Agilent 6220 accurate mass time of flight lc ms system

LM and LAM were extracted and purified as previously described [26 (link)], separated by polyacrylamide gel electrophoresis (PAGE) and stained using a SilverSnap kit (Pierce). Alternatively, purified LM/LAM was suspended in 10 mM ammonium carbonate/bicarbonate buffer (pH 8.16) and 4 µL was injected by direct infusion into an Agilent 6220 Accurate-Mass Time-of-Flight LC/MS system (Agilent Technologies, Santa Clara, CA, USA). The run was performed in negative ionization mode, with a mass range of 100–3000 Da. The reference nebulizer was set to 20 psig with a detection window of 100 ppm, a minimum height of 1000 counts, an acquisition rate of 0.63 spectra/s, and an acquisition time of 1589.4 ms/spectrum. The gas temperature was set to 325 °C with a drying gas of 7 L/min, dual ESI 3500 V, Fragmentor 150 V, and Skimmer 65 V. The flow rate was set to 0.25 mL/min, and a solvent consisting of 0.1 M formic acid, acetonitrile (1:1, v/v) was used to wash the lines for 25 min. Data were analysed using MassHunter and the length of the mannan chain was calculated, assuming the presence of a PI lipid anchor containing C16:1/18:1/16:0 fatty acids. Analyses were performed in triplicate.

A brief scheme of the 2D CMMC system is shown in Figure 1B. The system was conducted on an Agilent 1200 HPLC equipped with binary pumps (pumps 2 and 3) and a solvent degasser, autosampler, and column compartment controlled by an Agilent MassHunter Workstation (Agilent Technologies, Palo Alto, CA, USA). The in-house packed CMMC column was used as the first-dimension column, and the mobile phase was 10 mmol/L ammonia acetate at a flow rate of 0.2 mL/min (pump 1). A Capcellpak C18 column (100 mm × 3.0 mm, 3 µm, Shiseido, Japan) was applied for the second-dimension separation, and the mobile phase consisted of 0.1% formic acid (solvent A) and acetonitrile (solvent B) at 0.8 mL/min with a linear gradient elution program (10 to 60% B at 0 to 8 min; 60% B at 8 to 10 min; 60 to 10% B at 10 to 10.01 min;10% B at 10.01 to 13 min). A Rheodyne MXP9960-000 2-position/10-port switching valve (Rohnert Park, CA, USA) containing two 500 μL sample loops was used to connect the two dimensions. The output from the second-dimension column with a split ratio of 1:1 was introduced to the electrospray ionization (ESI) source of the Agilent 6220 Accurate-Mass Time-of-Flight LC/MS system (Agilent Technologies, Palo Alto, CA, USA). The detailed operation of the 2D system and MS conditions are described in our group's previous research 20 (link), 24 (link).