, MA, United States) using a polyphenyl column (Waters, Milford, MA, United States). The mobile phase consisted of a gradient of water and acetonitrile, supplemented with 0.1% trifluoroacetic acid. The released SpA (%) was then calculated according to Eq. 3.

Desalination of the peptides with TMT labels obtained from five groups was carried out using a C₁₈ column (Dr. Maisch, Germany). Next, the peptides were gathered and dried by vacuum. The 100 μg peptide samples were separated by reversed-phase HPLC (Thermo Scientific, United States) at pH = 10. Chromatographic column: 150 mm × 2.1 mm (waters, XBridge BEH C 18 XP Column); Mobile phases A: 10 mM ammonium formate aqueous solution, pH = 10; Mobile phases B: 10 mM ammonium formate, 90% ACN, 10% H₂O, pH = 10. Liquid phase gradient 120 min, mobile phase B: 5% for 2 min, 5–28% for 78 min, 28–50% for 12 min, 50–80% for 2 min, 80% for 4 min, 80–5% for 2 min and 5% for 20 min. One fraction was collected in 40 s intervals. A total of 180 fractions were collected and combined to obtain 20 fractions. The fractions were subjected to dried by vacuum and followed by storage at −80°C.