Sciex 5600 mass spectrometer

For LC-MS/MS profiling two biological replicates were used based on the high level of reproducibility between replicates. In previous studies most of the proteins had a reproducible peak between biological replicates (13 (link), 21 (link), 32 (link)). Ion exchange chromatography provided a high-resolution separation and 65 fractions were analyzed by mass spectrometry (analyzed on Sciex 5600 mass spectrometer). For the SEC and IEX profiling experiments that were analyzed to predict protein complex composition were analyzed on Sciex 5600 mass spectrometer. The SEC fractions that were analyzed to test for oligomerization changes in predicted AIMP1L-interactors by profiling the aimpl1 mutant were analyzed on Q Exactive mass spectrometer. For CoIP-MS pull downs three replicates were performed with antibodies against the protein of interest and negative controls and were analyzed on Q Exactive mass spectrometer.

Chemdraw 2020 software was used for compound structure drawing. The samples were separated with Waters BEH C18 column (2.5 μm, 2.1 mm × 150 mm) using Thermo Scientific (U3000) system consisting of SCIEX 5600 + mass spectrometer. The eluents were (A) 0.1% HCOOH-H2O and (B) acetonitrile. The gradient elution of the mobile phase was 95% (A) in 0–2 min, 95–5% (A) in 2–10 min, 5% (A) in 10–13 min, and 5–95% in 13–15 min at a flow rate of 0.3 mL/min. For UPLC‒MS analysis, the UPLC conditions were the same as above. Ultra-high and pure helium (He, 99.999%) was used as the collision gas, and high purity nitrogen (N2, 99.999%) was used as the nebulizing gas. Positive and negative ion modes ESI–MS and MS/MS were used for the detection. TOF MS parameters: source temperature: 500 °C (positive ion) and 450 °C (negative ion), scan range, m/z = 50–1200, scan accumulation time 0.2 s, product ion scan accumulation time 0.01 s; MS/MS parameters: high sensitivity mode with information-dependent acquisition (IDA), declustering potential (DP): ± 60 V, collision energy: 35 ± 15 eV.

This experiment used a SHIMADZU ultra-high performance liquid chromatograph (LC-30) (Tokyo, Japan) linked with a SCIEX5600 mass spectrometer (Tokyo, Japan) (Hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer), respectively, using electrospray ion source positive and negative ion mode. The SHIMADZU InerSustain C18 chromatographic column (100 mm × 2.1 mm, 2 µm) (Tokyo, Japan) was used to separate and detect the flavonoids in the purified and crude extracts of Acanthopanax senticosus. The working temperature was 30 °C, and the flow rate was 1 mL·min⁻¹. Solvent A was acetonitrile, and solvent B was 0.1% HCOOH-H₂O. The elution was completed using the following gradient: 0–2 min 95% B, 2–4 min 95–80% B, 4–12 min 80–75% B, 12–14 min 75–54% B, 14–26 min 54–0% B, 26–28 min 0% B, 28–29 min 0–95% B, 29–30 min 95% B.