FAs were extracted as described by Yang et al. [40 (link)]. Waters Oasis-HLB cartridges (30 mg/30 μm) were treated with ethyl acetate (1 mL), methanol (2 × 1 mL), and 95:5 v/v water/methanol containing 0.1% acetic acid (1 mL). We mixed 100 μL of plasma with 7 μL of internal standard solution (stock concentration: 500 nM), 10 μL of butylated hydroxytoluene (BHT; stock concentration: 2 mg/mL), and 120 μL of H2O:methanol (MeOH) (95:5) containing 0.1% acetic acid. The resulting samples (240 μL) were then loaded onto pre-treated cartridges and washed twice with 750 μL of H2O/MeOH (95:5) containing 0.1% acetic acid. The aqueous plug was pulled from the cartridges using a high vacuum, and the cartridges were then dried further under a low vacuum for about 20 min. Waters Oasis-HLB cartridges were eluted into tubes with 250 μL of methanol followed by 1 mL of ethyl acetate into 2 mL tubes containing 6 μL of 30% glycerol in MeOH, a trap solution. The samples were dried under nitrogen and then dissolved in 70 μL of methanol containing 20 nM of 1-cyclohexyl-dodecanoic acid urea. The samples were then vortexed for 5 min, transferred to autosampler vials with low-volume inserts, and stored at −20 °C until further analysis.
Oasis hlb cartridge
Manufactured by Waters Corporation
Sourced in United States, Germany, United Kingdom, Japan, France, Canada, Ireland, Morocco
Oasis HLB cartridges are solid-phase extraction (SPE) devices used for sample preparation in analytical chemistry. They are designed to capture and concentrate analytes of interest from liquid samples. The cartridges contain a polymeric sorbent with both hydrophilic and lipophilic (hydrophobic) characteristics, which enables the selective retention of a wide range of polar and non-polar compounds.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Promega (21 mentions)
Formic acid, by Merck Group (11 mentions)
Methanol, by Merck Group (7 mentions)
Speedvac, by Thermo Fisher Scientific (6 mentions)
Ltq orbitrap ms, by Thermo Fisher Scientific (5 mentions)
Ethyl acetate, by Merck Group (5 mentions)
Sequencing grade modified trypsin, by Promega (5 mentions)
Acetic acid, by Merck Group (5 mentions)
Acetonitrile, by Merck Group (5 mentions)
Formic acid, by Thermo Fisher Scientific (5 mentions)
Lysyl endopeptidase, by Fujifilm (4 mentions)
Byonic software program, by Protein Metrics (4 mentions)
Milli q system, by Merck Group (4 mentions)
Cellytic mem protein extraction kit, by Merck Group (4 mentions)
Ammonium acetate, by Merck Group (3 mentions)
Vacuum evaporation, by Thermo Fisher Scientific (3 mentions)
Iodoacetamide, by Merck Group (3 mentions)
Acetonitrile, by Thermo Fisher Scientific (3 mentions)
Trifluoroacetic acid, by Merck Group (3 mentions)
Milli q water purification system, by Merck Group (3 mentions)
276 protocols using oasis hlb cartridge
1
Plasma Fatty Acid Extraction Protocol
2
Quantification of Cimicoxib in Plasma and Milk
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The concentration of cimicoxib in the plasma samples was determined according to a validated HPLC method described in Jeunesse et al. [20 (link)]. Briefly, cimicoxib was extracted by a solid liquid extraction process using HLB Oasis cartridges (Waters). Separation was achieved by a reverse phase column with an octadecylsilane stationary phase (Merck Lichrospher 100 RP18e (125x4) mm, 5 μm) using a guard column (Merck Lichrospher 100 RP18e (4x4) mm, 5 μm). UV detection was performed at 242 nm. Within-day and day-to-day coefficients of variation were less than 9 % and the accuracy ranged from 94 to 103 %. The limit of quantification of the method was 0.01 μg/mL. For the milk samples, the method was validated using cow milk as blank matrix. Only the extraction was altered using dichloromethane as an extracting solvent. The specificity of the method was tested against co-extracted milk impurities and against demethylated cimicoxib which is the major metabolite of cimicoxib [30 ]. The limit of quantification in the milk samples was 0.03 μg/mL. Within-day and day-to-day coefficients of variation were less than 7 % and the accuracy ranged from 96 to 105 %.
