Ultra-pure water (Milli-Q®) was produced in the laboratory using an Elga Purelab Classic UV purification system from Veolia Water STI (Décines-Charpieu, France). Methanol (MeOH, LC-MS grade), acetonitrile (ACN, LC-MS grade), formic acid (FA, LC-MS grade), and ammonium acetate (AA, analytical reagent grade) were purchased from Fisher Scientific (Illkirch, France). Seven reference standards of peptides, including leucine encephalin, bombesin, [arg8]-Vasopressin, [ile]-angiotensin, bradykinin fragment 1–5, substance P, and bradykinin, were purchased from Merck (Molsheim, France). A detailed list of their physical properties can be found in Table S1 . DL-1,4-dithiothreitol (DTT, 99%) and iodoacetamide (98%), used in the enzymatic digestion of the six model proteins as reducing and alkylated agents, respectively, were purchased from Acros Organics (Geel, Belgium). Trypsin, human serum albumin (HSA), bovine serum albumin (BSA), β-casein, myoglobin, lysozyme, and cytochrome C were all purchased from Sigma-Aldrich (Steinheim, Germany). The sixty-seven reference standards of pharmaceuticals mentioned in this study were purchased from Sigma-Aldrich. A detailed list can be found in Table S2 .
Leucine encephalin
Manufactured by Merck Group
Sourced in Germany, United States
Leucine encephalin is a synthetic peptide that functions as a neurotransmitter and plays a role in pain management. It is commonly used in laboratory research and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Merck Group (3 mentions)
Formic acid, by Tianjin Kermel (3 mentions)
Acetonitrile, by Merck Group (2 mentions)
Iodoacetamide, by Thermo Fisher Scientific (1 mentions)
Myoglobin, by Merck Group (1 mentions)
Substance p, by Merck Group (1 mentions)
Bradykinin, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Cytochrome c, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Hydrobromic acid, by Thermo Fisher Scientific (1 mentions)
Ammonium dihydrogen phosphate, by Thermo Fisher Scientific (1 mentions)
Hplc supra gradient acetonitrile, by Biosolve (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Masslynx v4, by Waters Corporation (1 mentions)
Xevo g2 xs, by Waters Corporation (1 mentions)
Milli q gradient a10 system, by Siemens (1 mentions)
Zdf rats, by Charles River Laboratories (1 mentions)
Methanol, by Merck Group (1 mentions)
9 protocols using leucine encephalin
1
Comprehensive Analytical Workflow for Bioanalysis
2
Synthesis and Characterization of FDOPA
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For preparation of FDOPA the 6-trimethylstannyl-L-DOPA precursor was purchased from ABX (Germany). Dry chloroform >99 % for spectroscopy stabilized with amylene (stored on molecular sieves) and ammonium dihydrogen phosphate p.a. were obtained from Acros (Belgium) and 47 % hydrobromic acid, 25 % ammonia and diammonium hydrogenphosphate p.a. from Merck (Germany). A sterile solution for injection of ascorbic acid (100 mg/ml) was purchased from Centrapharm (Netherlands). A sterile 0.1 M sodium acetate solution, pH 4.7, was prepared in the hospital pharmacy. Reference compounds for calibration curves of FDOPA, L-DOPA and 6-hydroxy-DL-DOPA were purchased from respectively ABX (Germany) and Sigma Aldrich (Netherlands), HPLC supra gradient acetonitrile, used as solvent for LC-MS analysis, was obtained from Biosolve (Netherlands), ~98 % formic acid was bought from Fluka (Netherlands) and LC-MS grade water (>18.2 MΩ) was purchased from the Department of Clinical pharmacy & Pharmacology, UMCG. The reference compound leucine encephalin for calibration of the MS detector was purchased from Merck (Germany). For calibration of the MS, a solution was prepared of 200 μL 10 % formic acid, 100 μL 0.1 M sodium hydroxide (Merck, Germany) and 20 ml 80 % acetonitrile in water.
3
UPLC-MS/MS Analysis of Tryptic Peptides
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The trypsin digests were separated on an Acquity BEH C18 UPLC column (75 µm × 150 cm × 1.7 µm; Waters, Cheshire, UK) connected to a UPLC system for 90 min using LC-MS grade water in 0.1% formic acid (v/v; mobile phase A) and acetonitrile in 0.1% formic acid (mobile phase B). The separated peptides were analyzed for MS and MS/MS fragmentation on a Xevo-G2-XS (Waters, Milford, MA, USA), using an ESI source in positive mode. The scan time was set to 0.5 s in continuum mode, and the mass range was set to 50–2000 Da in TOF, with Leucine encephalin (200 pg/μL; Sigma-Aldrich, St. Louis, MO, USA) as an external calibrant. Mass spectra of the samples were acquired by MassLynx v4.0 software (Waters, Milford, MA, USA) and submitted for the identification and expression analysis of proteins.
4
Analytical-Grade Metabolite Extraction
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All reagents used from the pre-analytical to data acquisition stages were purchased from international suppliers in pure or analytical grade quality. Organic solvents used in metabolite extraction and data acquisition (acetonitrile and methanol) were of LC-MS grade, from Romil, SPS, (Cambridge, UK). Leucine encephalin and formic acid were acquired from Sigma Aldrich (Munich, Germany), while the water was purified using an in-house Milli-Q Gradient A10 system (Siemens, Fahrenburg, Germany).
