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

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).

Newly depolymerized large organic N contributes directly to amino acid uptake in young maize plants

“…After mixing and centrifugation, the aquatic phase was transferred through a Pasteur pipette lined with glass wool followed by 2 9 0.5 ml 0.1 M HCl into new test tubes and norleucine was added as internal standard. After freeze-drying and resuspension in 1 ml 0.01 M HCl, the sample was transferred to a polypropylene column containing 2 g Dowex 50w 9 8 cation exchange resin for separation of compounds with and without N. After eluting the amino acids with 2.5 M ammonium hydroxide solution, the sample was freeze-dried and derivatized according to Enggrob et al (2019).…”

“…After freeze‐drying and resuspension in 1 ml 0.01 M HCl, the sample was transferred to a polypropylene column containing 2 g Dowex 50w × 8 cation exchange resin for separation of compounds with and without N. After eluting the amino acids with 2.5 M ammonium hydroxide solution, the sample was freeze‐dried and derivatized according to Enggrob et al . ().…”

“…The > 100 kDa organic N was characterized for total C and N, bulk isotopic and amino acid specific composition, as described in Enggrob et al . () (Table ; Supporting Information Fig. S1).…”

“…Triple-labelled > 100 kDa organic N The protein-sized organic N (> 100 kDa) was produced from glasshouse-grown triple-labelled ( 14 C, 13 C, 15 N) white clover shoots (Enggrob et al, 2019). Briefly, white clover grown in sterile sand received a standard nutrient solution supplemented with 15 N-labelled ( 15 NH 4 ) 2 SO 4 (98 at%) and was C-labelled with 14 CO 2 and 13 CO 2 via labelled bicarbonate (for 13 C 99 at%) as described for 14 CO 2 by Rasmussen et al (2008).…”

“…The residue remaining on the filter after rinsing three times with 5 ml MilliQ water was used for the experiment with the resultant > 100 kDa organic N in the size > 100 kDa and < 0.45 lm (hereafter referred as '> 100 kDa organic N'). The > 100 kDa organic N was characterized for total C and N, bulk isotopic and amino acid specific composition, as described in Enggrob et al (2019) (Table 2; Supporting Information Fig. S1).…”