3
ECM Protein Identification by LC-MS/MS
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LC-MS/MS analysis was done on two different regions (biological duplicates) of each patient metastasis sample. ECM solubilization and digestion was done using methods previously described (21, 26 ). Briefly, the ECM-enriched pellet was reduced and solubilized in a 100 mmol/L ammonium bicarbonate solution containing 8 mol/L urea and 10 mmol/L dithiothreitol. Cysteines were alkylated by addition of 25 mmol/L iodoacetamide and proteins were deglycosylated with enzyme PNGaseF (New England BioLabs). Proteins were digested with Lys-C (Wako Chemicals USA, Inc.) followed by Trypsin (Sequencing Grade, Promega). Peptides were acidified with trifluoroacetic acid, desalted using 30 mg HLB Oasis Cartridges (Waters Corp.), eluted with 60% acetonitrile, 0.1% trifluoroacetic acid and concentrated in a Speed-Vac. Details of NanoLC-MS/MS analysis, Protein/peptide identification and Label-free Relative Protein Quantitation are described in Supplemental Materials and Methods.
4
Isolation of Urinary Endogenous Peptides
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Urine endogenous peptides were isolated by trichloroacetic acid (TCA) precipitation as described by Parker et al (10 (link)). Briefly, 700 µl of urine samples were concentrated by vacuum centrifugation using SpeedVac. Samples were concentrated to more accurately reproduce the methodology described by Parker et al (10 (link)), as the protocol was originally standardized for human plasma samples, which contain larger amounts of proteins/peptides than those normally found in urine. After concentration, samples were mixed 1:1 with PBS and urinary proteins were precipitated with 1 volume of 20% TCA for 1 h at 4°C. Samples were centrifuged at 16,000 × g for 10 min at 4°C and the peptide-containing supernatants were collected. Purified peptides were desalted using HLB OASIS cartridges (Waters), following manufacturer's instructions. Peptides were quantified using Pierce™ Quantitative Colorimetric Peptide Assay (Thermo Scientific, 23275) and stored at −20°C until analysis.
5
ECM Extraction and Proteomic Analysis
6
Quantification of Cyanobacterial Microcystins
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Methanol water and acetonitrile of LC-MS grade, as well as formic acid 99–100% a.r. were purchased from Sigma Aldrich Co. (St. Luis, U.S.A.). HLB OASIS cartridges were used for Solid Phase Extraction (Waters, Milford, MA, USA). For the molecular detection of cyanobacteria QIAamp DNA Mini Kit was purchased by Qiagen (Qiagen, USA), MyTaq™ Red Mix from BIOLINE (Bioline, UK) and NanoDrop (Shimadzu, Japan) was used for the evaluation of the quality and quantity of the extracted DNA. For screening a direct competitive microcystins (ADDA)-DM (direct monoclonal) ELISA kit was purchased from CD Creative Diagnostics (New York, USA). Certified reference materials (CRMs) of microcystin-LR, -RR and -YR were purchased from CIFGA (Lugo, Spain) and uncontaminated mussels’ tissue (CRM ZERO MUS) from National Research Council Canada (Newland and Labrador, Canada). The LC separation of the microcystins was carried out on a C18 reverse-phase column ACCLAIM POLAR ADVANTAGE II 3 μm 3 × 150 mm with guard column 2/pk and holder-coupler.
7
Wastewater Metabolite Profiling Workflow
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Raw wastewater samples collected from local wastewater treatment works, were filtered using GF/F glass microfibre filter 0.75 µm (Fisher Scientific, UK) followed by a solid phase extraction (SPE) using HLB Oasis® cartridges Water, UK) to reduce the matrix effect and to concentrate each sample by 400fold. SPE procedure was as follows: 100 mL of filtered wastewater were loaded onto Oasis HLB cartridges, which were preconditioned with 2 mL MeOH followed by 2 mL H2O. After loading, the cartridges were dried for 30 min and analytes were eluted with 4 mL MeOH. Extracts were then dried under a gentle nitrogen stream using a TurboVap evaporator (Caliper, UK, 40•C). Dry extract was then reconstituted in 250 µL 80:20 H2O:MeOH, transferred to polypropylene vials and analysed using Dionex Ultimate 3000 HPLC coupled with a Bruker Maxis HD Q-TOF according to the procedure described above.
