Agilent nano lc esi qtof ms system

N-glycan samples were reconstituted in nanopure water for analysis, randomized, and analyzed using an Agilent nanoLC/ESI-Q-TOF-MS system (Agilent Technologies). Samples were introduced into the MS with a microfluidic chip, consisting of enrichment and analytical columns packed with PGC and a nanoelectrospray tip. A binary gradient was applied to separate and elute glycans at a flow rate of 0.3 μl/min: (A) 3% (v/v) acetonitrile and 0.1% (v/v) formic acid in water and (B) 90% (v/v) acetonitrile in 1% (v/v) formic acid in water. MS spectra were acquired at 1.5 s per spectrum over a mass range of m/z 600 to 2000 in positive ionization mode. Mass inaccuracies were corrected with reference mass m/z 1221.991. Collision-induced dissociation (CID) was performed with nitrogen gas using a series of collision energies (Vcollision) dependent on the m/z values of the N-glycans, based on the equation: Vcollision= slope (m/z) + offset; where the slope and offset were set at (1.8/100 Da) V and −2.4 V, respectively.

Purified glycosphingolipid samples were reconstituted in methanol : water (1 : 1, v/v) and analyzed using an Agilent nano-LC/ESI QTOF MS system (Agilent, CA). Samples were loaded onto the enrichment column packed with ZORBAX C18 (5 μm) at 3 μL min^–1. Separation was carried out on the analytical column at a constant flow rate of 0.3 μL min^–1 using a binary gradient as follows: (A) 20 mM ammonium acetate and 0.1% (v/v) acetic acid in water; (B) 20 mM ammonium acetate and 0.1% (v/v) acetic acid in methanol : isopropanol (85 : 15, v/v); 70–75% (B), 0–1 min; 75–85% (B), 1–4 min; 85–100% (B), 4–40 min; 100% (B), 40–55 min; 100–70% (B), 55–58 min; 70% (B), 58–60 min. Per acquisition, one MS scan was followed by five MS/MS scans in data-dependent mode. Collision-induced dissociation was performed using a series of collision energies based on optimized conditions according to the formula V_collision = m/z (1.2/100 Da) + 12.