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13 protocols using xcalibur software version 4

1

Metabolomics Workflow for D. nervosa

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The Xcalibur software version 4, (Thermo Fisher Scientific, San Jose, CA, USA) was used to acquire the raw data including the full-scan MS and MS2 data, which were processed by the Compound Discover version 3 using the metabolomics workflow templates to detect the differential components between the microdialysates before and after application of D. nervosa. The detailed parameters of metabolomics workflow template were as follows: The minimum peak intensity was set as 10000; the maximum element counts were C30 H60 O20 S4 N10 Cl4; the mass tolerance of MS and MS2 was within 5 and 10 ppm, respectively; and differential analysis was selected for postprocessing.
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2

Identification of Chlorogenic Acid Derivatives in Inula cappa

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LC-MS data analysis was performed using Xcalibur software version 4 (Thermo Fisher Scientific, San Jose, California, USA). The raw data including the full-scan MS and MS2 data were processed by the Compound Discover 3.0 using the expected compounds predicted method [11 (link)] based on the metabolism workflow templates to detect the chlorogenic acid derivatives constituents of Inula cappa. Finally, candidates for CGA were characterized based on the diagnostic fragment ions, retention time, and bibliography.
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3

Polyamine Extraction and Quantitation

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For polyamine extraction, snap-frozen tissues were pulverized on dry ice using mortar and pestle and subjected to 5% trichloroacetic acid (TCA) extraction using 20–25 mg pulverized tissue or 20 µl whole blood and a final extract volume of 750 µl. Quantitative HPLC–MS/MS-based determination of spermidine, putrescine, ornithine and spermine was performed as described before by employing stable-isotope labeled internal standards26 (link).
Samples were incubated on ice (1 h) and centrifuged at 10,000 g at 4 °C (10 min). 150 µl supernatant was mixed with 800 µl double distilled water, 125 µl sodium carbonate buffer (1 M, pH 9) and 25 µl isobutyl chloroformate, then incubated at 35 °C (15 min). The solution was centrifuged at 10,000 g (1 min) and the resulting supernatant was measured in the Ultimate 3000 HPLC system (Thermo Fisher Scientific, USA) coupled to a triple-quadrupole mass spectrometer, a Quantum TSQ Ultra AM controlled by Xcalibur Software version 4.0 (both Thermo Fisher Scientific, USA).
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4

Peptide Identification by Nano-UPLC-MS/MS

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Each peptide sample was dissolved in formic acid (FA, 10%) and 5 µL was injected into a nano-ACQUITY UPLC system (Waters, Milford, MA, USA). Peptides were separated on a 1.7 mm BEH C18 column (Waters, Milford, MA, USA) at a flow rate of 300 nL/min. Peptide elution was achieved with a linear gradient (solution A: H2O (95%), CH3CN (5%), FA (0.1%); solution B: CH3CN (95%), H2O (5%), FA (0.1%)); 15–50% B over 180 min). MS and MS/MS data were acquired with an LTQ-Orbitrap XL (ThermoFisher, Waltham, MA, USA). The fifteen most intense doubly and triply charged peptide ions were chosen by the Xcalibur software version 4.0 (ThermoFisher, Waltham, MA, USA.) and fragmented. The resulting MS data were processed to generate peak lists for protein identifications.
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5

Cholesterol Quantification in Lysosomes

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The lysosomal samples were extracted from anti-HA magnetic beads with methanol and ethyl acetate with d7-cholesterol (2 μg per sample), and further derivatized with nicotinic acid to improve the mass spectrometric detection sensitivity of cholesterol. Measurement of cholesterol was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer. Data processing was conducted with Xcalibur™ Software version 4.0 (Thermo Fisher Scientific). A quality control (QC) sample was prepared by pooling the aliquots of the study samples and was used to monitor the instrument stability. The QC was injected six times in the beginning to stabilize the instrument and was injected every four study samples to monitor the instrument performance. The data was accepted if the coefficient variance (CV) of cholesterol in QC sample was < 15%. The data was reported as the peak area ratio of cholesterol to d7-cholesterol.
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6

