18o water

Manufactured by Cambridge Isotopes

Sourced in United States

18O-water is a stable isotope of water enriched with the oxygen-18 isotope. It is a colorless, odorless liquid with the chemical formula H2^18O. 18O-water is used as a research tool in various scientific applications, including tracer studies, metabolic research, and environmental analysis.

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Lab products found in correlation

Glu c, by Promega (3 mentions) Chymotrypsin, by Promega (2 mentions) Lys c, by Promega (2 mentions) Arg c, by Promega (2 mentions) Pepmap rslc c18 column, by Thermo Fisher Scientific (2 mentions) Endo h, by Promega (2 mentions) Proteoiq, by Premier Biosoft (2 mentions) Fusion instrument software, by Thermo Fisher Scientific (2 mentions) Proteome discoverer 1, by Thermo Fisher Scientific (2 mentions) Trypsin, by Promega (2 mentions) Alpha lytic protease, by New England Biolabs (1 mentions) Endoglycosidase h, by Promega (1 mentions) Simplyblue safestain, by Thermo Fisher Scientific (1 mentions) Iodoacetamide, by Merck Group (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Pngase f, by Promega (1 mentions) Asp n, by Promega (1 mentions) Iodoacetic acid, by Merck Group (1 mentions) Guanidine hydrochloride, by Merck Group (1 mentions) Bradykinin, by Merck Group (1 mentions)

8 protocols using 18o water

Glycosylation Profile Analysis of RC1 and RC1-4fill

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The glycosylation profiles of RC1 and RC1–4fill were determined
as previously described [REF]. Briefly, samples were denatured with Lys-C
(Promega), Arg-C (Promega), Glu-C (Promega), and chymotrypsin (Promega).
Following digestion, the samples were deglycosylated by Endo-H (Promega) and
PNGaseF (Glyko®, Prozyme) in the presence of ¹⁸O-water
(Cambridge Isotope Laboratories). The resulting peptides were separated on an
Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and analyzed using an
Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo
Fisher Scientific) with a 240-min linear gradient consisting of 1–100%
solvent B over 180 min at a flow rate of 200 nL/min. Full MS scans were acquired
using the Fusion instrument software (v2.0, Thermo Fisher Scientific), and the
resulting spectra were analyzed and filtered using SEQUEST (Proteome Discoverer
1.4, Thermo Fisher Scientific) and ProteoIQ (v2.7, Premier Biosoft). Site
occupancy was calculated using spectral counts assigned to the
¹⁸O-Asp-containing (PNGaseF-cleaved) and/or HexNAc-modified
(EndoH-cleaved) peptides and their unmodified counterparts.

Escolano A., Gristick H.B., Abernathy M.E., Merkenschlager J., Gautam R., Oliveira T.Y., Pai J., West AP J.r., Barnes C.O., Cohen A.A., Wang H., Golijanin J., Yost D., Keeffe J.R., Wang Z., Zhao P., Yao K.H., Bauer J., Nogueira L., Gao H., Voll A.V., Montefiori D.C., Seaman M.S., Gazumyan A., Silva M., McGuire A.T., Stamatatos L., Irvine D.J., Wells L., Martin M.A., Bjorkman P.J, & Nussenzweig M.C. (2019). Immunization expands HIV-1 V3-glycan specific B-cells in mice and macaques. Nature, 570(7762), 468-473.

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Glycosylation Profile Analysis of RC1 and RC1-4fill

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Gel Electrophoresis and Protein Digestion

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4–15% Mini-PROTEAN^® TGX^™ Precast Protein Gels, 15-well, 15 μl (Bio-Rad); SimplyBlue^™ SafeStain (Invitrogen); dithiothreitol (Sigma); iodoacetamide (Sigma); alpha lytic protease (New England BioLabs); chymotrypsin (Athens Research and Technology); AspN (Promega); Glu-C (Promega); trypsin (Promega); endoglycosidase H (Promega); PNGaseF (Promega); 18O water (Cambridge Isotope Laboratories).

