Accq tag method

Identity and purity of the final product were confirmed by ESI–MS (CEINGE, Naples, Italy -Figure S4) and amino acid analysis (CiCT, Barcelona, Spain, Table S2). The amino acid analysis was carried out by dissolving the polypeptide in water (1% w/v phenol). An aliquot of α-aminobutyric acid (AABA) solution (2.5 mM) and concentrated HCl (37%) were added to obtain a final concentration of 6 M HCl. The final solution was transferred to hydrolysis tubes. Hydrolysis was performed at 110 °C for 16 h. Then, the samples were evaporated by using a rotary evaporator, resuspended in 20 mM of HCl, and filtered. Aliquots of the filtered solutions were derivatized with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate, according to the Waters AccQ-Tag method. Final analytical determination was carried out. AccQ-Tag derivatized amino acids were analyzed by HPLC with UV detection (λ = 254 nm) on a WATERS 600 HPLC gradient system equipped with a WATERS 2487 UV detector (WATERS, Milford, MA, USA.

Biomass samples were first hydrolysed at 105°C for 24 h with 6 M HCl in vacuum sealed glass ampoules. After hydrolysis, samples were evaporated under vacuum, re-dissolved in 20 mM HCl, and filtered. Amino acid content of an aliquot of the filtrate is then determined using the AccQ Tag method (Waters Corp., Milford MA, USA). Derivatisation was carried out using the AccQ Fluor reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) according to the method specifications. Once derivatised, amino acids were separated and analysed using a Waters Nova-Pak C18 (4 μm, 3.9 × 150 mm) in a HPLC gradient system (Waters 600) provided with an UV detector (Waters 2487). Detection was performed at 254 nm and α-amino-N-butyric acid (AABA) was used as internal standard.

Protein sources, basal diet and fecal samples were dried until constant weight in an air-forced oven at 65°C, 1-mm milled, and analyzed in duplicate, according to official methods (26 ), as previously described (27 (link)). Samples were analyzed for dry matter (DM; ID 934.01), ash (ID 942.05), ether extract (EE; ID 920.39), and Kjeldahl N (ID 990.03; in fresh feces samples). Crude protein (CP) was calculated as Kjeldahl N × 6.25. Gross energy (GE) analysis was performed with an adiabatic bomb calorimeter (Werke C2000, IKA, Staufen, Germany). The basal diet was also analyzed for neutral detergent fiber (with α-amylase, without sodium sulfite, and expressed exclusive of residual ash, NDF) (28 (link)), and for starch (in 0.5-mm milled samples) (29 ) contents.
Amino acids were determined as described by Aragão et al. (30 (link)). Briefly, samples were hydrolyzed with 6 M HCl solution at 116°C for 48 h. Precolumn derivatization was performed according to the AccQ Tag method (Waters, Milford, MA, United States) using the Waters AccQ Fluor Reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) and the analyses carried out by ultra-high-performance liquid chromatography on a Waters reversed-phase amino acid analysis system with norvaline as the internal standard. Peaks were then analyzed with EMPOWER software (Waters). The analyses were carried out in duplicate.

Each sample was analyzed using three separate LC-MS/MS methods using two different LC systems (Thermo Scientific ICS-5000+ ion chromatography and Thermo Ultimate 3000). Each was coupled directly to a Q-Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer with a HESI II electrospray ionization source (Thermo Scientific, San Jose, CA). Full details for each method are provided in the “Supplementary information” section (Supplementary Methods). Briefly, for method 1, anion-exchange chromatography coupled with mass spectrometry (IC-MS/MS) was performed as recently published [31 (link)]. Reversed phase C18 column analysis was performed for both methods 2 and 3. For method 2, the samples were used underivatized (same as in method 1) [31 (link)], while for method 3, samples were derivatized prior to analysis using a modified version of the Waters AccQ-Tag method designed for amino-acid analysis [35 (link)].

The extracts were derivatized with the Waters AccQ•Tag method, in accordance with the manufacturer’s protocol. The analysis was performed using a 1290 Infinitely UHPLC system from Agilent Technologies (Waldbronn, Germany) with G4220A binary pump, G1316C thermostated column compartment, and G4226A autosampler with G1330B autosampler thermostat coupled to an Agilent 6490 triple quadrupole mass spectrometer equipped with a jet stream electrospray source operating in the positive ion mode^{63 (link)}. The amino acid multiple-reaction-monitoring (MRM) transitions were optimized using MassHunter MS Optimizer software (Agilent Technologies Inc., Santa Clara, CA, USA), and the data were quantified using MassHunter Quantitation software B07.01 (Agilent Technologies); the amount of each amino acid was calculated on the basis of the calibration curves.

Juveniles fasted for 14 h before sampling. Samples of diets and juveniles were stored at −30 °C at the beginning and end of the experiment. Due to the small size of juveniles, proximate composition and fatty acid profile were made on whole body. Analyses were performed in duplicate by Analiza Calidad laboratory (Burgos, Spain) following Commission Regulation (EC) 152/2009. Moisture was determined by drying at 105 °C, crude protein according to the Kjeldahl method, crude lipid by extraction with light petroleum and further distillation, ash by calcination at 550 °C, and gross energy according to EU regulation 1169/2011. The content of carbohydrates was calculated by subtracting moisture, protein, lipid, and ash content from the wet weight.
Fatty acid profiles were determined by hydrophilic interaction chromatography (HPLC) using the AccQTag method from Waters (Milford, MA, USA).
From whole-body fatty acid content (g kg⁻¹), the following indices were calculated:
ΣPUFA/ΣSFA = sum of polyunsaturated fatty acids × sum of saturated fatty acids⁻¹EPA + DHA = eicosapentaenoic acid + docosahexaenoic acid
Σn − 6/Σn − 3 = sum of omega 6 fatty acids × sum of omega 3 fatty acids⁻¹

Nodule metabolites (amino acids, organic acids, soluble sugars) were extracted by the alcoholic extraction method and resuspended in water as described in Brouquisse et al. (1991) (link). Sucrose was determined enzymatically (Velterop and Vos, 2001 (link)) at 340 nm using a microplate spectrophotometer. Succinate and malate were determined by anion exchange HPLC (Dionex) with conductivity detection (Moing et al., 1998 (link)). Free amino-acids were analyzed by HPLC using the AccQ.Tag method from Waters (Milford, MA) with fluorescence detection (Moing et al., 1998 (link)). Three independent biological replicates have been performed with three technical replicates per biological assay.