Guanosine 5 monophosphate

The qualitative analysis of monophosphate nucleotides was performed as reported by Yamaoka et al. [19 (link)]. LC–MS was performed under the following conditions: instrument, Waters UPLC™ and system with an electrospray ionization (ESI) interface and a quadrupole mass detection system (Waters, Milford, MA, USA); software, MassLynx^TM; column, ACQUITY UPLC™ Column HSS T3 (2.1 mm ID and 100 mm length, 1.8-μm particle size) (Waters, Milford, MA, USA). The ESI source was operated at 120 °C with a desolvation temperature of 450 °C, 800 L/h desolvation gas flow rate, and a capillary voltage set at 3.5 kV. The cone voltage was 30 V and collision energies 30 eV. Integration and quantitation were performed using the Waters MassLynx^TM software. Detection of these compounds was performed in the negative ionization. Monophosphates nucleotides (xanthosine 5’-monophosphate (XMP), cytidine 5’-monophosphate (CMP), uridine-5’-monophosphate (UMP), thymidin-5’-monophosphate (TMP), adenosine-5’-monophosphate (AMP), inosine 5’-monophosphate (IMP), guanosine-5’-monophosphate (GMP)), were purchased from Sigma–Aldrich (Wrocław, Poland). All data were obtained in triplicate. The results were expressed as mg/100 g of dry weight (d.w.).

Adenosine 5′-monophosphate (AMP), uridine 5′-monophosphate (UMP), guanosine 5′-monophosphate (GMP), cytidine 5′-monophosphate (CMP), and ribose 5′-monophosphate (rMP) were purchased as disodium salts from Sigma-Aldrich (Bangalore, India) and used without further purification. The phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). All other reagents used were of analytical grade and purchased from Sigma-Aldrich (Bangalore, India).

Eighty dried samples of T. matsutake were collected in accordance with official sampling requirements [19 ] from the mountain areas of Xiaojin County, Jiulong County, Yajiang County, Kangding County, Muli County, and Lixian County in Sichuan Province, China. Fifteen authentic standards of adenosine (A), cytidine (C), guanosine (G), inosine (I), thymidine (T), uridine (U), xanthosine dehydrate (X), 2′-deoxyadenosine (dA), 2′-deoxycytidine (dC), 2′-deoxyguanosine (dG), 2′-deoxyuridine (dU), adenosine 5′-monophosphate (AMP), cytidine 5′-monophosphate (CMP), guanosine 5′-monophosphate (GMP), and uridine 5′-monophosphate (UMP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Milli-Q water purification system was used to prepare ultra-pure water for the UPLC analysis (Millipore, Bedford, MA, USA). The solvent ammonium acetate, acetonitrile, and diethylamine with LC-MS grade for UPLC-MS analysis were also purchased from Sigma-Aldrich (Sigma-Aldrich, St.Louis, MO, USA). Other chemicals and solvents of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Sources of mononucleotides: adenosine 5′-monophosphate (Sigma-Aldrich), uridine 5′-monophosphate (P-L Biochemicals), guanosine 5′ monophosphate (Sigma-Aldrich), cytidine 5′-monophosphate (Sigma-Aldrich). We used ultra-pure deionized water (Milli-Q) from a Millipore system to prepare 10 mM solutions of the nucleotides. All solutions were filtered using Pierce™ Protein Concentrators PES, 3K MWCO, 0.5 mL, centrifuged at 14,000 rpm.
Mica was purchased from SPI Supplies (Mica grade v-4). Scotch tape was used to repeatedly cleave the mica until a complete cleavage was achieved after 3–5 times. In addition to the controls described in the text, we ran blind tests with wet-dry cycles using only Milli-Q water to verify the cleaning procedures. This assured that the polymers and rings were not due to an unknown contaminant in the water.