5
ZDF Rat Diabetes Research Protocol
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The ZDF rats were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Metformin was purchased from Sino-American Shanghai Squibb Co., Ltd. (NO. 1305089, Shanghai, China). Glimepiride was purchased from Sanofi Pharmaceutical Co., Ltd. (NO. 3JB070, Beijing, China). Rat insulin ELISA Kits 10-1250-01 were obtained from Mercodia AB Co., Ltd. (NO. 21759, Uppsala, Sweden). Quo-Test A1C HbA1c Reagent Kits were purchased from Quotient Diagnostics Ltd. (NO. 020166, Surrey, UK). Kits used to detect triglyceride and total cholesterol were obtained from German Desai Diagnosis System Co., Ltd. (Frankfurt, Germany). Acetonitrile and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany). Distilled water was purchased from Watson’s Food & Beverage Co., Ltd. (Guangzhou, China). Leucine encephalin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid was purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). All other reagents and chemicals were of analytical grade.
6
Chemicals and Materials for Bioassays
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The following chemicals and materials were obtained from the indicated suppliers: Acetonitrile (Merck, Darmstadt, Germany); formic acid (Merck, Darmstadt, Germany); Leucine encephalin (Sigma-Aldrich, St. Louis, MO, USA); Lipopolysaccharides (LPS; Sigma-Aldrich); Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA); fetal bovine serum (FBS; Wisent, Saint-Jean-Baptiste, QC, Canada); trypsin (Biosharp, Hefei, China); Dimethyl sulfoxide (DMSO; Sigma-Aldrich); and Amentoflavone (Shunbo, Shanghai, China). Assay kits for malondialdehyde (MDA), superoxide dismutase (SOD) and NO were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
7
Quantitative Analysis of Metabolites
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Ultrapure water (18.2 MΩ) was prepared using a Milli-Q water purification system (Millipore, Billerica, MA, United States). Acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany). Formic acid (LC/MS grade) was purchased from Fisher Scientific (Spain). Leucine encephalin was supplied by Sigma-Aldrich (St. Louis, MO, United States). Cyclic AMP, cyclic GMP, retinoic acid, L -arginine, L -isoleucine, L -methionine, stearic acid, L -phenylalanine, L -valine, L -histidine, stearic acid, and uridine were purchased from Sigma-Aldrich (St. Louis, MO, United States). LysoPC (18:0) and LysoPC (17:0) were purchased from Avanti Lipids Polar, Inc. (Alabaster, AL, United States). Other chemicals were all analytical grade. FastQuant RT (With gDNase) was obtained from TianGen Biotech (Beijing, China). SYBR TransStart Green qPCR SuperMix was purchased from TransGen Biotech (Beijing, China). The positive control drug, Ginkgo Biloba extract 761 (EGb 761) was purchased from Dr. Willmar Schwabe GmbH & Co. KG.
8
Rapid Cancer Tissue Profiling via REIMS
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Tissues
were cauterized ex vivo using a monopolar handpiece
(iKnife disposable device, Waters Research Center (WRC), Budapest,
Hungary) equipped with a 1.7 mm diameter needle electrode, connected
to an electrosurgical heat-generator (Force FX, Covidien), operated
in cut pure mode. The generated vapors were aspirated though the REIMS
interface via a venturi pump built-in into the mobile Xevo G2-XS Q-ToF
mass analyzer (Waters Corporation, Wilmslow, U.K.). Isopropanol (Honeywell)
containing leucine-encephalin (Sigma-Aldrich) was infused at 150 μL/min
for lock mass correction.26 (link) Acquisition
parameters of REIMS data were: negative ionization mode, mass range: m/z 100–1500, scan time: 1 s. Mass
resolution for [LeuEnk–H]− was around 40000.
After REIMS analysis, the remaining tissue was either fixed in formalin
and embedded in paraffin, or frozen in liquid nitrogen. Hematoxylin
and eosin (H&E) stained sections were prepared for histopathology
review. The tissue surrounding the defect from the REIMS procedure
was analyzed to deduce the tissue components of the evaporated tissue.
The most substantial tissue component was taken as representative
for the metabolic profile(s) generated from the tissue defect.
were cauterized ex vivo using a monopolar handpiece
(iKnife disposable device, Waters Research Center (WRC), Budapest,
Hungary) equipped with a 1.7 mm diameter needle electrode, connected
to an electrosurgical heat-generator (Force FX, Covidien), operated
in cut pure mode. The generated vapors were aspirated though the REIMS
interface via a venturi pump built-in into the mobile Xevo G2-XS Q-ToF
mass analyzer (Waters Corporation, Wilmslow, U.K.). Isopropanol (Honeywell)
containing leucine-encephalin (Sigma-Aldrich) was infused at 150 μL/min
for lock mass correction.26 (link) Acquisition
parameters of REIMS data were: negative ionization mode, mass range: m/z 100–1500, scan time: 1 s. Mass
resolution for [LeuEnk–H]− was around 40000.
After REIMS analysis, the remaining tissue was either fixed in formalin
and embedded in paraffin, or frozen in liquid nitrogen. Hematoxylin
and eosin (H&E) stained sections were prepared for histopathology
review. The tissue surrounding the defect from the REIMS procedure
was analyzed to deduce the tissue components of the evaporated tissue.
The most substantial tissue component was taken as representative
for the metabolic profile(s) generated from the tissue defect.
9
Analytical Quantification of Adamantane Derivatives
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All solvents and water were purchased from Fisher Scientific Õ (Leicestershire, UK) and were LC-MS/MS grade. Leucine encephalin, 1-adamantylamine and internal standard 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) were brought from Sigma-Aldrich (TX, USA). Reference drugs used for UPLC-Tof were purchased from Cerillant Õ Sigma-Aldrich, LGC Õ (Luckenwalde, Germany) and Purechemicals.net (London, UK).
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