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D4 l alanine

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D4-L-alanine is a stable isotope labeled amino acid. It is used as a research tool in analytical and biochemical applications.

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Lab products found in correlation

Z2 coulter counter, by Beckman Coulter (1 mentions) Zorbax sb c8, by Agilent Technologies (1 mentions) 6 well plate, by BD (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions)

3 protocols using d4 l alanine

1

Measuring Sphingolipid Metabolism in Cells

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SL labeling assays and SPT activity measurements were performed in vivo in cells. SPTLC1-KO cells expressing SPTLC1-ALS and -HSAN1 variants were plated at 200,000 cells/mL in 6-well plates. Cells were grown for 48 hours to 70% confluence in DMEM/10% FBS growth media. For standard labeling assay, the medium was replaced with L-serine–free DMEM (Genaxxon Bioscience) containing 10% FBS, 1% P/S, and D3-15N-L-serine (1 mM unless indicated otherwise) and 2 mM D4-L-alanine (Cambridge Isotope Laboratories). Cells were grown further in the labeling media for 16 hours. C6-Cer, when used in activity assays, was added together with the labeling media. For siRNA-mediated knockdown in HEK293 cells and fibroblasts, the assay was started 72 hours after transfection. For lipid analysis, HEK293 cells were harvested in ice-cold PBS. Fibroblasts were harvested by trypsinization (500 μL). Finally, 50 μL of cells were counted (Z2 Coulter Counter, Beckman Coulter) and cell pellets were frozen at –20°C until extraction.
Lone M.A., Aaltonen M.J., Zidell A., Pedro H.F., Morales Saute J.A., Mathew S., Mohassel P., Bönnemann C.G., Shoubridge E.A, & Hornemann T. (2022). SPTLC1 variants associated with ALS produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins. The Journal of Clinical Investigation, 132(18), e161908.
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2

Deuterium-Labeled Peptide Synthesis

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[D4]L-alanine from Cambridge Isotope Laboratories (Tewksbury, MA) was modified with an N-terminal Fmoc protecting group and was recrystallized from Ethyl acetate/hexane (80:20). Other protected amino acids and Rink amide resin, including arginine whose side chain was additionally protected with 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protecting group, were purchased from Anaspec (Freemont, CA), Bachem (Torrance, CA), Sigma Aldrich and NovaBiochem (MillaporeSigma, Burlington, MA). Peptides were synthesized on a model 433A solid-phase peptide synthesizer (Applied Biosystems/Thermo Fisher Sci.; Foster City, CA) using a 0.1 mmol scale FastMoc chemistry. Two deuterium-labeled alanines were incorporated per peptide, with 50% and 100% isotope abundance levels, to distinguish the 2H NMR signals based on relative intensities. Following cleavage from the resin using a trifluoroacetic acid (TFA) based cocktail, the resulting C-termini amidated peptides were precipitated with methyl-tert-butyl-ether/hexane (50:50). The peptides were purified by reversed-phase high-performance liquid chromatography on a 5 μm octylsilica Zorbax SB-C8 or SB300-C3 column (9.4 mm × 250 mm) (Agilent Technologies, Santa Clara, CA), as described in [32 (link), 49 (link)]. The molecular mass of peptide and distributions of deuterium were confirmed by MALDI-TOF mass spectrometry (Figure S1).
Lipinski K., McKay M.J., Afrose F., Martfeld A.N., Koeppe RE I.I, & Greathouse D.V. (2019). Influence of Lipid Saturation, Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes. Chembiochem : a European journal of chemical biology, 20(21), 2784-2792.
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3

Isotope Labeling of Serine and Alanine

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Cells (250,000) were seeded in 2 ml fresh medium in 6-well plates (BD Falcon) and cultured for 2 days, reaching approximately 70%–80% confluence. The medium was exchanged for L-serine- and L-alanine-free DMEM (Genaxxon Bioscience), containing 10% FBS (Thermo Fisher Scientific; FSA15043) and 1% penicillin and streptomycin (100 units/ml and 0.1 mg/ml, respectively; Millipore Sigma-Aldrich). Two hours after medium exchange, isotope-labeled (2,3,3)-d3-15N-L-serine (1 mM) and (2,3,3,3)-d4-L-alanine (2 mM) was added (Cambridge Isotope Laboratories).
Karsai G., Steiner R., Kaech A., Lone M.A., von Eckardstein A, & Hornemann T. (2021). Metabolism of HSAN1- and T2DM-associated 1-deoxy-sphingolipids inhibits the migration of fibroblasts. Journal of Lipid Research, 62, 100122.
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