300sb c18

Monosaccharide derivatives were analyzed in Agilent 1000 series HPLC (Agilent Technologies, Model No.1100). A reversed-phase C₁₈ column (ZORBAX 300 SB-C18, 5 μm, 4.6 × 250 mm, USA) and 1-butylamine-phosphoric acid-tetrahydrofuran mobile phase system consisting of solvents A and B were used for this analysis. Solvent A comprises of 0.2% 1-butylamine, 0.5% phosphoric acid, and 1% tetrahydrofuran in water, and solvent B consisted of equal parts of solvent A and acetonitrile. The separations were carried out at 24 °C using a flow rate of 1 mL/min, and 20 mL of each sample was injected. An UV detector was used to detect the derivatized monosaccharides. The gradient program was used according to [21 (link)].

C. elegans sperm was suspended in PBS solution, disrupted by sonication for 8 s (repeated three times at intervals of 8 s), mixed with SDS loading buffer and heated for 10 min. After centrifugation, the supernatant was loaded on a 12% SDS-PAGE gel for separation. The resulting gel was silver stained using a Fast Silver Stain kit (Beyotime, Shanghai, China). The major sperm protein (MSP) band was removed prior to tryptic in-gel digestion, according to standard protocols. The tryptic peptides were extracted using 60% acetonitrile in 0.1% formic acid, dried in vacuum and then resuspended in 0.1% formaic acid for mass spectrometry analysis. LC-MS/MS analysis was performed in a LTQ-orbitrap XL (ThermoFinnigan, San Jose, CA) coupled online with an Eksgent Nano 2D LC system. Peptides mixture was first loaded on a trap column (300SB-C18, 5 × 0.3 mm, 5 μm particle) (Agilent Technologies, Santa Clara, CA), and then analysed using a self-packed capillary C18 column (75 μm i.d. × 150 mm, 3 μm particle, C18 resin), and eluted with a gradient of 4-35% of Buffer B (0.5% formic acid in acetonitrile) in Buffer A (0.5% formic acid in water) at a flow rate of ~300 nl/min for 120 min. Data-dependent scanning was incorporated to select the 10 most abundant ions from a full-scan mass spectrum (mass range 200–1800 Da) for fragmentation by collision-induced dissociation.

The standard LC-MS/MS and database searching were performed according to the protocol described previously (Sun et al., 2015 (link)). Briefly, LC-MS analysis was carried out using a Surveyor MS Pump Plus HPLC system coupled to a Thermo Fisher Finnigan LTQ linear ion trap mass spectrometer (Thermo Fisher Corporation, San Jose, CA, USA) using nano-electrospray ionization. Tryptic peptides were loaded onto a trap column (300SB-C18, 5 × 0.3 mm, 5 μm particle size; Agilent Technologies, Santa Clara, CA, USA) connected through a zero dead volume union to the self-packed analytical column (C18, 100 × 0.1 mm, 3 μm particle size; SunChrom, Germany). The peptides were then separated by linear gradient elution involving 0–45% B over 55 min followed by 45–100% B over 10 min (B is 80% acetonitrile, 0.1% formic acid) at a flow rate of 500 nL/min. MS data were analyzed using SEQUEST against the National Center for Biotechnology Information (NCBI) human protein database and the results filtered, sorted, and displayed using Bioworks 3.2. Returned protein lists were filtered using the parameters: Peptide Xcorr value >1.90 (for +1 charge), >2.75 (for +2 charge), >3.75 (for +3 charge); peptide Delt CN >0.1; protein probabilities <0.001. At least two unique peptides were required for each identified protein.

Product formation in AdoMet cleavage and hydroalkylation assays was quantified using a Q-TOF LC/MS (Agilent 6545 mass spectrometer coupled to an Agilent Infinity 1260 liquid chromatography system) and a Zorbax reversed-phase column (300SB-C18, 3.5 μm, 2.1 × 50 mm, Agilent). Solvent A was H₂O with 0.1% acetic acid, and solvent B was acetonitrile with 0.1% acetic acid. Flow rate was 0.4 mL/min. The LC method for all assays was as follows: 0–2 min, 1% B; 2–4 min, gradient from 1 to 50% B; 4–6 min, gradient from 50 to 100% B; 6–7 min, 100% B; 7–8 min, gradient from 100 to 1% B.