After analysis, data extracted from the Bruker system were processed with MetID software (Advanced Chemistry Development, Inc., ACD/Labs, UK) in order to predict metabolite structures. However, the software predicts a large number of possible metabolites, of which a rather small number is actually observed in in vitro experiments. We therefore developed a systematic workflow as presented in Figure 1 to limit false positive measurements.
After analysis, data extracted from the Bruker system were processed with MetID software (Advanced Chemistry Development, Inc., ACD/Labs, UK) in order to predict metabolite structures. However, the software predicts a large number of possible metabolites, of which a rather small number is actually observed in in vitro experiments. We therefore developed a systematic workflow as presented in Figure 1 to limit false positive measurements.
8
Festival Urine Profiling by HPLC-QTOF
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Seven pooled urine samples were collected from a UK festival event. They came from five different urinals sampled on three different days. Solid phase extraction (SPE) was performed on pooled urine samples using HLB Oasis® cartridges Water, UK) to reduce the matrix effect and to concentrate each sample by 4-fold. SPE procedure was as follows: 2 mL of pooled urine were loaded onto Oasis HLB cartridges, which were preconditioned with 2 mL MeOH followed by 2 mL H2O. After loading, the cartridges were dried for 30 min and analytes were eluted with 4 mL MeOH. Extracts were then dried under a gentle nitrogen stream using a TurboVap evaporator (Caliper, UK, 40•C). Dry extract was then reconstituted in 500 µL 80:20 H2O:MeOH, transferred to polypropylene vials and analysed using Dionex Ultimate 3000 HPLC coupled with a Bruker Maxis HD Q-TOF according to the procedure described above.
9
Protein quantification and digestion
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The protein concentration in the vitreous and depleted plasma was measured using BCA methods according to the protocol provided by the manufacturer. Aliquots of 100 μg protein were reduced, alkylated, and digested according to the protocol. Briefly, 60 μL 9 M urea (5.4054 g) and 30 mM dithiothreitol (DTT; 0.04628 g) in 10 mL 100 mM Tris-base (pH 8.0) were added to each sample and incubated for 30 min at 37°C. Each sample was allowed to cool at room temperature before 9 μL 500 mM iodoacetamide (IAA; 0.0925 g/mL) was added. The solution was incubated for 20 min at room temperature. To dilute the urea from 6 M to 0.6 M, 771 μL 100 mM Tris buffer (pH 8.0) was added to each sample. Each sample was then digested with trypsin (Promega, Madison, WI, USA) at a protein-to-enzyme ratio of 50 : 1 at 37°C overnight. To quench the digestion reaction, 50 μL 0.1% TFA was added. The digested peptide mixture was applied onto an HLB Oasis cartridge (Waters Milford, MA, USA) for desalting, and peptides were eluted using 1 mL 60% ACN with 0.1% FA solution.
10
MRM-MS Analysis of Hypothalamic ACBD7
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Ability of hypothalamic cell to process ACBD7 in vivo has been evaluated using multiple reactions monitoring mass spectrometry (MRM-MS) analysis of hypothalamic explant (Proteomic Centre Facility of the Laval University, Québec City, Canada, QC). Briefly, fresh MBH hypothalamic explants were solubilized in extraction buffer containing 0.5% of sodium deoxycholate, 50 mM of 1,4-dithiothreitol and protease inhibitor. After sonication, half of the volume was precipitated by incubating with acetone (-20°C, 12 hr) and the remaining half part was purified using Amicon cartridge (cut-off 10 KDa). Both fractions were then combined and purified using HLB Oasis cartridge (Waters, Mississauga, Canada, ON). After adjustment of the sample concentration with formic acid solution (0.1%), 100 fmol/µl of exogenous synthetic peptide was added to each sample in order to normalize each analysis.
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