The influence of hydrolysis and derivatization on the determination of amino acid content and isotopic ratios in dual labeled (13 C, 15 N) white clover.

BACKGROUND

The cycling of peptide and protein-bound amino acids (AAs) is important for studying the rate limiting steps in soil N turnover. A strong tool is stable C and N isotopes used in combination with compound-specific isotope analysis (CSIA), where a prerequisite for analysis is appropriate methods for peptide and protein hydrolysis and appropriate methods for derivatization of AAs for analysis by gas chromatography (GC).

METHODS

We examined the efficiency of a standard acidic hydrolysis (6 M HCl, 20h at 110°C) and a fast acidic hydrolysis (6 M HCl, 70 min at 150°C) on the recovery of AAs from a protein standard (Bovine Serum Albumin). The best methods were used on dual-labeled (13 C and 15 N) clover shoot and root juice, divided into four molecular weight (Mw) size fractions. We used NAIP (N-acetyl isopropyl esterification) derivatization for GC/C-IRMS analysis of AA standards.

RESULTS

The NAIP derivatization gave very low LODs (< 2 pmol) and LOQs ranging from 0.55 to 4.89 pmol. Comparing the concentrations of individual AAs in hydrolyzed versus un-hydrolyzed clover juice samples of the low Mw size fraction (< 1 kDa) showed a significant decline in concentration (p < 0.03) for seven AAs after hydrolysis. Despite the decline in AA concentration, we found a linear connection between the obtained atomic fraction (13 C/total carbon and 15 N/total nitrogen) for individual AAs of hydrolyzed versus un-hydrolyzed samples.

CONCLUSIONS

The methodology distinguished differences in atomic fractions across AAs, in individual AAs in Mw size fractions, and between shoot and root samples of experimentally labeled white clover. Specifically, the method separated L-glutamate (Glu) and glutamine (Gln). Thus, for a broader use in plant and soil ecology, we present an optimized methodology for GC/C-IRMS analysis of AAs from organic nitrogen samples enriched with 13 C and 15 N - AA stable isotope probing (SIP).