Amino acid standard solution

Table S1 lists the twenty-five cultivars of highland barley that were used in this study: four two-rowed yellow highland barley (YT1-4), nine six-rowed yellow highland barley (SY1-9), and six-rowed purple hulless barley (PS1-12). They were planted at Lhasa, Tibet (altitude: 3658 m, oxygen content: 15.09%, mean temperature: 18.60 °C) in 2017. Whole grains were ground using a whirlwind mill (CT293, FOSS, Hillerod, Denmark) equipped with a 0.5 mm screen. Grains and grain flour were stored in double-layer self-sealing bags at 4 °C.
γ-Aminobutyric acid (GABA), putrescine (Put), spermidine (Spd), spermine (Spd), amino acid standard solution, sodium acetate, β-mercaptoethanol polyvinyl pyrrolidone (PVP), and pyridoxal 5-phosphate (PLP) were from Sigma Aldrich (Saint Louis, MO, USA). The HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Waltham, MA, USA). All other chemicals and reagent used in the experiments were analytical grade.

The amino acid profile was determined according to the method described by Moore and Stein [22 (link),23 ,24 ]. The samples were hydrolysed in liquid 6 mol/L HCl containing 0.5% phenol at 110 °C for 24 h under an argon atmosphere. After lyophilization, the obtained hydrolysates were dissolved in sodium citrate buffer (pH 2.2) and filtered through a 0.45 μm syringe filter. An amino acid analyser with a strong cation ion exchanger was used. Amino acids were eluted with a sodium-citrate buffers gradient and spectrophotometrically detected at 570 and 440 nm after post-column derivatization with ninhydrin, according to the standard manufacturer’s protocol (Amino acid analyser AAA400. User manual. Ingos s.r.o. Praha 2007). Sulphur-containing amino acids were analysed as oxidation products (after performing acid oxidation followed by the standard hydrolysis procedure with HCl); therefore, asparagine and glutamine were determined as aspartic and glutamic acids. Tryptophan was not determined, as it is destroyed during acid hydrolysis. For calibration of the amino acid analyser, the amino acid standard solution was used (Sigma, St. Louis, MO, USA). Data processing was performed using the software of the chromatographic device (Chromulan, Pikron, Czech Republic).

The amino acids profile of crayfish abdominal samples were estimated by ion exchange chromatography using an automatic amino acid analyzer Biochrom 30+ (Biochrom, Cambridge, UK), according to Spackman et al. [42 (link)]. The Biochrom 30+ analyzer provides accurate quantitative analysis of amino acid mixtures and the technique was based on amino acid separation using strong cation exchange chromatography, followed by the ninhydrin colour reaction and photometric detection at 570 nm, except for proline, which was detected at 440 nm. Samples were previously hydrolysed in 6M HCl (Merck, Germany) at 110 °C for 24 h. After hydrolysis, the samples were cooled to room temperature (20 °C) and dissolved in 25 mL of loading buffer (pH 2.2) (Biochrom, Cambridge, UK). Samples were then filtered through a 0.22 μm pore size PTFE filter and the filter residue was transferred into a vial (Agilent Technologies, Santa Clara, CA, USA). The filtrate was then stored in a refrigerator and analyzed. The amino acid peaks were identified by comparison of retention times with the retention times of standard amino acids purchased from Sigma Aldrich (Amino Acid Standard Solution (Sigma-Aldrich, St. Louis, MI, USA)). The results were expressed as mass of amino acid (g) in 100 g of sample.