A liquid chromatographic/mass spectrometric method to evaluate 13C and 15N incorporation into soil amino acids

Amino acids are highly associated with biogeochemical cycling and represent an important potential source and sink of carbon (C) and nitrogen (N) in terrestrial ecosystems. Tracing the isotope dynamics of amino acids can improve the understanding of the origin and transformation of amino acids in soil matrix at process-levels; hence, the liquid chromatographic/mass spectrometric (LC/MS) method to evaluate C-13 or N-15 enrichment in amino acids is necessary to be established. Laboratory incubations of a Mollisol (sampled from 0-20 cm) were conducted with either (NH)-N-15 (4) (+) plus glucose or NH (4) (+) plus U-C-13-glucose as substrates. The substrates were added weekly until the soils were sampled after 1 and 4 weeks, respectively. The soil samples were then ground to < 0.25 mm and hydrolyzed to release amino acids. After being purified, the amino acids were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and then identified by LC/MS. The air-dried original soil was used as control and analyzed in the same assay. The amino acids were quantified based on the total current ion chromatograms. To fractionate and quantify the C and N isotopes in the amino acids, the intensities of the quasi-molecular peaks and the minor fragments were measured under both scan and selective ion monitoring modes. The intensities of the corresponding isotope fragments in amino acids increased significantly after labile substrate addition, indicating the N-15 and C-13 incorporation into soil amino acids, including both free and proteinaceous or peptide forms during the incubations. The synchronous utilization of glucose was dominant to structure amino acid skeleton and the conversion from NH (4) (+) to multi-nitrogen-containing amino acids exhibited the same pattern. The isotope enrichment in the amino acids was calculated according to the relative intensity increase of the isotope fragment and expressed as atom percentage excess (APE). The amount of isotope-labeled amino acids was furthermore differentiated from the native portion based on both APE and the concentration of individual compounds. The isotope-based LC/MS technique was useful to assess N-15 and C-13 enrichment in soil amino acids at relative high levels. The quantification of isotope incorporation makes it possible to evaluate the C or N turnover velocity of individual amino acids induced by the available substrates. Furthermore, the differentiation between the labeled and unlabeled amino acids was quite helpful to investigate the synthesis-decomposition dynamics of amino acid pools and explore the pathways of biological transformation of soil amino acids.