What Is L-Lysine-¹³C₆,¹⁵N₂ Dihydrochloride?

L-Lysine-¹³C₆,¹⁵N₂ dihydrochloride is a synthetic, isotopically enriched derivative of the proteinogenic amino acid L-lysine. All six carbon atoms of the lysine backbone are replaced with carbon-13 (¹³C), and both nitrogen atoms — the α-amino group and the ε-amino group — are replaced with nitrogen-15 (¹⁵N). The resulting compound is supplied as its dihydrochloride salt to enhance aqueous solubility and chemical stability under standard laboratory storage conditions.

Because lysine is an essential amino acid that mammals cannot synthesize de novo, exogenously supplied heavy lysine is efficiently incorporated into newly synthesized proteins during cell culture without metabolic scrambling to other amino acids — a key advantage over some other labeled residues used in isotope labeling experiments.

The compound is catalogued by Aladdin Scientific under SKU L1444084 and is cross-referenced to MCE-TJY catalog number HY-W009762S.

Isotopic Properties and Mass Spectrometry Significance

The simultaneous incorporation of ¹³C and ¹⁵N isotopes confers an approximate +8 Da mass shift relative to the monoisotopic mass of unlabeled L-lysine. This shift is sufficiently large to resolve heavy and light peptide pairs in virtually all modern high-resolution mass spectrometers, including Orbitrap, Q-TOF, and triple-quadrupole instruments, without spectral overlap with natural isotope envelopes of the light counterpart.

The dual-label strategy (¹³C₆,¹⁵N₂) provides superior accuracy compared to single-label variants. The presence of both carbon-13 and nitrogen-15 labels minimizes the risk of isotopic cross-contamination between heavy and light channels, improving the signal-to-noise ratio of quantitative measurements and enabling precise relative quantification of protein expression changes across experimental conditions.

High isotopic purity is critical: residual unlabeled amino acid in the heavy reagent would introduce systematic underestimation errors in quantitative ratios. Researchers should verify isotopic enrichment certificates (typically ≥98 atom% for each isotope) supplied with each lot.

SILAC Protocol and Cell Culture Integration

Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is the primary application for L-Lysine-¹³C₆,¹⁵N₂ dihydrochloride. In a typical duplex SILAC experiment, one cell population is cultured in standard medium containing light (¹²C,¹⁴N) lysine, while a second population is cultured in medium containing the heavy labeled lysine (¹³C₆,¹⁵N₂). After at least five to six cell doublings — sufficient for near-complete (>97%) isotopic incorporation — the two populations are mixed 1:1 by total protein mass, co-digested with trypsin (which cleaves after lysine and arginine residues), and analyzed by LC-MS/MS.

Key protocol considerations include:

• Dialyzed serum: Standard FBS contains free amino acids that dilute heavy label uptake; dialyzed FBS (molecular weight cut-off ≤10 kDa) should be used to deplete endogenous lysine.
• Arginine co-labeling: For complete proteome coverage, heavy L-arginine (e.g., ¹³C₆ or ¹³C₆,¹⁵N₄) is typically combined with heavy lysine to label all tryptic peptides.
• Incorporation rate verification: A pilot experiment measuring the heavy-to-light ratio of a reference protein or total proteome digest is recommended before full-scale experiments.
• Concentration matching: The concentration of heavy lysine in the medium should match that of the light counterpart to avoid differential effects on cell growth or metabolism.

L-Lysine-¹³C₆,¹⁵N₂ dihydrochloride is also applicable as a spike-in internal standard for absolute quantification workflows and for validating targeted proteomics assays (e.g., SRM/MRM, PRM).

Handling, Storage, and Quality Considerations

Storage: Store the compound at −20 °C in a tightly sealed container, protected from light and moisture. The dihydrochloride salt form is hygroscopic; exposure to ambient humidity can lead to weight gain and apparent concentration errors when preparing stock solutions. Desiccated storage and rapid resealing after each use are recommended.

Stock solution preparation: Dissolve in ultrapure water or 0.1 M HCl to yield a concentrated stock (e.g., 50–100 mM). Filter-sterilize (0.22 µm) before addition to cell culture medium. Avoid repeated freeze-thaw cycles; aliquot stocks for single use.

Quality parameters to verify:

• Isotopic enrichment: ≥98 atom% ¹³C and ≥98 atom% ¹⁵N (confirm via certificate of analysis)
• Chemical purity: ≥98% (HPLC)
• Identity: confirm by NMR or high-resolution MS

Safety: The compound presents low acute toxicity under normal laboratory handling. Follow standard laboratory safety practices: wear appropriate PPE (gloves, lab coat, eye protection), avoid inhalation of dust, and dispose of waste according to local regulations. Refer to the Safety Data Sheet (SDS) for comprehensive hazard information.