Luna nh2 column

The amount of free hydroxyproline was quantified using tandem mass spectrometry (LC-MS/MS) in wound tissues similar to our recent rat study [20 (link)]. Metabolite extraction was performed with a modified Bligh Dyer method [21 (link)]. Extracted metabolites from the aqueous phase were dried in a CentriVap Concentrator (Labconco, Kansas city, MO, USA) and then stored at -80°C until analysis. Protein pellets were used to normalize extracted metabolite quantity by using a bicinchoninic acid assay (G-Biosciences, St. Louis. MO, USA). A hydroxyproline (Sigma-Aldrich, St. Louis, MO, USA) standard curve was obtained by running hydroxyproline solution at 1 × 10⁻³, 1 × 10⁻⁴, 1 × 10⁻⁵, 1 × 10⁻⁶, 1 × 10⁻⁷, 1 × 10⁻⁸, 1 × 10⁻⁹ M concentrations. For hydroxyproline detection, hydrophilic interaction liquid chromatography was performed on a Micro200 LC (Eksigent, Redwood, CA, USA) with a Luna NH₂ column (3 μ, 100Å, 150mm by 1.0mm, Phenomenex, Torrance, CA, USA). Samples were analyzed on a 5600+ TripleTOF Mass Spectrometer (AB SCIEX, Framingham, MA, USA) and (132.10 → 86.09) m/z transition was used for hydroxyproline detection.

The cellular concentration of Cx-SAM was quantified with an isotope ratio-based approach (17 (link)), using ¹⁵N-labeled isotopic standard of Cx-SAM. The isotopic standard (chemical formula = C₁₆H₂₂¹⁵N₆O₇S, [M+H] = 449.1171) was prepared by methanol extraction of the ligand from the recombinant CmoA, which had been purified from cells grown in M9 media supplemented with ¹⁵NH₄Cl as the sole nitrogen source, followed by HPLC purification as described previously (14 (link)). Approximately 6 × 10¹⁰ wild-type E. coli K-12 cells grown in 10-ml LB media were pelleted by centrifugation. Metabolites were extracted with 1 ml of methanol:acetonitrile:water (40:40:20) with 0.1-mM FA, and 90 nM of an isotopic standard of Cx-SAM. After 20-min incubation on ice, the lysed cells were centrifuged at 16 000g for 20 min at 4°C. The sample was subjected to LC-MS analysis (Agilent 1200 HPLC coupled to LTQ Orbitrap Velos mass spectrometer; ESI positive ion mode detection; Phenomenex Luna NH2 column, bead size of 5mm, pore size of 100 Å, 150 × 2 mm) using a gradient from 75 to 2% of acetonitrile and 0.1% FA over 30 min. The amount of cellular Cx-SAM was quantified by comparing the intensities of chromatographic peaks with those from the isotopic standard, assuming the intracellular volume of E. coli is ∼1 μm³ (19 (link),20 (link)).

To carry out the quantification of amino acids, potassium and sodium, 14 samples per group (cold treated and non-treated) were used. Each sample consisted of a pool (1 mL) of the hemolymph collected from 3–5 individuals.
Free amino acids were determined by liquid chromatography coupled by triple quadrupole mass spectrometry (LC-TQ system, Evoq-Elite Bruker, Germany). From each sample, 100 µL were taken, diluted in Milli-Q water up to 100 mL and filtered with 0.45 µm nylon filters. A final volume of 5 µL was then injected into the LC-TQ system using a Phenomenex Luna NH2 column (150 mm × 2.1 mm, 3 µm).
Potassium and sodium hemolymph levels were determined by inductively coupled plasma mass spectrometry (ICP-MS). We took 100 µL from each sample, which were diluted in Milli-Q water up to 50 mL. An aliquot (5 mL) was continuously aspirated by a peristaltic pump and a final volume of 1 mL was then introduced into the ICP-MS system (Agilent 7700e, Santa Clara, CA, USA).