ADAMTSL5 11mer peptide (20 nM) was incubated with purified recombinant ERAP1 Hap2 or Hap10 (2μg) at 37°C in 50 mM Tris-HCl (pH 8.0) for up to 2 h. Reactions were stopped by the addition of 0.5% trifluoroacetic acid. Each sample was analyzed for peptide digestion by RP-HPLC (Shimadzu) on a 2.1 mm × 250 mm C18 column (Vydac) over a gradient of 18-34% acetonitrile and a flow rate of 0.25 ml/min. Peptide peaks were analyzed using LC Solutions software (Shimadzu). Synthetic peptides (20 nM) and buffer only samples were run and analyzed in identical conditions to establish the ADAMTSL5 peptide series retention times and the absence of cross-contamination (Fig. 5A ).
Lc solutions software
Manufactured by Shimadzu
Sourced in Japan
LC Solutions software is a comprehensive data acquisition and analysis solution designed for Shimadzu's liquid chromatography (LC) instruments. The software provides a user-friendly interface for instrument control, data processing, and reporting functions.
Automatically generated - may contain errors
Lab products found in correlation
Dgu 20a3 degasser, by Shimadzu (3 mentions)
Lcms 8050 triple quadrupole mass spectrometer, by Shimadzu (2 mentions)
Nexera x2 ultra hplc, by Shimadzu (2 mentions)
Centrifuge 5804, by Eppendorf (2 mentions)
Hplc vial, by Agilent Technologies (2 mentions)
0.22 μm syringe filter unit, by Merck Group (2 mentions)
Rezex roa, by Phenomenex (2 mentions)
Gel filtration molecular weight standard, by Bio-Rad (2 mentions)
Corresponding guard column, by Tosoh (2 mentions)
Lc20 ad isocratic pump system, by Shimadzu (2 mentions)
Sil 20a autosampler, by Shimadzu (2 mentions)
Spd 20a uv detector, by Shimadzu (2 mentions)
Zic hilic, by Merck Group (2 mentions)
Lc 20ad pump, by Shimadzu (2 mentions)
Rp hplc, by Shimadzu (1 mentions)
Lc 2010cht hplc system, by Shimadzu (1 mentions)
Luna c18, by Phenomenex (1 mentions)
High performance liquid chromatography system, by Shimadzu (1 mentions)
Tskgel bioassist g3swxl column, by Tosoh (1 mentions)
Prominence ultra fast liquid chromatography hplc system, by Shimadzu (1 mentions)
20 protocols using lc solutions software
1
ADAMTSL5 Peptide Digestion Assay
2
Quantitative Analysis of Vitamins
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The multi-vitamins stock solution was prepared by weighing in a 100 mL volumetric flask 5 mg of vitamin B12; 12.5 mg of vitamin B2; 25 mg each of vitamins B1, B6 and B3. Forty millilitres (40 mL) of water was then added and the solution was shaken vigorously before adding 4 mL of 2 M NaOH. After complete dissolution of the vitamins, 50 mL of 1 M phosphate buffer (pH 5.5) was added and the solution made up to the mark with water. Stock standard solutions were prepared daily. Different concentrations of the standards were injected into the HPLC to obtain the peak areas. Peak areas were plotted against concentration for each vitamin to make specific calibration curves. The Shimadzu LC-2010CHT HPLC system was used with conditions according to Moreno et al. [25 (link)]. A volume of 20 µL for each sample was injected into the HPLC equipped with a C18 reversed-phase column (250 × 4.6 mm, 4 μm), 0.05 M ammonium acetate (solvent A)–methanol (solvent B) 92.5:7.2 as mobile phase at 1 mL/minute flow rate. A diode array detector was used to scan from 200 to 500 nm and LC-solutions software (Shimadzu, Japan) was used to integrate the peak areas for each vitamin. After the run, the peak area of each unknown sample was obtained, and concentrations were calculated using the calibration curves.
3
HPLC Analysis of Metabolites
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The chemical composition of samples was assessed on a Shimadzu HPLC (Shimadzu Scientific Instruments) equipped with an RID-10A refractive index detector. 30 μL injections were run on an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) at 50∘C with 0.01 N H 2SO4 mobile phase and a flow rate of 0.6 ml/minute. External standards were run approximately daily containing acetate, butyrate, formate, glucose, lactate, propionate, and succinate at 8 concentrations between 0.1 mM to 20 mM. Due to the complexity of the chromatogram, the identity and area of retained peaks was curated manually, assisted by the LC Solutions Software (Shimadzu Scientific Instruments) Standard curves were fit using weighted regression (inverse square of the concentration), and, for all compounds except propionate, without an intercept.
