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UC Davis Plant Sciences


Amino Acid Sample Preparation

Acid Hydrolysis

Acid hydrolysis is performed to liberate individual amino acids from proteinaceous samples. Dry, homogenized sample materials are placed in new borosilicate vials with heat- and acid-resistant caps, 0.5mL (animal tissues) or 2mL (plant tissues) of 6M hydrochloric acid added, and vial threads wrapped with PTFE-tape. Vials are then flushed with N2, sealed, and placed in an oven at 150°C for 70 minutes. After cooling, 200µL of heptane:chloroform (6:5, v:v) is added to the acid hydrolysates of sample materials, the vials briefly vortexed, and the organic layer discarded in order to remove any remaining lipophilic compounds prior to drying. Samples are then dried in a heating block at 60°C under a gentle stream of N2. For samples of purified proteins, such as collagen, the lipid removal step is not performed. For samples with a significant inorganic matrix, additional clean-up steps may be required, including cation-exchange chromatography.

 

Ethanolic (80%) extraction of free amino acids is also available, please contact us for details.

 

13C in amino acids
Hydrolysates (<10µmoles total) are first dissolved in 200µL 0.4M hydrochloric acid; for a variety of matrices this maintains a pH<1, regardless of sample matrix. pH control is critical in the use of chloroformates as derivatization reagents[1]. 100µL of each solution is placed in a GC vial with a 350µL fixed insert. 20µL of the internal reference solution (typically L-norleucine), 50µL of methanol, and 30µL of pyridine are added, and the reagents briefly vortexed. To each vial, 15µL of the derivatizing reagent, methyl chloroformate, are added, the vials vortexed and set aside. All reagents are present in excess relative to the substrate. After ten minutes of rest at room temperature, 100µL of chloroform are added and the solution vortexed until an emulsion forms. The solution is set aside until the emulsion separates into aqueous and organic layers, usually in less than five minutes. The organic layer is transferred into a new GC vial with 300µL fixed insert. Prepared samples may be stored at -20°C for up to 2 weeks, but are typically analyzed within 24 hours of preparation.

 

The resulting methoxycarbonyl methyl esters are then suitable for GC-C-IRMS analysis. Importantly, these derivatives contain very little exogenous carbon from the derivatization reagents, facilitating the δ13C analysis of amino acids. The amino acids suitable to derivatization as methoxycarbonyl methyl esters include: Ala, Asp, Glu, Gly, Ile, Leu, Lys, Met, Pro, Thr, Tyr, and Val. This method is not suitable for His, Hyp, or Ser. Measurement error is variable between amino acids, but is generally ≈ ±1‰, after accounting for the influence of exogenous carbon1.
Fig1

Fig. 1.Derivatization reaction of valine with methyl chloroformate in the presence of methanol and pyridine (simplified from Chen et al. 2010). 

 

15N in amino acids
First, the sample acid hydrolysates (<10µmoles total) and 20µL of the internal reference solution are combined and then dried under a stream of nitrogen. 1mL of 1.85M acidified isopropanol is then added to each reaction vial and the solution heated at 100°C for 1h. The remaining isopropanol is evaporated under nitrogen at 40°C. Chloroform is added (250µL) and evaporated under nitrogen in a cold block to remove remaining excess reagents. The partial derivatives are then acetylated with a mixture of acetic anhydride, trimethylamine, and acetone (1mL; 1:2:5, v/v/v; 10min., 60°C) and the reagents evaporated under nitrogen gas in a cold block (0°C). Once dry, ethyl acetate is added (2mL), along with a saturated NaCl solution (1mL), and the solution vortexed. Following phase separation, the aqueous phase is discarded and the ethyl acetate removed under nitrogen gas in a cold block (0°C). Trace water is removed with two additions of chloroform (1mL). Finally, ethyl acetate is added (100µL) and the N-acetyl isopropyl esters transferred to a GC vial with insert. Prepared samples may be stored up to 12 weeks at -20°C prior to analysis, but are generally analyzed within 24 hours of preparation.

 

The resulting N-acetyl amino acid isopropyl esters are then suitable for GC-C-IRMS analysis. The amino acids suitable to derivatization by this method include: Ala, Asp, Glu, Gly, His, Hyp, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val. This method is not well suited for the analysis of Ile. Measurement error is variable between amino acids, but is generally <±1‰ [2, 3].

 

General considerations
Following acid hydrolysis, some amino acids cannot be detected by GC-C-IRMS. They include glutamine and asparagine, which are converted to their respective acid forms (i.e. Glu, Asp). Tryptophan is destroyed during acid hydrolysis, while cysteine is only partially released as cystine. Note that many of these amino acids may be measured using MCF as free amino acids, just not from acid hydrosylates. Arginine cannot be derivatized by nearly all techniques, regardless of experimental context. Please be aware that sample materials are inherently variable in amino acid composition (Figs. 2-4) and not all amino acids may be measureable from all samples. Further, variation in the amino acid composition of samples will impact relative measurement error among the individual amino acids.

 

 

Fig2

 Fig. 2. Chromatograph of δ15N analysis of N-acetyl amino acid isopropyl ester derivatives from mammal skin.

 

Fig3

 Fig. 3. Chromatograph of δ15N analysis of N-acetyl amino acid isopropyl ester derivatives from bone collagen.

 

Fig4

 Fig. 4. Chromatograph of δ15N analysis of N-acetyl amino acid isopropyl ester derivatives from oak flour.

 

 

References

1R.G. Walsh, S. He, and C.T. Yarnes. 2014.Compound-specific δ13C and δ15N analysis of amino acids: a rapid, chloroformate-based method for ecological studies. Rapid Commun. Mass Spectrom. 28: 96–108. [doi: 10.1002/rcm.6761]


2C.T. Yarnes and J. Herszage. 2017. The relative influence of derivatization and normalization procedures on the compound-specific stable isotope analysis of nitrogen in amino acids. Rapid Communications in Mass Spectrometry 31: 693-704. [doi: 10.1002/rcm.7832]


3A. K. Styring, A. Kuhl, T. D. J. Knowles, R. A. Fraser, A. Bogaard, R. P. Evershed. Practical considerations in the determination of compound-specific amino acid δ15N values in animal and plant tissues by gas chromatography-combustion-isotope ratio mass spectrometry, following derivatisation to their N-acetyl isopropyl esters. Rapid Communications in Mass Spectrometry 26: 2328-2334. [doi: 10.1002/rcm.6322]

 




 
e-mail: sif@ucdavis.edu | phone: 530-752-8100 | fax: 530-752-4361
UC Davis Stable Isotope Facility | Department of Plant Sciences
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