Phospholipid separation by polar headgroup was performed on a Thermo Fisher Dionex UltiMate-3000 RSLC system. The separation of lipids was performed on a PVA-Sil column (150 × 2.1 mm I.D., 120 A) from YMC Europe GmbH thermostated at 35 °C. Chromatographic method was adapted from Ramos et al. [48 (link)]. For fatty acid quantification, phospholipids were digested and methylated using the one pot procedure described in [49 (link)]. Methylated fatty acids were analyzed in TraceGC Ultra coupled to an ITQ900 from Thermo Fisher equipped with an Agilent DB-5 capillary column. Further details are given in SI.
Pva sil column
Manufactured by YMC
Sourced in Japan
The PVA-Sil column is a type of chromatography column used for the separation and purification of various chemical compounds. It is designed with a stationary phase consisting of polyvinyl alcohol (PVA) coated silica particles.
Automatically generated - may contain errors
Lab products found in correlation
Xevo tq s mass spectrometer, by Waters Corporation (3 mentions)
Acquity uplc h class, by Waters Corporation (2 mentions)
Trace gc ultra, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 rslc system, by Thermo Fisher Scientific (1 mentions)
Itq 900, by Thermo Fisher Scientific (1 mentions)
Db 5 capillary column, by Agilent Technologies (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Qtrap 6500, by AB Sciex (1 mentions)
Api 3000, by AB Sciex (1 mentions)
Masslynx 4, by Waters Corporation (1 mentions)
7 protocols using pva sil column
1
Phospholipid Separation and Fatty Acid Analysis
2
Lipid Extraction and Analysis from Liver Tissue
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Homogenates were subjected to lipid extraction as described by Matyash et al. [23 (link)]. Briefly, frozen liver tissue samples were homogenized with a TissueLyser II (Qiagen) with 5 mm stainless steel beads for three 1-min cycles at 30 Hz. We added 500 μl of PBS per 5–10 mg of tissue and lipids were extracted by adding 5 ml of cold tert-butyl methyl ether/methanol (TMBE/MeOH) (10/3; v/v) solution containing butylated hydroxytoluene (BHT) (50 μg/ml) and 50 μl of internal standard solution with appropriate concentrations and incubated for 10 min before vortexing the mixture. We added an additional 2 ml of PBS and the homogenates were incubated for 15 min, with occasional mixing, followed by centrifugation and the collection of the upper organic phase. The extracted lipid phase containing TAG, DAG, and ceramides was separated by hydrophilic interaction chromatography (HILIC) on a PVA-Sil column (vinyl alcohol polymerized silica (5 μm) support; L 250 mm X ID 4 mm) (YMC, Kyoto, Japan). The PVA-Sil column was fitted to HPLC equipment (Shimadzu LXR) coupled to an electrospray ionization (ESI) source of a triple-quadrupole mass spectrometer QTRAP 6500 (AB Sciex). Ceramide species were quantified according to published procedures [24 (link),25 (link)], and neutral lipids (DAG and TAG) were quantified by mass spectrometry as described by Leiker et al. [26 (link)].
3
Triacylglycerol Quantification in Muscle Tissues
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The triacylglycerols (TAG) of the Bligh and Dyer extract (Bligh and Dyer, 1959 (link)) were prepared from homogenized muscles in water and separated on a normal phase PVA-Sil column (Polymerised Vinyl Alcohol silica (5 μ) support; L 250 mm X ID 4 mm) (YMC, Kyoto, Japan). The PVA-Sil column was fitted on a Agilent 1200 HPLC equipment coupled to the electrospray ionization (ESI) source of a triple-quadrupole mass spectrometer (API3000, AB Sciex). TAG species were quantified by integration of molecular ions (as ammonium ion adducts) from full scan mass spectra and application of standard curves that relate the responses of known amounts of reference standards to that for a single internal standard as described elsewhere (Hutchins et al., 2008 (link)).
