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Subsequently, samples were concentrated to dryness in vacuum before dissolving in 150 L of 80% methanol (precooled at -20C), then centrifuged again at 13 500 g and 4C for 10 min to obtain the supernatant for high-performance liquid chromatography Ultimate 3000 (Thermo Fisher Scientific, USA)-tandem mass spectrometry Q Exactive Focus (Thermo Fisher Scientific, USA) (LC-MS/MS) analysis ( 2.3.2 Extraction of Total Metabolites All samples were thawed at 4C, and 100 L of each sample were pipetted to new 2 mL tubes

Step 5 Gently Swirl if Necessary If portions of the peptide remain undissolved after several minutes, gently swirl or slowly rotate the vial between your fingers

Note on solvent selection: Bacteriostatic water is strongly preferred over plain sterile water for research vials that will be accessed multiple times

A slow injection is less painful

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