Acquity uplc pump

The Morgan–Elson assay was used in order to estimate the N-acetyl-D-glucosamine content released after chitinase treatment, as previously reported [17 (link),20 (link)].
Preparation of the samples for ESI-MS: the demineralized organic scaffolds of A. wolffgangi and E. gibbosa were hydrolysed in 6 M HCl for 24 h at 50 ^oC. After hydrolysis samples were filtrated with 0.4 μm filter and freeze-dried to remove the excess of HCl. The dried samples were dissolved in deionized water for analysis. All ESI-MS measurements were performed on Waters TQ Detector ACQUITYuplc mass spectrometer (Waters, USA) equipped with ACQUITYuplc pump (Waters, USA) and BEHC18 1.7 μm, 2.1 × 50 mm UPLC column. Nitrogen was used as nebulizing and desolvation gas. Graphs were generated using Origin 8.5 for PC.

Skin was divided into dermis and epidermis (on ice, by scalpel, with the aid of visual inspection at ×40 magnification) as previously described (17 (link), 20 (link)). Skin (5–30 mg) and plasma samples (∼1 ml) were extracted using ice-cold methanol (15%, v/v), and 12-hydroxyeicosatetraenoic acid (HETE)-d₈, prostaglandin (PG)B₂-d₄, 8 (9)-epoxy eicosatrienoic acid-d₁₁, and 8,9-dihydroxyeicosatrienoic acid-d₁₁ (20 ng each/sample; Cayman Chemicals, Ann Arbor, MI, USA) were used as internal standards. The extracts were semipurified using solid-phase extraction cartridges (C18-E; Phenomenex, Macclesfield, United Kingdom) to eliminate matrix effects. Ultraperformance liquid chromatography with electrospray ionization and tandem mass spectrometry (UPLC/ESI-MS/MS) analysis was performed on an Acquity UPLC pump (Waters, Wilmslow, United Kingdom) coupled to an electrospray ionization triple quadrupole mass spectrometer (Xevo TQ-S; Waters) as previously described (17 (link), 31 (link), 32 (link)). A detailed list of the multiple reaction monitoring transitions and collision energy settings is provided in Supplemental Table S1. Results are expressed as picograms per milligram protein (skin) or picograms per milliliter (plasma).

Extracted lipids were analysed by multiple reaction monitoring (MRM) using ultraperformance liquid chromatography with electrospray ionisation and tandem mass spectrometry (UPLC/ESI–MS/MS), with an Acquity UPLC pump (Waters, Wilmslow, United Kingdom) coupled to an electrospray ionisation triple quadrupole mass spectrometer (Xevo TQ-S; Waters). Autosampler temperature was 8 °C; column temperature was 30 °C; solvent flow rate was 0.3 mL/min and a BEH C8 1.7 µm 2.1 × 100 mm reverse phase column was used (Waters, Wilmslow, United Kingdom). Solvent gradients used for analysis of ceramides or SM using mobile phase A (0.1% formic acid in LC/MS grade water) and mobile phase B (methanol containing 0.1% formic acid) are described in Supplementary Table S1. Electrospray ionisation was performed in positive mode using the following settings: capillary voltage, 3.5 kV; source temperature, 100 °C; cone voltage, 30 V; desolvation gas temperature, 450 °C. A full list of MRMs and collision energies is provided in Supplementary Tables S2 and S3. Ceramide and SM data were analysed using semi-quantitation against class-specific deuterated internal standards, and normalised against protein content.