4
Chromatographic Separation of PFAM and NAG
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PFAM and NAG standards were prepared in a mixture at 1 mM concentration of each standard. The mixture was separated via normal phase chromatography utilizing a YMC PVA-Sil column (4.6 × 250 mm, 5 μm particle size). Gradient elution is carried out starting at 95% mobile phase A (heptane with 0.5% v/v methyl-tert-butyl-ether) and increasing linearly to 50% mobile phase B (methyl-tert-butyl-ether with 10% v/v 2-propanol and 0.2% v/v acetic acid) over 40 min with a flow rate of 1 mL/minute. Fractions were collected at 1 min intervals with an injection volume of 200 μL and the times corresponding to NAG and PFAM elution were determined by reversed phase chromatography and MS/MS detection.
5
Quantitative Skin Lipid Analysis
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Extraction of barrier lipids from the SC was carried out using a modified Bligh and Dyer procedure61 (link) as described by Boiten et al.62 (link). To determine the amount of material extracted from the SC, the SC was dried and weighed before as well as after the extraction procedure. The obtained lipid extract was dissolved in a suitable volume of heptane:chloroform:methanol (95:2.5:2.5 v:v:v). The lipids were analyzed and quantified using normal phase liquid chromatography - mass spectrometry (LC-MS) analysis, as described by Boiten et al.62 (link). In brief, SC lipid extracts were separated on a PVA-Sil column (5 μm particles, 100 × 2.1 mm ID; YMC, Kyoto, Japan) with an Acquity UPLC H-class (Waters, Milford, MA, USA). CERstotal were detected by an XEVO TQ-S mass spectrometer (Waters). LC-MS measurements occurred in full scan mode with settings for time between 1.25–8.00 minutes at m/z 350–1350 and for time between 8.0–12.5 minutes at m/z 500–1350.
6
SC Lipid Extraction and Quantification
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Extraction of total lipids from isolated SC of FTMs after 14 and 28 days was cultured, and NHS was performed with an adjusted Bligh and Dyer method as described by Boiten et al. (2016 ). To determine the weight percentage of lipids in the SC, dry SC weight was measured before and after extraction. The lipids were analysed and quantified using normal phase liquid chromatography–mass spectrometry according to the method described by Boiten et al. The sample concentration of SC extracts was set at 0.3 mg/ml, and 5 μl was injected. SC lipid extracts were separated on a PVA‐Sil column (5‐μm particles, 100 × 2.1‐mm i.d.; YMC, Kyoto, Japan), and CERstotal were detected with an Acquity UPLC H‐class (Waters, Milford, MA, USA) coupled to an XEVO TQ‐S mass spectrometer (Waters). Measurements were performed in full scan mode from 1.25–8.00 min between m/z 350 and 1,350 and from 8.0 to 12.5 min between m/z 500 and 1,350. Ceramide N(24deuterated)S(18) was used as internal standard (ISTD). Predicted response factors for quantification were calculated using the response over mass (Figure S2 ). For ceramides AS, NP, and NS, the signal was corrected for overlap of unsaturated ceramides containing two naturally abundant 13C. Nomenclature of the CER subclasses is followed according to Motta et al. (1993 ).
7
Stratum Corneum Lipid Profiling
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Stratum corneum was isolated using trypsin digestion steps and stored until use in an inert environment, as described before [57 (link)]. Lipids were extracted using a modified Bligh and Dyer extraction procedure, as reported before [19 (link),58 (link)]. The dry weight of the SC was determined before and after lipid extraction using a microbalance. Extracted lipids were evaporated and reconstituted in an appropriate volume of heptane/chloroform/methanol (95/2.5/2.5 v/v/v) to obtain a 0.3 mg/mL concentration. Ceramide N(24deuterated)S(18) (Evonik Industries, Essen, Germany) was added as the internal standard (ISTD). Liquid chromatography coupled to mass spectrometry (LC–MS) analyses were performed as described by Boiten et al. [19 (link)]. In brief, a 5 µL sample volume was injected into the ultra-pressure liquid chromatograph (UPLC) (Waters, Milford, MA, USA) equipped with a PVA-Sil column (5 µm particles size, 100 × 2.1 mm i.d., YMC, Kyoto, Japan). Detection occurred via a XEVO TQ-S mass spectrometer (Waters), measuring full scan m/z between 350 and 1200 atomic mass units. Data were analyzed with Masslynx software (Masslynx 4.1, Waters, Milford, MA, USA), corrected for ISTD, and quantified using a 3D response model according to methods as described before [19 (link)].
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