Deuterium oxide (D2, 99.8%), H218O (18 O, 97%), and 2H218O (D2, 98%; 18 O, 97%) were purchased from Cambridge Isotope Laboratories (Andover, MA). Methanol (high purity) was purchased from Honeywell Burdick & Jackson® (Muskegon, MI). Acetone (HPLC grade) and sodium hydroxide (10 N, 30% w/w, Certified) were purchased from Fisher Scientific (Fairlawn, NJ). MethElute™ derivatization reagent (0.2 M Trimethylanilinium hydroxide in methanol, (pH ≥ 10) was purchased from Thermo Scientific (Waltham, MA) and used as received. Oasis® HLB µElution plates (30 µM) were purchased from Waters Corporation (Milford, MA). GC headspace vials (10 mL) and high temperature screw top caps were purchased from Agilent Technologies (Santa Clara, CA). Standard material of 2′-deoxyadenosine monohydrate (purity > 99%) was purchased from Sigma-Aldrich (St Louis, MO). Gases used for GC-MS/MS analysis were helium (Grade 5.5 purity), nitrogen (Ultra high purity), isobutane (Matheson 99.99% purity) and methane (Research Grade purity) and were purchased from Roberts Oxygen Company (Rockville, MD). Gas purifiers purchased from Agilent Technologies were used to remove hydrocarbons and moisture from the gases.
Oasis hlb elution plate
Manufactured by Waters Corporation
Sourced in United States, Germany
The OASIS® HLB µElution Plate is a solid-phase extraction (SPE) product designed for sample preparation and purification. It is a 96-well plate format that utilizes hydrophilic-lipophilic-balanced (HLB) sorbent to extract and concentrate analytes from liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
Gemini c18 column, by Phenomenex (5 mentions)
Trypsin, by Promega (5 mentions)
Agilent 1200 infinity, by Agilent Technologies (3 mentions)
Sequencing grade trypsin, by Promega (3 mentions)
Milli q system, by Merck Group (2 mentions)
Acetic acid, by Carl Roth (2 mentions)
Tmt10plex isobaric label reagent, by Thermo Fisher Scientific (2 mentions)
Tmt16plex isobaric label reagent, by Thermo Fisher Scientific (2 mentions)
Deuterium oxide, by Cambridge Isotopes (1 mentions)
Hplc grade acetone, by Thermo Fisher Scientific (1 mentions)
Methelute derivatization reagent, by Thermo Fisher Scientific (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Membraneslide 1.0 pen, by Zeiss (1 mentions)
Adhesivecap 500 clear, by Zeiss (1 mentions)
Unison uk amino column, by Imtakt (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Thermomixer shaker, by Eppendorf (1 mentions)
1200 infinity hplc system, by Agilent Technologies (1 mentions)
Superdex peptide pc 3.2 30 column, by GE Healthcare (1 mentions)
Trypsin, by Serva Electrophoresis (1 mentions)
18 protocols using oasis hlb elution plate
1
Isotopic Tracer Analysis Protocol
2
Synthesis and Characterization of Phenol Metabolites
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Sulfates and glucuronides of (poly)phenol standards were obtained from Toronto Research Chemicals (Toronto, ON, Canada). Kaempferol-3-O-ß-d -glucuronide was obtained from Extrasynthese (Genay, France). The 1-methylpyrogallol-O-sulfate, 2-methylpyrogallol-O-sulfate, 4-methylcatechol-O-sulfate, 4-methylgallic-3-O-sulfate, catechol-O-sulfate, pyrogallol-O-1-sulfate, pyrogallol-O-2-sulfate and vanillic acid-4-O-sulfate were kindly provided by Dr Claudia Nunes dos Santos and Dr Rita Ventura, and their synthesis has been described elsewhere [18 (link)]. The 2-, 3- and 4-hydroxyhippuric acids were purchased from Enamine (Kiev, Ukraine). Remaining compounds were obtained from Sigma-Aldrich Co. (Steinheim, Germany). Acetic acid was from Carl Roth (Karlsruhe, Germany) and Oasis HLB µElution plates (2 mg sorbent per well, 30 µm) were from Waters (Eschborn, Germany). Milli-Q system (Merck KGaA, Darmstadt, Germany) ultrapure water was used. Unless otherwise stated, all chemicals and reagents were obtained from Sigma-Aldrich Co. (Steinheim, Germany).
3
Proteomic Profiling of Glomerular Tissue
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Ten-micrometre-thick FFPE sections were deparaffinized, rehydrated, stained, and scanned with ScanScope XT Aperio. Selected FFPE sections were mounted on preirradiated polyethylene naphthalate slides (MembraneSlide 1.0 PEN, Carl Zeiss MicroImaging GmbH), and a total area of approximately 2 million μm2 dissected glomeruli for each sample tissue was isolated using a PALM Microbeam System (P.A.L. M, Bernried, Germany) and pressure catapulted into a tube cap (AdhesiveCap 500 clear, Zeiss). Microdissected FFPE glomeruli were stored at − 20 °C until peptide extraction. Then, they were resuspended in 10 μl of lysis buffer (0.1 M Tris pH 8, 0.1 M dithiothreitol [DTT], 4% sodium dodecyl sulfate). A filter-aided sample preparation (FASP) protocol based on trypsin digestion was used to extract the proteins [18 (link)]. Digested peptides were eluted and desalted using Oasis HLB µElution plates (Waters, Milford, MA), dried by a vacuum centrifuge, and rehydrated in 2% acetonitrile (ACN) and 0.1% formic acid (FA). NanoLC-ESI-LTQ Orbitrap Elite was used for tandem mass spectrometry.
4
Viltolarsen Bioanalysis by LC-MS/MS
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Plasma concentrations of viltolarsen were determined by a different bioanalytical method for each animal species and humans based on LC-MS/MS equipped with a Unison UK-Amino column (3-μm particle size; 2.0 × 75 mm or 3.0 × 150 mm; Imtakt, Kyoto, Japan). The lower limits of quantification were 0.5 µg/mL (mouse), 0.04 µg/mL (cynomolgus monkey), 0.02 µg/mL (dog), and 0.0191 µg/mL (human), and the linearity of the calibration curves was confirmed for all methods. The mobile phases 0.1% formic acid and acetonitrile were used with appropriate elution gradients. The column was operated at 0.5 or 0.6 mL/min and 25°C or 40°C and the autosampler temperature was set at 20°C or 23°C. Compounds were quantified in positive-ion multiple-reaction-monitoring mode by applying the parent-to-product transitions viltolarsen m/z 1155 → 112 and propranolol m/z 260 → 116. All plasma samples with internal standard or standard solutions underwent solid-phase extraction on Oasis HLB µElution Plates (Waters, MA, USA), and the eluates (1 to 10 µL) were subjected to LC-MS/MS analysis.
5
Synthesis of Hydroxyhippuric Acids
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(Steinheim, Germany) and 2-, 3-and 4-hydroxyhippuric acids were purchased from Enamine (Kiev, Ukraine). Acetic acid was from Carl Roth (Karlsruhe, Germany) and Oasis HLB µElution plates were from Waters (Eschborn, Germany). Milli-Q system (Merck KGaA, Darmstadt, Germany) ultra pure water was used. Unless otherwise stated, all chemicals and reagents were obtained from Sigma-Aldrich Co. (Steinheim, Germany).
6
Quantitative Proteomic Workflow using TMT Labeling
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Captured proteins were reduced in 10 mM DTT in 50 mM HEPES pH 8.5 at 56 °C for 30 min and alkylated with 20 mM 2-chloroacetamide in 50 mM HEPES pH 8.5 for 30 min at room temperature in the dark. Samples were prepared using the SP3 protocol11 (link). Proteins were digested by trypsin (Promega) at 37 °C overnight using an enzyme-to-protein ratio of 1:50. Peptides were labeled with TMT10plex Isobaric Label Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. For further sample clean up, an OASIS HLB µElution Plate (Waters) was used. Offline high pH reverse phase fractionation was carried out on an Agilent 1200 Infinity high-performance liquid chromatography system, equipped with a Gemini C18 column (3 μm, 110 Å, 100 × 1.0 mm2, Phenomenex).
7
Quantitative TMT-based Proteomic Workflow
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All reagents were prepared in 50 mM HEPES, pH 8.5 (Biomol, 05288.100). Cysteines were reduced using dithiothreitol (56°C, 30 min 10 mM; Biomol, 04010.25). Samples were cooled to 24°C and alkylated with iodoacetamide (room temperature, in the dark, 30 min, 10 mM; Merck, 8.04744.0100). Subsequently, the samples were prepared for LC-MS/MS using the SP3 protocol [37 ], digested with trypsin (enzyme to protein ratio, 1:50; Promega, V5111) at 37°C overnight. TMT10plex™ Isobaric Label Reagent (Thermo Fisher Scientific, 90111) was added to the samples according the manufacturer’s instructions. Labeled peptides were cleaned up using OASIS® HLB µElution Plate (Waters, Milford, MA, USA). Offline high pH reverse phase fractionation was performed using an Agilent 1200 Infinity high-performance liquid chromatography (HPLC) system, equipped with a Gemini C18 column (3 μm, 110 Å, 100 × 1.0 mm; Phenomenex, Torrance, CA, USA). The solvent system consisted of 20 mM ammonium formate (pH 10.0; Sigma-Aldrich, 78314-500ML-F) as mobile phase (A) and 100% acetonitrile (Fisher Chemicals, A955-1) as mobile phase (B). This was performed at the Proteomics Core Facility at EMBL Heidelberg, Germany.
8
In-Gel Tryptic Digestion of Proteins
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Coomassie blue stain was removed from gel pieces by incubating for 30 min at 37°C in 50 mM triethylammonium bicarbonate (TEAB)-acetonitrile (1:1). Gel pieces were then dehydrated with acetonitrile at room temperature, followed by reduction/alkylation using dithiothreitol (DTT) and iodoacetamide. Gel pieces were dehydrated with acetonitrile and rehydrated in a solution of trypsin (10 µg/ml) in 0.05% AcOH. The digestion was carried out at 37°C overnight. Peptides were extracted at 37°C for 30 min using the extraction buffer (50% acetonitrile and 3.3% trifluoroacetic acid [TFA]). All the steps outlined above were carried out on a thermomixer shaker (Eppendorf) unless stated otherwise. Extracts were then dried using a speed vacuum concentrator and resuspended in 0.1% TFA. Salts were removed using an Oasis HLB µElution plate (Waters) before LC/MS-MS analysis.
9
Cysteine Reduction and Alkylation for TMT Proteomics
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Proteins from inputs and RNase eluates of eRIC were incubated with DTT (10 mM in 50 mM HEPES pH 8.5) for 30 min at 56 °C for the reduction of disulfide bridges in cysteines. Reduced cysteines were alkylated with 2-chloroacetamide (20 mM in 50 mM HEPES pH 8.5) at room temperature for 30 min in the dark. Samples were further processed using the SP3 protocol90 (link),91 (link) and digested with trypsin (sequencing grade, Promega) using an enzyme-to-protein ratio of 1:50 for overnight at 37 °C. Peptides were then recovered by collecting supernatant on a magnet and combining with a second elution wash of beads with HEPES buffer. Subsequently, peptides were labeled with TMT16plex Isobaric Label Reagent (#A44521, Thermo Fischer Scientific) according to the manufacturer’s instructions. Samples were combined for the TMT16plex and further cleaned up using an OASIS® HLB µElution Plate (Waters). Offline high pH reverse phase fractionation was carried out on an Agilent 1200 Infinity high-performance liquid chromatography system, equipped with a Gemini C18 column (3 μm, 110 Å, 100 ×1.0 mm, Phenomenex)92 (link).
10
Peptide Sample Preparation for MS
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Beads were thawed to room temperature (RT) and centrifuged at 2500 g for 2 min and the H2O was removed. The beads were then resuspended in 40 μl Trypsin buffer (50 mM Tris, 1 mM CaCl2, pH8), 4 μl of 0.1 M DTT was added, and the samples were heated to 95 °C for 5 min. The samples were then cooled to RT and 5 μl of 200 mM iodoacetamide was added, and the samples were incubated, shaking at RT for one hour. 0.8 μl of 0.1 M DTT was added to quench the remaining iodoacetamide, and samples were incubated shaking for 10 min. The pH was adjusted to approximately pH8 with 0.5 M Tris, 2 μg of Trypsin (Promega, V5111) was added to each sample and they were incubated shaking at 37 °C overnight (o/n). Following this, 5 μl of 10% trifluoroacetic acid was added to each sample and the peptide solutions were cleaned up with an Oasis HLB µElution plate (2 mg sorbent; Waters). Following elution, samples were frozen at − 80 and freeze-dried.
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