Hypersep hypercarb spe cartridge

LLO-released oligosaccharides were resuspended in 500 µL of water (LC–MS grade, Fisher Chemical) and then purified using carbograph columns (Hypersep™ Hypercarb™ SPE Cartridges, 200 mg, 3 mL, ThermoFisher Scientific). First, the columns were conditioned by successive washing with 3 mL of 1 M sodium hydroxide (Laboratory reagent grade, Fisher scientific), 6 mL of water (LC–MS grade, Fisher Chemical), 3 mL of acetic acid 30% (v/v) Acros Organics) and finally 3 mL of water (LC–MS grade, Fisher Chemical). These steps may be carried out under vacuum (5 mmHg) using a Manifold. Then, the columns were successively washed with 3 mL of 50% acetonitrile in water (v/v) (LC–MS grade, Fisher Chemical), 6 mL of 5% acetonitrile in water (v/v) and finally with 6 mL of water (LC–MS grade, Fisher Chemical). The LLO-released oligosaccharide samples were resuspended in 500 µL of water (LC–MS grade, Fisher Chemical) and were loaded into the column. After washing with 3 mL of water and 3 mL of 5% acetonitrile in water (v/v), LLO-released oligosaccharides were eluted by 4 × 0.5 mL of 50% acetonitrile in 0.1 M of TFA (v/v). The eluted fractions were recovered and were lyophilized in glass tubes prior to permethylation.

MALDI-TOF/TOF-MS of N-glycans were performed as described previously (24 (link)). Briefly, cell lysates were denatured with UREA, DTT and IAM, and digested with PNGase F (New England BioLabs; Ipswich, MA, USA). Released N-glycans were collected, desalted with HyperSep Hypercarb SPE cartridges (Thermo Fisher Scientific, Bellefonte, PA, USA), lyophilized, and subjected to MALDI-TOF/TOF-MS (UltrafleXtreme, Bruker Daltonics; Bremen, Germany) analysis in positive-ion mode. Mass signals were analyzed and annotated with GlycoWorkbench software (http://code.google.com/p/glycoworkbench) (28 (link)). Relative proportion was calculated by adding the relative intensity of the same type of N-glycans.

The N-glycans were released by PNGase F glycosidase (New England Biolabs, Beverly, MA) according to previous protocols (25 (link), 26 (link)). Briefly, 200 μg of isolated glycoproteins were concentrated and desalted by adding to a size-exclusion spin ultrafiltration (Amicon Ultra-0.5 mL 10,000 MW cut off, Millipore). Then, the obtained glycoproteins were denatured with 8M urea (Sigma-aldrich), 10 mM DTT, and 10 mM IAM (Sigma-Aldrich). Followed by the exchange of buffer into 40 mM NH₄HCO₃ via using 10 K centrifugal ultrafiltration. After that, 5 μL of PNGase F (NEB) were added into ultrafiltration and incubated over night with shaking at 37°C. The reaction was stopped by incubating the mixture at 80°C for 5 min. After centrifuge, the released N-glycans were collected and purified with HyperSep Hypercarb SPE cartridges (25 mg, 1 mL; Thermo Scientific) according to the manufacturer recommendation. The N-glycans were eluted by 0.5 mL of elution solution (50% (v/v) acetonitrile with 0.1% (v/v) TFA). The purified N-glycans were collected and lyophilized.

The isolation and purification of N-glycans were performed based on previously described methods (Qin et al., 2017 (link); Yang et al., 2020b (link)). The glycoproteins were concentrated and desalted by Amicon Ultra-0.5 3 KDa ultrafiltration units (Millipore, United States) and then denatured by the addition of 8 M urea, 10 mM DTT, and 10 mM IAM. The denatured glycoproteins were exchanged to 40 mM NH₄HCO₃ buffer and incubated with trypsin (37°C, overnight). The trypsin in the mixture was inactivated by heating (80°C, 5 min) and then the PNGase F (New England Biolabs, Beverly, MA) was added to release the N-glycans (37°C, overnight). Subsequently, the digest was subjected to HyperSep Hypercarb SPE cartridges (25 mg, 1 mL; Thermo Scientific) to remove peptides. The purified N-glycans were collected and lyophilized.