Kirsten Lønne Enggrob1, Thomas Larsen2,3, Mogens Larsen4, Lars Elsgaard1, Jim Rasmussen1. 1. Department of Agroecology, Aarhus University, Foulum, Denmark. 2. Max Planck Institute for the Science of Human History, Kahlaische Str. 10, 07745, Jena, Germany. 3. Leibniz-Laboratory for Radiometric Dating and Stable Isotope Research, Christian-Albrechts Universität zu Kiel, Kiel, Germany. 4. Department of Animal Science, Aarhus University, Foulum, Denmark.
Abstract
RATIONALE: The cycling of peptide- and protein-bound amino acids (AAs) is important for studying the rate-limiting steps in soil nitrogen (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, 20 h 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/combustion-isotope ratio mass spectrometry (C-IRMS) analysis of AA standards. RESULTS: The NAIP derivatization gave very low limits of detection (LODs) (< 2 pmol) and limits of quantification (LOQs) ranging from 0.55 to 4.89 pmol. Comparing the concentrations of individual AAs in hydrolyzed versus unhydrolyzed 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 unhydrolyzed 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).
RATIONALE: The cycling of peptide- and protein-bound amino acids (AAs) is important for studying the rate-limiting steps in soil nitrogen (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, 20 h 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/combustion-isotope ratio mass spectrometry (C-IRMS) analysis of AA standards. RESULTS: The NAIP derivatization gave very low limits of detection (LODs) (< 2 pmol) and limits of quantification (LOQs) ranging from 0.55 to 4.89 pmol. Comparing the concentrations of individual AAs in hydrolyzed versus unhydrolyzed 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 unhydrolyzed 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).