All organic solvents and reagents were purchased commercially and used without further purification. FA standards and AMP+ MaxSpec Kit were purchased from Cayman Chemical (Ann Arbor, MI). Charge-tagging PB reagents, including 2-, 3-, and 4-acpy, 1-(5-(fluoro)pyridin-2-yl)ethanone (5-F-2-acpy), 1-(5-(trifluoromethyl)pyridin-2-yl)ethanone (5-CF3-2-acpy), and 2-benzoylpyridine (bzpy) were purchased from Bidepharm (Shanghai, China). 3-Acetyl-1-methylpyridinium (CH3-py) was purchased from Sigma-Aldrich (St. Louis, MO). Pooled normal human plasma with anticoagulant lithium heparin added was obtained from Innovative Research, Inc. (Novi, MI). Plasma samples from T2D patients and normal control were supplied by the specimen bank of Dongfeng Hospital of Hubei University of Medicine. The human studies abided by the Declaration of Helsinki principles. All the procedures related to these samples were compliant with relevant ethical regulations approved by the Ethical Review Board of Tsinghua University (IRB No. 2017007). Yak milk powder was purchased from market (Liaoyuan Dairy LTD, Gansu, China). HPLC grade ACN, methanol (MeOH), and isopropanol (IPA) were purchased from Fisher Scientific Company (Ottawa, ON, Canada).
Amp maxspec kit
Manufactured by Cayman Chemical
Sourced in United States
The AMP+ MaxSpec Kit is a laboratory equipment product designed for the detection and quantification of adenosine monophosphate (AMP) in various sample matrices. The kit provides the necessary reagents and components to perform the analysis.
Automatically generated - may contain errors
Lab products found in correlation
Methanol meoh, by Thermo Fisher Scientific (1 mentions)
Isopropanol ipa, by Thermo Fisher Scientific (1 mentions)
Hplc grade acn, by Thermo Fisher Scientific (1 mentions)
Fa standards, by Cayman Chemical (1 mentions)
X500r q tof mass spectrometer, by AB Sciex (1 mentions)
Lc 20ad system, by Shimadzu (1 mentions)
Raw 264, by Merck Group (1 mentions)
Methyl tert butyl ether (mtbe), by Merck Group (1 mentions)
Hplc grade isopropanol, by Thermo Fisher Scientific (1 mentions)
Cay10566, by Merck Group (1 mentions)
Hypercarb column, by Thermo Fisher Scientific (1 mentions)
1 hydroxybenzotriazole, by Merck Group (1 mentions)
Waters micro mass zq 4000, by Waters Corporation (1 mentions)
4 protocols using amp maxspec kit
1
Charge-Tagging of Fatty Acids
2
Fatty Acid Profiling by LC-MS
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Lipids were extracted by the Folch method and followed by alkaline hydrolysis to liberate fatty acids. For identification and relative quantification of fatty acids, chemical derivatization of samples spiked with internal standard was performed using N-(4-aminomethylphenyl) pyridinium (AMPP) (AMP + MaxSpec Kit, Cayman Chemical, MI, USA). AMPP-derivatized fatty acids were analyzed by reverse-phase (RP) liquid chromatography mass spectrometry (LC-MS). For determination of C = C locations in fatty acids, C = C-specific derivatization based on Paternò–Büchi reaction was conducted offline. The reaction solution was collected in vials for subsequent reversed-phase liquid chromatography (RPLC)-MS analysis. All LC-MS analyses were conducted on a LC-20AD system (Shimadzu, Kyoto, Japan) connected with an X500R QTOF mass spectrometer (Sciex, Toronto, CA).
3
Lipid Extraction and Analysis in RAW 264.7 Macrophages
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High-performance LC (HPLC) grade iso-propanol (IPA), acetonitrile (ACN), methanol (MeOH), and water were purchased from Fisher Scientific. Methyl tert-butyl ether (MTBE), formic acid (HCOOH), ammonium formate, and 2-acetylpyridine (2-acpy) were purchased from Sigma Aldrich. Lipid standards were purchased from Avanti Polar Lipids. AMP+ MaxSpec Kit was purchased from Cayman Chemical.
RAW 264.7 macrophages (American Type Culture Collection (ATCC); Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and collected by centrifugation. The cell pellets were washed, frozen in liquid nitrogen, and stored at −80°C. For direct inhibition of enzyme activity, RAW 264.7 macrophages were treated for 72 h with 60 μM FADS2 inhibitor (SC26196, purchased from Sigma-Aldrich) and 2.5 μM SCD1 inhibitor (CAY10566, purchased from Sigma-Aldrich), while the control group was treated with DMSO.
RAW 264.7 macrophages (American Type Culture Collection (ATCC); Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and collected by centrifugation. The cell pellets were washed, frozen in liquid nitrogen, and stored at −80°C. For direct inhibition of enzyme activity, RAW 264.7 macrophages were treated for 72 h with 60 μM FADS2 inhibitor (SC26196, purchased from Sigma-Aldrich) and 2.5 μM SCD1 inhibitor (CAY10566, purchased from Sigma-Aldrich), while the control group was treated with DMSO.
4
Derivatization and LCMS Quantification of SCFAs
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Samples were derivatized with the AMP + MaxSpec Kit (Cayman Chemical, Ann Arbor, USA, 710000) according to the manufacturer's instructions. Briefly, the samples were derivatized by adding 20 l of acetonitrile/dimethylformamide, 20 l of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide, 10 l of 1-hydroxybenzotriazole (Sigma-Aldrich), and 30 l of the AMP + reagent. The reaction mixture was incubated for 30 min at 60°C. Samples were stored at -20°C before analysis. AMP + -derivatized SCFAs were separated on a Hypercarb column (3 m, 100 × 2.1 mm; Thermo Fisher Scientific, 35003-031030) using a gradient of solvent A (5% acetonitrile and 0.1% formic acid) and solvent B (100% acetonitrile). After 5 min of 100% solvent A, the proportion of solvent B was increased to 100% within 15 min at a flow rate of 200 l/min at 30°C. Quantification of SCFA was performed by LC-ESI-MS (Alliance HT Waters 2790 with Waters Micromass ZQ 4000, Waters, Eschborn, Germany, 176004001) using the selected ion recording mode for the AMP + signals 227 (acetic acid), 241 (propionic acid), 255 (butyric acid), and 269 (valeric acid) (Sigma-Aldrich). For absolute quantification, an SCFA standard calibration mixture (WSFA-2; Merck, Darmstadt, Germany, 46975-U) was used.
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