4
Quantification of Plasma Melatonin Levels
5
HPLC Quantification of Organic Acids
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The organic acid quantification was based on Rawi et al. (2021) (link). The fermentation sample from each sampling period was pipetted into a 2 mL microcentrifuge tube for centrifugation (Centrifuge-5804, Eppendorf) at 13000 rpm for 10 min to obtain a clear supernatant. The supernatant was filtered through a 0.22 μm syringe filter unit (Millipore) into an HPLC vial (Agilent Technologies, Cheshire, United Kingdom). Prominence Series Liquid Chromatography (Shimadzu Corp., Japan) using a reverse phase ion-exclusion C12 column (Rezex ROA, Phenomenex) was used for SCFA analysis. Analytes were detected using a UV detector at a wavelength of 210 nm. The isocratic mobile phase used was 0.25 mM sulphuric acid (H2SO4). A 15 μL sample was injected into the heated column (40°C) programmed to run in isocratic elution at a flow rate of 0.5 mL/min for 40 min. The peaks and response factors within the sample were calibrated and calculated using the LC Solutions software (Shimadzu). The standard solution contained of 12.5, 25, 50, 75, 100, 125, 150, 175, and 200 mM acetate, butyrate, propionate, and lactate.
6
IgG1 Fc Glycoform Characterization
7
SEC Analysis of Monoclonal Antibody
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SEC was performed as described,30 using a Shimadzu Prominence ultra-fast liquid chromatography HPLC system. 10 µL of mAb (10 µg total protein) was injected and separated by a TSKgel G4000SWXL column (8 µm particle size, 7.8 mm ID × 30 cm) with the corresponding guard column operated at ambient temperature (Tosoh Biosciences) using a 30-minute run time. Gel filtration molecular weight standards (Bio-Rad, Hercules, CA) were injected as controls. Data were analyzed using LC-Solutions software (Shimadzu, Kyoto, Japan).
8
Size-exclusion Chromatography with MALS
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Experiments were conducted on a system comprising a Wyatt HELEOS-II multiangle light scattering detector and a Wyatt rEX refractive index detector linked to a Shimadzu liquid chromatography (LC) system (SPD-20A UV detector, LC20-AD isocratic pump system, DGU-20A3 degasser, and SIL-20A autosampler). Experiments were conducted at room temperature (20 ± 2 °C). Solvents were filtered through a 0.2 µm filter prior to use, and a 0.1 µm filter was present in the flow path. The column was equilibrated with >2 CV of buffer (50 mM NaPi, 300 mM NaCl pH 7.4) before use and buffer was infused at the working flow rate until baselines for UV, light scattering, and refractive index detectors were all stable. The sample injection volume was 100 µL of protein at 6 mg mL−1 in 50 mM NaPi buffer, 300 mM NaCl pH 7.4. Shimadzu LC Solutions software was used to control the LC and Astra V software for the HELEOS-II and rEX detectors. The Astra data collection was 1 min shorter than the LC solutions run to maintain synchronization. Blank buffer injections were used as appropriate to check for carryover between sample runs. Data were analyzed using the Astra V software. Molecular weights were estimated using the Zimm fit method with degree 1. A value of 0.158 was used for protein refractive index increment (dn/dc).
9
Quantification of Treosulfan and EBDM
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Treosulfan and S, S-EBDM levels in plasma were measured using a Shimadzu-Nexera X2 ultra HPLC coupled with an LCMS-8050 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). The parameters were adjusted to yield maximum multiple reaction monitoring signals (Table S1 ). Chromatographic separation of the analytes was done using Zorbax Eclipse Plus C18 (100 mm × 2.1 mm, 3.5 μm; Agilent, CA) protected with a C18 guard column from the same source using isocratic elution with a mobile phase 0.01 M ammonium formate buffer at a flow rate of 0.4 mL/minute maintained at 40°C. The total run time was 5.5 minutes (Figure S1 ). The chromatograms were analyzed using LC Solutions software (Shimadzu, Kyoto, Japan).
10
Quantification of Globin Chains in iEPCs
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Globin chain analysis was performed using a previously reported method [44 (link)], with minor modifications. Briefly, 3 million differentiated iEPCs were resuspended in 100 µL of distilled water and frozen and thawed thrice in −80 °C. The cell lysate was centrifuged at 14,000× g for 10 min at 4 °C, and the supernatant was transferred to vials for injection into the HPLC system. Globin chains were quantified using a Shimadzu UFLC consisting of binary gradient pumps, an autosampler, and a column oven coupled with UV detection (all equipment from Shimadzu, Kyoto, Japan), and the data were analyzed using LC Solutions software (Shimadzu, Kyoto, Japan). Chromatographic separation of the analytes was done using an Aeris Widepore 3.6 lm XB-C18 25 cm, 4.6 mm column behind a Security Guard UHPLC Widepore C18 4.6 mm guard column (Phenomenex, Torrance, CA, USA). The gradient method was used for elution with mobile phases (Solvent A-0.1% trifluoroacetic acid (TFA, Sigma-Aldrich, St. Louis, MO, USA), pH 3.0, and 40% Solvent B-0.1% TFA in acetonitrile (Sigma-Aldrich, St. Louis, MO, USA)) at a flow rate of 1.0 mL/min and column temperature maintained at 70 °C. The total run time was eight minutes. The UV detection was set at 190 nm for globin chain detection.
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