Unipoint

Purification and pyridylamination of oligosaccharides were performed as described [19 (link), 42 (link), 43 (link)]. Pyridylated (PA)-N-oligosaccharides were separated into neutral N-oligosaccharides and mono−/di−/tri−/tetra-sialylated N-oligosaccharides through an anion-exchange column (Mono Q5/50GL, GE Healthcare) using HPLC and DE52-packed column (Whatman, GE Healthcare) [44 (link)]. Sialylated PA-N-glycans were treated with neuraminidase at 37 °C for 14 h in 50 mM ammonium acetate (pH 5.0) to cleave sialic acids, followed by heating at 100 °C for 5 min and filtering through 0.2 μm spin filter (Ultrafree-MC LG, Millipore). Neutral PA-N-oligosaccharides were analyzed using HPLC as described [19 (link), 42 (link), 43 (link)]. N-oligosaccharide structures were determined by calculating the mannose unit value from NP-HPLC (Takara Bio) and the glucose unit value from RP-HPLC (Takara Bio), as described [20 (link), 45 (link)], also by comparison with known standards and sequential exoglycosidase digestion (see below). PA-N-glycans were quantified as described [44 (link)], and HPLC chromatogram data were analyzed using Unipoint (Gilson), LC station (Shimadzu), and Empower2 (Waters). The workflow of N-oligosaccharides analysis and separation by HPLC is shown in Additional file 2: Figure S1B.

Quantitative analyses of 5-MCA-NAT were performed with a HPLC instrument (Gilson, Middleton, WI, USA) composed of a solvent delivery pump (305 model), UV-visible detector (118 model), and controller software (UniPoint, all by Gilson). The injector was equipped with a 20 µl loop (7125 Rheodyne, Berkeley, CA, USA). The chromatographic separation was achieved as described previously [12] (link) by a reversed phase protocol with a Mediterranea Sea18 column (25 cm×4 mm, 5 µm particle size; Teknokroma, Barcelona, Spain). The mobile phase was a mixture of methanol (Panreac, Barcelona, Spain) and ultrapure milliQ water (40∶60 v/v). The flow rate was set at 0.8 ml/min and the eluent was monitored at 244 nm.

HPLC data were exported from the Gilson Unipoint software that operates the machinery to OriginPro2016. Peak areas were calculated in OriginPro2016 using the inbuilt Peak Analyzer tool with the baseline mode set to asymmetric least squares smoothing.
To determine the percentage of conversion of fed PA-B to PA-A in the Fig. 2 dataset, samples were first adjusted for their methanol controls, to account for any inherent production of either compound by the strains. Percentage conversion was calculated as the amount of PA-A produced divided by the total pseudomonic acids produced (PA-A and PA-B).