Chmielewski D., Wilson E.A., Pintilie G., Zhao P., Chen M., Schmid M.F., Simmons G., Wells L., Jin J., Singharoy A, & Chiu W. (2023). Integrated analyses reveal a hinge glycan regulates coronavirus spike tilting and virus infectivity. Research Square.

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IgG1 Antibody Characterization Protocol

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All chemicals were reagent grade or above.
Guanidine hydrochloride (GndHCl), ethylenediaminetetraacetic acid
(EDTA), dithiothreitol (DTT), iodoacetic acid (IAA), trifluoroacetic
acid (TFA), acetonitrile (ACN), HPLC-grade water, and bradykinin were
from Sigma-Aldrich (St. Louis, MO). Sequencing grade trypsin, GluC,
and Asp-N were from Roche (Indianapolis, IN). ¹⁸O-water
(97%) was from Cambridge Isotope Laboratories (Andover, MA). Recombinant
monoclonal IgG1 antibody (antistreptavidin immunoglobulin gamma 1)
was produced in Chinese hamster ovary (CHO) cells (Amgen, Thousand
Oaks, CA), purified according to standard manufacturing procedures,
formulated at a concentration of 30 mg/mL in 50 mM sodium acetate
at pH 5.2, and stored at −70 °C.

Liu M., Zhang Z., Cheetham J., Ren D, & Zhou Z.S. (2014). Discovery and Characterization of a Photo-Oxidative Histidine-Histidine Cross-Link in IgG1 Antibody Utilizing 18O-Labeling and Mass Spectrometry. Analytical Chemistry, 86(10), 4940-4948.

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Synthesis and Characterization of 18O-NAM

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This compound was synthesized as described previously.^{39 (link),55} Briefly, 3-cyanopyridine (100 mg, 0.96 mmol) and 1,1′,3,3′-tetramethylguanidine (5.1 mg, 0.04 mmol) were dissolved in 200 μL ¹⁸O-water (97%, Cambridge Isotope Laboratories). The solution was heated at 100°C for 1 h before water and 1,1′,3,3′-tetramethylguanidine were removed by lyophilization. After crystallization from benzene, ¹⁸O-NAM was obtained (102 mg, 0.82 mmol, 85% yield). The incorporation of ¹⁸O was determined to be 96.5% by MALDI-TOF.

Tran A., Yokose R, & Cen Y. (2018). Chemo-enzymatic Synthesis of Isotopically Labeled Nicotinamide Riboside. Organic & biomolecular chemistry, 16(19), 3662-3671.

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Synthesis of 18F-FLT Radiotracer

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¹⁸F-FLT was synthesized in a TRACERlab MXFDG (GE Healthcare) using an ABX-FLT kit (ABX) [14 (link)]. No-carrier-added ¹⁸F-fluoride was produced via the ¹⁸O(p,n)¹⁸F reaction from > 98% enriched ¹⁸O-water (Cambridge Isotope Laboratories) on an RDS eclipse RD/HP medical cyclotron (Siemens/CTI). The radiochemical purity of the final product was > 99%, and the yield was 12.3 ± 4.6% (end of synthesis, n = 17).

Tsujikawa T., Tasaki T., Hosono N., Mori T., Makino A., Kiyono Y., Zanotti-Fregonara P., Yamauchi T, & Okazawa H. (2019). 18F-FLT PET/MRI for bone marrow failure syndrome-initial experience. EJNMMI Research, 9, 16.

