2 4 6 trihydroxyacetophenone monohydrate

Enzymatic release of N-linked glycans from HIV-1 gp145 was carried out following the method of Küster et al. [32 (link)] with minor modifications. Briefly, excised bands containing 20 μg of gp145 were incubated with 5000 units of PNGase F (NEB) for 24 hrs. at 37°C [32 (link)]. For the analysis of oligosaccharides containing sialic acid, 20 μg of gp145 in solution were incubated with 50 units of α2–3,6,8,9 NEUA (NEB) for 24 hrs. at 37°C, prior to PNGase F digestion. Subsequently, glycans were extracted into LC-MS water in an ultrasonic bath. The extracted glycans were then dried in a vacuum concentrator and dissolved with water. For MALDI-ToF analysis, glycans were co-crystallized with a solution of 50% acetonitrile/50% 20 mM ammonium citrate containing 134 mM of 2′,4′,6′-Trihydroxyacetophenone monohydrate (Sigma-Aldrich). MALDI-ToF MS analysis of the N-glycans was performed in the reflector positive and negative ion mode using a 4800 Plus MALDI ToF/ToF Analyzer (AB Sciex) that was calibrated externally using the Calmix 5 Opti-ToF High-Resolution TIS Calibration Insert (AB Sciex). N-glycan assignments were carried out using GlycoWorkbench software and confirmed by MALDI-ToF post-source decay spectra (S2 Fig) [33 (link)].

Polyethylene glycol (PEG, M_w ∼ 1500 Da) was supplied by Fluka Chemie
GmbH (Buchs, Switzerland). Polypropylene glycol (PPG, M_w ∼ 1010 Da, M_w/M_n 1.04) was purchased from PSS Polymer Standards
Service GmbH (Mainz, Germany). Polytetrahydrofuran of M_w ∼ 1000 Da and M_w ∼
1400 Da (denoted as PTHF1000 and PTHF1400, respectively) was obtained
from Royal DSM N.V. (Heerlen, The Netherlands). Nylon-6 was purchased
from Scientific Polymer Products Inc. (M_w ∼ 25,000 Da, Oregon, USA). Polystyrene (PS, M_w ∼ 1300 Da, M_w/M_n 1.10) was purchased from Thermo Fisher Scientific
GmbH (Kandel, Germany). Polybutylene terephthalate (PBT, M_w ∼ 16,150 Da). ULC-MS-grade methanol (MeOH), ethanol
(EtOH), and n-hexane were acquired from Biosolve
B.V. (Valkenswaard, The Netherlands). Tetrahydrofuran (THF) and hexafluoroisopropanol
(HFIP, ≥99% purity) were supplied by Sigma-Aldrich Chemie B.V.
(Zwijndrecht, The Netherlands). All organic solvents
were used without further purification. 2,5-Dihydroxybenzoic acid
(DHB, 98% purity), dithranol (DT, ≥90% purity), 2′,4′,6′-trihydroxyacetophenone
monohydrate (THAP, ≥99.5% purity), silver trifluoroacetate
salt (AgTFA, 98% purity), and trifluoroacetic acid (TFA, ≥99%
purity) were purchased from Sigma-Aldrich.

Trifluoroacetic acid (TFA), acetonitrile (ACN), sinapinic acid (SA), caffeic acid (CA), 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHAH), ferulic acid (FA), 2,4,6-trihydroxyacetophenone monohydrate (THAP), 2-(4-hydroxyphenylazo)benzoic acid (HABA), 2,6-dihydroxyacatophenone (DHAP), 9-aminoacridine (9-AA) and dithranol (INN) from Sigma–Aldrich (Dorset, UK) were used.
14 g of nutrient agar (Fisher Scientific Ltd. Loughborough, UK) was dissolved and mixed thoroughly in a bottle containing 500 mL of water. This bottle was then autoclaved at 121 °C for 15 min and subsequently used for the bacterial cultures.