Lipidomic Analysis by LC-MS/MS

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The lipids were analyzed by LC-MS/MS using a Thermo Vanquish HPLC (Thermo Fisher Scientific, Germering, Germany). MS was performed with heated ESI source in positive and negative mode, respectively. The spray voltage was set to 3.5 kV for positive and −2.8 kV for negative mode, and ion transfer capillary was 325°C. Nitrogen was used as both sheath gas and auxiliary gas and was set to 35 and 15 arbitrary units, respectively, and the auxiliary gas temperature was 250°C. For MS/MS, higher-energy collision dissociation (HCD) with nitrogen gas and step collision energy (NCE) of 20, 30, 50 for positive mode, and 20, 30, and 50 for negative mode were used to present a broader range of fragment ions and collected as much informative data as possible. MS data were acquired in the scan range of m/z 80–1200 and were processed using Xcalibur software version 4.0 (Thermo Scientific, San Jose, CA, United States).
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7

Nanoliquid Chromatography-MS/MS Peptide Analysis

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The resulting peptides were analysed by online nanoliquid chromatography coupled to an MS/MS instrument (Ultimate 3000 RSLCnano and Q-Exactive HF, Thermo Fisher Scientific) using a 360-min gradient for proteome-wide analysis and a 200-min gradient for interactome characterization. For this, peptides were sampled on a 300 μm × 5 mm PepMap C18 precolumn and separated in a 200 cm µPAC column (PharmaFluidics) or a 75 μm × 250 mm C18 column (Aurora Generation 2, 1.7 µm, IonOpticks) for, respectively, proteome-wide and interactome analyses. MS and MS/MS data were acquired using Xcalibur software version 4.0 (Thermo Scientific).
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8

UHPLC-Q Exactive MS Analysis Protocol

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The chromatographic system (Dionex UltiMate 3000 UHPLC system, Dionex Softron GmbH, Germering, Germany) consisted of a binary pump (HPG-3400RS), an autosampler (WPS-3000RS), a degasser (SRD-3400), and a column oven (TCC-3000RS). Detection was carried out on a quadrupole/orbital ion trap Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Analytes were separated on a reversed-phase Ascentis Express C18 column (2.1 mm × 100 mm, 2.7 µm) from Supelco (Bellefonte, PA, USA). The LC-MS system was equipped with a heated electrospray ionization source (HESI-II) and Xcalibur software, version 4.0 (Thermo Fisher Scientific, San Jose, CA, USA).
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9

Cholesterol Quantification in Lysosomes

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The lysosomal samples were extracted from anti-HA magnetic beads with methanol and ethyl acetate with d7-cholesterol (2 μg per sample), and further derivatized with nicotinic acid to improve the mass spectrometric detection sensitivity of cholesterol. Measurement of cholesterol was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer. Data processing was conducted with Xcalibur™ Software version 4.0 (Thermo Fisher Scientific). A quality control (QC) sample was prepared by pooling the aliquots of the study samples and was used to monitor the instrument stability. The QC was injected six times in the beginning to stabilize the instrument and was injected every four study samples to monitor the instrument performance. The data was accepted if the coefficient variance (CV) of cholesterol in QC sample was < 15%. The data was reported as the peak area ratio of cholesterol to d7-cholesterol.
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10

High-Resolution Shotgun Proteomics Analysis

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The dried peptides were dissolved in 5% ACN with 0.1% FA immediately before analysis and loaded onto a 150 mm x 75 µm PepMap RSLC capillary analytical C18 column with 2 μm particle size (LC Packings) using Ultimate 3000 HPLC system (Dionex, Idstein, Germany). Peptide separation was done at a flow rate of 0.300 µl/min, and the solvents gradient started at 3 min and was ramped to 60% Buffer B (90% ACN + 0.03% FA) over 60 min. Eluted peptides were analyzed using a Q Exactive HF mass spectrometer with a nanoelectrospray ionization source (ESI) (Thermo Fisher Scientific, Bremen, Germany). The mass spectrometer was externally calibrated and operated in positive and data-dependent modes. Survey MS scans were conducted in the 200–2000 m/z range, with a resolution of 120,000. The top ten most intense ions were fragmented with 30 eV collision energy on an HCD collision cell and analyzed with a resolution of 30,000. A 10 s dynamic exclusion window was applied, and an isolation window of 4 m/z was used to collect suitable tandem mass spectra. The obtained data were acquired with the Xcalibur software version 4.1 (Thermo Fisher Scientific, Bremen, Germany).
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