Ultimate 3000 liquid chromatography system

An equal amount (TMT1 = 40 μg, TMT2 = 40 μg, TMT3 = 35 μg) of each sample was digested with trypsin (2.5 μg trypsin per 100 μg protein; 37°C, overnight), labelled with Tandem Mass Tag (TMT) 6 or 10 plex reagents according to the manufacturer's protocol (Thermo Fisher Scientific) and the labelled samples pooled.
100 μg aliquots of pooled samples were evaporated to dryness, resuspended in 5% formic acid and then desalted using a SepPak cartridge according to the manufacturer's instructions (Waters). Eluates from the SepPak cartridge was again evaporated to dryness and resuspended in buffer C (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Scientific). In brief, samples were loaded onto an XBridge BEH C18 Column (130 Å, 3.5 μm, 2.1 mm × 150 mm, Waters) in buffer C and peptides eluted with increasing gradient of buffer D (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0 to 95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC–MS/MS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific).

The bile acid quantification included four primary bile acids (α-MCA, β-MCA, CA, and UDCA), four secondary bile acids (ω-MCA, chenodeoxycholic acid [CDCA], deoxycholic acid [DCA], and lithocholic acid [LCA]), and seven conjugated bile acids (tauro-β-muricholic acid [T-β-MCA], tauroursodeoxycholic acid [TUDCA], taurocholic acid [TCA], glycocholic acid [GCA], taurochenodeoxycholic acid [TCDCA], taurodeoxycholic acid [TDCA], and taurolithocholic acid [TLCA]). Briefly, 20 mg of raw feces was dissolved in 200 μL of 70% d3-cholic acid aqueous solution containing 2 ppm d4-cholic acid as an internal standard, and it was homogenized using an ultrasonicator for 30 min. The sample solutions were then centrifuged at 18,000 × g for 5 min. LC-MS analysis was performed using an UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific, Dreieich, Germany) coupled with a high-resolution Q Exactive Plus instrument equipped with an ESI source (Thermo Fisher Scientific) and an Acquity HSS T3 (2.1 by 100 mm, 1.7 μm) column (Waters, Milford, MA, USA).

Pull-down samples were reduced (10 mM TCEP 55 °C, 1 h), alkylated (18.75 mM iodoacetamide, room temperature, 30 min), digested on the beads with trypsin (2.5 μg trypsin; 37 °C, overnight), and then labelled with Tandem Mass Tag (TMT) six-plex reagents according to the manufacturer's protocol (Thermo Fisher Scientific, Loughborough LE11 5RG, UK), and the labelled samples were pooled.
The pooled sample was evaporated to dryness, resuspended in 5% formic acid, and then desalted using a SepPak cartridge according to the manufacturer's instructions (Waters, Milford, Massachusetts, USA). The eluate from the SepPak cartridge was again evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 μm, 2.1 mm × 150 mm, Waters, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM Ammonium Hydroxide in acetonitrile, pH 10) from 0% to 95% over 60 min. The resulting fractions (4 in total) were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Scientific).

In order to evaluate a possible variation in skin hydration during the study, the content of NMFs was measured, as NMFs are hydrophilic markers of stratum corneum [28 (link)]. Skin surface samples were taken for NMFs analysis on D0, D0 + 4 h, D7, D14 and D24 by rubbing a 5-cm line on the skin with two swabs (Dryswab^® MWE, Medical Wire & Equipment, Wiltshire, UK) previously soaked in an aqueous non-ionic surfactant solution (QIMA, Labège, France—proprietary method), using a standardised, previously validated, sampling technique (unpublished data). The swab heads were then removed, placed in dry Eppendorf tubes (Safe-Lock Tubes 1.5 mL, Eppendorf AG, Hamburg, Germany) and frozen at −20 °C until analysis.
NMFs of interest (urocanic acid (cis/trans-UCA), pyrrolidone carboxylic acid (PCA) and serine were extracted using an aqueous solution, then analysed by a LC/MS system (UltiMate 3000 liquid chromatography system (ThermoScientific, Sunnyvale, CA, USA) coupled with a MSQ Plus Mass detector (ThermoScientific, Sunnyvale, CA, USA). Results are expressed in µg per sample.

Immuno-isolated samples were reduced (10 mM TCEP, 55 °C for 1 h), alkylated (18.75 mM iodoacetamide, RT for 30 min) and then digested from the beads with trypsin (2.5 µg trypsin; 37 °C, overnight). The resulting peptides were then labelled with TMTpro sixteen-plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific) and the labelled samples were pooled and desalted using a SepPak cartridge, according to the manufacturer’s instructions (Waters). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) before fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 µm, 2.1 mm × 150 mm, Waters) in buffer A and peptides were eluted with an increasing gradient of buffer B (20 mM Ammonium Hydroxide in acetonitrile, pH 10) from 0 to 95% over 60 min. The resulting fractions (6 in total) were evaporated to dryness and resuspended in 1% formic acid before analysis by nano-LC MSMS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific).

Aliquots of 50 µg of each sample were digested with trypsin (1.25 µg trypsin; 37 °C, overnight), labelled with Tandem Mass Tag (TMTpro) sixteen plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, LE11 5RG, UK) and the labelled samples pooled.
An aliquot of 200ug of the pooled sample was desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 µm, 2.1 mm × 150 mm, Waters, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM Ammonium Hydroxide in acetonitrile, pH 10) from 0 to 95% over 60 min. The resulting fractions (20 in total) were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific).

Aliquots of 100 μg of 10 samples per experiment were digested with trypsin (2.5 μg trypsin per 100 μg protein; 37°C, overnight), labeled with TMT10plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, United Kingdom) and the labeled samples pooled.
An aliquot of 50 μg of the pooled sample was evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 μm, 2.1 mm × 150 mm, Waters, United Kingdom) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM Ammonium Hydroxide in acetonitrile, pH 10) from 0 to 95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific).