Tryptic digests (50 μg) were de-sialylated with neuraminidase (New England BioLabs, Ipswich, MA) at 37°C using pressurized accelerated digestion (barocycler) technology (Pressure Biosciences, South Easton, MA). Completion of the de-sialylation reaction was confirmed by LC-MS/MS analysis. De-sialylated glycopeptides were evaporated in a vacuum concentrator (Labconco, Kansas City, MO) and dissolved in mobile phase A (2% ACN, 0.1% FA) for LC-MS/MS measurements. For the analysis of site occupancy, aliquots of the tryptic digests (50 μg) were heated at 99°C for 10 mins to deactivate trypsin. Five μl of deglycosylation buffer G7 (New England BioLabs, Ipswich, MA) were added to 50 μg of each tryptic digest and the samples were evaporated in a vacuum concentrator (Labconco) and dissolved in 50 μl of 18O water for deglycosylation with 0.5 μl PNGase F (New England BioLabs) using a 1 hour barocycler assisted protocol as described previously (24 (link)).
Vacuum concentrator
Manufactured by Labconco
Sourced in United States
The Vacuum Concentrator is a laboratory equipment designed to evaporate solvents from liquid samples under reduced pressure. It is used to concentrate and dry various types of samples, such as proteins, peptides, nucleic acids, or other compounds, by removing the solvent through gentle heating and vacuum application.
Automatically generated - may contain errors
Lab products found in correlation
Cryolys precellys 24 sample homogenizer, by Bertin Technologies (3 mentions)
Methoxyamine hydrochloride in pyridine, by Merck Group (2 mentions)
Trypsin, by Promega (2 mentions)
Sonic 5, by Polsonic (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Minilys homogenizer, by Bertin Technologies (1 mentions)
Recombinant peptide n glycosidase f pngase f, by Roche (1 mentions)
Octyl β d glucopyranoside, by Merck Group (1 mentions)
Pierce quantitative colorimetric peptide assay, by Thermo Fisher Scientific (1 mentions)
S trap micro spin column, by Protifi (1 mentions)
Xevo tq xs system, by Waters Corporation (1 mentions)
Oasis primehlb spe columns, by Waters Corporation (1 mentions)
Powergen 700, by Thermo Fisher Scientific (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
Microcentrifuge tube, by Eppendorf (1 mentions)
Hemoglobin from bovine blood, by Merck Group (1 mentions)
Hypoxanthine, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Avantor (1 mentions)
Neutral protease, by Solarbio (1 mentions)
Acetic acid, by Merck Group (1 mentions)
58 protocols using vacuum concentrator
1
Automated De-sialylation and Deglycosylation
2
Lipid Extraction and Fractionation Protocol
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The extraction was carried out based on the ratio between dry mass and water loss [35 (link),36 (link)] using 2:2:1 methanol: chloroform: water according to Kim et al. [37 (link)]. The hydrophilic fraction was removed from the extract and dried under vacuum using vacuum concentrator (Labconco, Kansas, USA) for 24 h.
3
Simultaneous Extraction of Xenobiotic and Endogenous Metabolites from Earthworms
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Collected worms were all flash-frozen and ground in liquid nitrogen using mortar and pestle, then kept at −80 °C until extraction. To simultaneously extract TPHP metabolites and endogenous metabolites of earthworm, 600 μl ice cold methanol (LC-MS grade)/ultrapure water (volume ratio 4:1) containing internal standard N-9-Fmoc-L-glycine (20 mg/l, Sigma Aldrich, CAS 29022-11-5, >98%) was added to 50 mg worm tissue. The mixture was homogenized with three zirconium oxide beads (2.8 mm diameter) at medium speed for 60 s and two times using a Minilys homogenizer (Bertin Technologies, France). Then, the mixture was vortexed for 5 min, placed on dry ice for 15 min, and centrifuged at 14000 rpm for 10 min at 4 °C. The supernatant was transferred to a sample vial and analysed by LC-QTOF. An aliquot of 50 μl was dried in a vacuum concentrator (Labconco, MO, USA) at 4 °C for further derivatization and analysis via GC-MS.
4
Urine Metabolite Extraction and Derivatization
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Metabolites from urine were extracted using a previously reported procedure with a modification [26 (link)]. Each urine sample was thawed on ice for 60 min and vortexed for 1 min. An aliquot of 10 μL of urine was added into a labeled 1.5 mL micro-centrifuge tube containing 990 μL of pure methanol. The mixture was thoroughly vortexed for 5 min and then centrifuged at 16,100× g for 10 min at 4 °C. The supernatant was transferred into a 1.5 mL micro-centrifuge tube and completely dried using a vacuum concentrator (Labconco, Kansas City, MO, USA). The dried sample was derivatized in two steps. The sample was derivatized with 5 μL of 40 mg/mL methoxyamine hydrochloride in pyridine (Sigma-Aldrich, St. Louis, MO, USA) for 90 min at 30 °C for methoximation, and then with 45 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (Fluka, Buchs, Switzerland) for 30 min at 37 °C for silylation. 2 μL of mixture of fatty acid methyl esters including methyl forms of C8, C9, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, and C30 was added to the derivatized sample as a retention index marker.
5
Purification and Deglycosylation of SARS-CoV-2 S Protein
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Clinical specimens collected from infected patients containing SARS-CoV-2 were grown in cell culture using Vero E6 cells following a reported procedure [21 ] and, as used in a previous study [12 (link)], were the source of an originating-like strain. Following chemical and heat inactivation and filtration, the virus was precipitated with polyethylene glycol precipitation of virus was performed after filtration through a 300-K molecular weight cut-off (MWCO) filter (Pall Corporation, Cheltenham, Victoria). The retentate was reconstituted in buffer (50 mM ammonium bicarbonate), sonicated (3 × 30 min) and then deglycosylated following the addition of 1.2 units of recombinant peptide-N-glycosidase F (PNGaseF) (Roche Diagnostics, North Ryde, Sydney, Australia) and 5 mM octyl β-D-glucopyranoside (Sigma Aldrich–Merck, Castle Hill, Sydney, Australia). The released viral proteins were separated by SDS-PAGE and the S-protein (at some 150 kDa) was excised from the gel. The gel plug was transferred into 25 mM ammonium bicarbonate solution containing 10% v/v acetonitrile (ACN) and 10 mM dithiothreitol (DTT) (10 mM) and heated for 30 min at 60 °C. The gel plug was washed three times with 25 mM ammonium bicarbonate in 50% acetonitrile and then dried in a vacuum concentrator (Labconco Corporation, Kansas City, MI, USA).
6
Protein Extraction from MCF-7 Cell Lines
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MCF-7/WT and MCF-7/DOX-1 cells were lysed with a lysis solution of 8 M urea (161-0731, Bio-Rad, USA, 100 mM Tris-HCl, pH 8, 161-0719, Bio-Rad, Hercules, CA, USA). Then, the mixture was centrifuged at 12,000× g and 4 °C for 15 min. Next, cold acetone (−20 °C, 1000201000, Merck, Germany) in a ratio of 1:8 (v/v) was added to the supernatant, and the mixture was vortexed and stored at −20 °C for 60 min. Afterward, the mixture was again centrifuged for 15 min at 12,000× g and 4 °C. The obtained pellet was dried in a vacuum concentrator (Labconco, Kansas City, MO, USA).
7
Retina Protein Peptide Preparation
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Aliquots of 50µg of retina protein lysate were used for peptide preparation using the S-Trap method in accordance with previous protocol [53 (link)]. Briefly, the lysate was reduced in 20 mM Dithiothreitol (DTT) at 95 °C for 10 min, and then alkylated in 40 mM iodoacetamide (IAA) in the dark for 10 min at RT. Next, the solution was acidified with 12% aqueous phosphoric acid and diluted with S-Trap protein binding buffer. The S-Trap micro spin column (ProtiFi, Farmingdale, NY, USA) was used to trap the protein in the membrane, and protein was digested with trypsin (1:25, w/w, trypsin: protein) on the filter at 47 °C for 1 h. After digestion, the peptides were eluted with consecutive reagents: (1) 50 mM TEAB, (2) 0.2% aqueous formic acid (FA), and (3) 50% Acetonitrile (ACN) containing 0.2% FA. The eluted solution was dried out with a vacuum concentrator (Labconco, Kansas City, MO, USA) at 4 °C. The peptides were resuspended in 0.1% FA and measured with Pierce Quantitative Colorimetric Peptide Assay (Cat no.23275, Thermo, Waltham, MA, USA) following the manufacturer’s instruction. The peptide sample was normalized with 0.1% FA to a final concentration of 0.5 µg/μL for mass spectrometry identification and quantification.
8
Reduction, Alkylation, and Tryptic Digestion of MAB1
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The purified MAB1 samples were dried down using a vacuum concentrator (LABCONCO, Kansas City, MO). The dried samples were re-suspended in 100 mM Tris-HCl containing 8 M urea and 10 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl), and then incubated at 37°C for 30 minutes. The reduced cysteine residues were alkylated with 10 mM iodoacetamide at room temperature for 30 minutes in the dark. Following alkylation, the urea concentration was diluted to 1.25 M prior to digestion. Trypsin (Promega, Sunnyvale, CA) was added the samples at an enzyme: substrate ratio of 1:10 and incubated at 37°C for 4 hours. Digestion was terminated by addition of 20% formic acid. The digested samples were stored at -80°C until analysis.
9
Serum Protein Deglycosylation and Tryptic Digestion
10
Targeted Metabolomics of Plant Phytochemicals
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Baicalin, wogonin, 13-HPOT, OPDA, OPC-8, and MeJA standards were purchased from Desite Biotech (Chengdu, Sichuan Province, China). Standard stock solutions (0.1 mg ml−1) were prepared following Geng et al. (2023) (link).
The frozen samples were crushed in a mixer mill (MM 400, Retsch) containing small steel balls in liquid nitrogen for 1.5 min at 30 Hz. Then, 1 g of sample was extracted three times with 10 ml of 50% methanol in an ultrasonicator bath for 30 min and centrifuged at 12,000 × g for 10 min to remove debris. The supernatant was collected and concentrated with a vacuum concentrator (Labconco, Kansas City, KS, USA) at 4°C until it was 2 ml. The concentrated supernatant was filtered through a 0.2-μm filter before injection. UPLC was performed with the Acquity H system (Waters Corp., Milford, MA, USA). MS was performed with the Xevo TQ-XS system (Waters Corp). Separation was achieved with a 150 × 2.1-mm, 3-µm C18-120 column (Shimadzu, Tokyo, Japan) and the following gradient: 0.1% formic acid in water (A) vs. 0.1% formic acid in acetonitrile (B) run at 0.3 ml min−1 and a column temperature of 40°C (0 min, 95% B; 15 min, 95% B; 16 min, 95% B; 17 min, 5% B; 20 min, 5% B).
The frozen samples were crushed in a mixer mill (MM 400, Retsch) containing small steel balls in liquid nitrogen for 1.5 min at 30 Hz. Then, 1 g of sample was extracted three times with 10 ml of 50% methanol in an ultrasonicator bath for 30 min and centrifuged at 12,000 × g for 10 min to remove debris. The supernatant was collected and concentrated with a vacuum concentrator (Labconco, Kansas City, KS, USA) at 4°C until it was 2 ml. The concentrated supernatant was filtered through a 0.2-μm filter before injection. UPLC was performed with the Acquity H system (Waters Corp., Milford, MA, USA). MS was performed with the Xevo TQ-XS system (Waters Corp). Separation was achieved with a 150 × 2.1-mm, 3-µm C18-120 column (Shimadzu, Tokyo, Japan) and the following gradient: 0.1% formic acid in water (A) vs. 0.1% formic acid in acetonitrile (B) run at 0.3 ml min−1 and a column temperature of 40°C (0 min, 95% B; 15 min, 95% B; 16 min, 95% B; 17 min, 5% B; 20 min, 5% B).
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