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Isotope Labeling of Nicotinonitrile via Oxidation

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In a pressure tube, placed under vacuum and then filled with argon, [¹⁵N,¹³C]nicotinonitrile (0.306 g, 0.0029 mol) was added, followed by manganese oxide (0.05 g; 0.6 mmol) and [¹⁸O]water (from Cambridge Isotope Laboratories, 97 atom % ¹⁸O) (0.350 mL, 0.019 mol, 6.5 equiv.) added from a syringe under an argon atmosphere. The tube was closed with a threaded PTFE cap, immersed in an oil bath and heated therein at 70–85°C overnight. The tube was allowed to cool down to room temperature and the content of the tube was dissolved in a 1:1 DCM/acetonitrile mixture. The solution was filtered twice through a filter paper and the filtrate was concentrated under reduced pressure to give a white solid (0.280 g, 78%). ¹H NMR (D₂O), δ, ppm: 7.48 (dd, 1H, ³J_HH= 8.0 Hz, ³J_HH= 5.1 Hz, H5), 8.14 (dddd, 1H, ³J_HC= 8.0 Hz, ³J_HH= 3.9 Hz, ⁴J_HH= 2.2 Hz, ⁴J_HH= 1.9 Hz, H4), 8.61 (apparent d, 1H, ³J_HH= 4.5 Hz, H6), 8.83 (br s, 1H, H2). ¹³C NMR (D₂O), δ, ppm: 124.16 (d, ³J_CC=3.5 Hz, C5), 129.28 (dd, ¹J_CC=64.5 Hz, ²J_NC=7.1 Hz, C3), 136.43 (²J_CC=2.0 Hz, C4), 147.61 (d, ²J_CC=3.6 Hz, C2), 151.79 (C6), 170.75 (d, ¹J_NC=17.6 Hz, ¹³C(O)−¹⁵N). HRMS found: 127.05930. Calculated for ¹³C¹²C₅H₇¹⁵N¹⁴N¹⁸O (M+1): 127.05992.

Shats I., Williams J.G., Liu J., Makarov M.V., Wu X., Lih F.B., Deterding L.J., Lim C., Xu X., Randall T.A., Lee E., Li W., Fan W., Li J.L., Sokolsky M., Kabanov A.V., Li L., Migaud M.E., Locasale J.W, & Li X. (2020). Bacteria boost mammalian host NAD metabolism by engaging the deamidated biosynthesis pathway. Cell metabolism, 31(3), 564-579.e7.

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Protein Reduction, Alkylation, and Trypsin Digestion

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For each sample, 3 mg of protein was reduced in 5 mM dithiothreitol (DTT) at 56 °C for 30 min and alkylated in 15 mM iodoacetamide (IAA) at room temperature for 30 min in the dark. Pure proteins were obtained via trichloroacetic acid/acetone precipitation. For trypsin digestion, protein pellets were resuspended in 50 mM ammonium bicarbonate (ABC) buffer and proteolytically cleaved with 20 μg of sequencing-grade trypsin (Promega Corporation, Madison, WI, USA) at a ratio of 50:1 (protein:enzyme, w/w) at 37 °C overnight. The digested peptides were dried using a speed-vac and immediately re-dissolved in 50 µL of 50 mM calcium chloride, followed by the addition of trypsin (Promega Corporation, Madison, WI, USA) at a ratio of 50:1 (protein:enzyme, w/w). For enzymatic labeling, peptides were differentially labeled with either ¹⁶O or ¹⁸O water (97%, Cambridge Isotope Laboratories, MA, USA) containing 20% acetonitrile and incubated for 24 h at 37 °C. Labeling was stopped using 1% TFA. Peptide concentration was measured using a quantitative colorimetric peptide assay kit (Thermo Fisher Scientific). For comparison, two labeled samples were mixed in equal amounts and dried.

Lee E.H., Chung J.W., Sung E., Yoon B.H., Jeon M., Park S., Chun S.Y., Lee J.N., Kim B.S., Kim H.T., Kim T.H., Choi S.H., Yoo E.S., Kwon T.G., Kang H.W., Kim W.J., Yun S.J., Lee S, & Ha Y.S. (2022). Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways. International Journal of Molecular Sciences, 23(21), 13240.

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