Acclaim 120

High-performance liquid chromatography (HPLC) was used for the SNS quality control by an Agilent Technologies 1260 Infinity system with a 1260 DAD VL detector. A C18 column (Thermo Acclaim120, 4.6 mm × 250 mm, 5 mm) was used for the analysis, and the column temperature was maintained at 30 °C. The mobile phase was composed of water containing 0.1% phosphoric acid (A) and acetonitrile (B). The linear gradient elution program for the mobile phase was as follows: 0–35 min, 5–-26% B; 35–80 min, 2650% B; 80–90 min, 50–65% B; 90–105 min, 65–85% B; 105–110 min, 85–5% B. At 210 nm, monitoring was carried out. The injection volume was 10 μL.

Samples analysis was performed using a system comprised of a UHPLC Vanquish pump, Vanquish Autosampler, Vanquish UHPLC + column compartment, and ISQ EC mass spectrometer (ThermoFisherSci.), in conjunction with ThermoFisherScientificDionexChromeleon 7 Chromatography Data System software. A C18 column (4.6 mm ID X 100 mm L, 5 µm, 120 Å; Acclaim 120; ThermoFisherSci.) was used with a 0.25 mL/min. flow rate and 0.5 µL injection volume; the autosampler needle was washed with 10% (v/v) methanol (10 s) after each sample injection. Mobile phases A and B consisted of 10 mM ammonium acetate and LC-MS grade acetonitrile, respectively. The linear gradient mobile phase flow was characterized by an increase in acetonitrile from 50% to 80% between 0 to 4 min, followed by a decrease to 50% between 4 to 8 or 15 min. Preliminary results guided selection of the following mass spectrometric parameters: vaporizer temperature (VT) = 250 °C; ion transfer tube temperature (ITT) = 200 °C; optimum sheath gas pressure (SGP) = 25 psig; auxiliary gas pressure (AGP) = 2 psig; sweep gas pressure (SWGP) = 0.5 psig; and negative mode, which resulted in an extracted Gln-FMOC chromatogram at m/z 367.1. Column and autosampler temperatures were maintained at 35 °C and 15 °C, respectively. This method represented here as Gen-MS-I.

The analysis was conducted in two parts. First, baseline samples were randomized into batches, with each batch containing 20 experimental samples with roughly equal numbers from each group and a set of quality control (QC) samples. These QC samples were duplicates of a commercial, standard human serum sample (Sigma), a duplicate of one of the samples in the batch, and a replicate from a previously run duplicate sample. Subsequently, paired samples where a 28d sample was available were analysed, including a repeat of the baseline sample along with its 28d paired sample. Randomisation and batch structure were the same across both studies.
All samples were analysed by Liquid Chromatography-Selected Reaction Monitoring-Mass Spectrometry (LC-SRM-MS) using the method described in (24 (link)). Using a 6495 triple-quadrupole mass spectrometer with an electrospray ion source (Agilent), connected to an Agilent Infinity 1200 Series liquid chromatography system, 4 μL of each sample was injected directly onto a C18 column (250 mm × 2.1 mm I.D., Thermo Scientific Acclaim 120, 3 μm particle size) held at 50°C. Peptides were eluted at a 250 μL/min flow rate using the previously described gradient (24 (link)). Six acquisition windows were used for the eluting peptides, where one window was acquired for both FHL-1 and FHR-2.

TNB (1,3,5-tris(1-naphthyl)benzene, C₃₆H₂₄, CAS No. 7059-70-3, Hotspot Biotechnology, Weifang, China) was purchased and purified by sublimation under reduced pressure. The final purity of 0.997 (mole fraction) was determined by HPLC using a Dionex Ultimate 3000 chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a UV detector (254 nm) and a Dionex Acclaim 120 chromatographic column (C18-bonded silica, 5 μm, 120 Å, 4.6 mm × 250 mm), and 100% acetonitrile was used as an eluent. The benzene used for solution calorimetry (C₆H₆, CAS No. 71-43-2) was purified according to [31 ]. Its final purity determined by gas chromatography (Agilent 7890 B, Santa Clara, CA, USA) exceeded 0.999 (mass fraction). The characterization of the samples and auxiliary compounds used for the calibration of the instruments is provided in Table S1.

LC separations
were performed
using Dionex Ultimate 3000 series UHPLC (Thermo Fisher Scientific)
coupled to an Orbitrap Q-Exactive Plus Mass Spectrometer (Thermo Fisher
Scientific). The used column was Acclaim 120 (C18, 5 μm, 120
Å, 4.6 mm × 100 mm) (Thermo Fisher Scientific). The mobile
phases were A, H₂O, and B, AcN, both with 0.1% formic acid.
The flow rate used in all LC–MS experiments was 1 mL·min^–1, and sample elution was performed by using the gradient
from 5 to 95% of B over 6.5 min. 10 μL of sample injection was
set. Ionization was performed using an electrospray ion source operating
in positive ion mode with a capillary voltage of 3.80 kV and a capillary
temperature of 400 °C. The sheath gas, auxiliary gas, and sweep
gas flow rates were set at 75, 20, and 1 (arbitrary units), respectively.
The auxiliary gas temperature was set at 500 °C. For each studied
mass, a set of MS/MS spectra were acquired using an isolation width
of 1.0 and 5.0 m/z, and a screening of collision
energies (from 10 to 40 HCD) was carried out. All MSⁿ data
were analyzed using the Qual Browser embedded in the Thermo Fisher
Scientific Xcalibur program and the FreeStyle software.