Comprehensive structural assignment of glycosaminoglycan oligo- and polysaccharides by protein nanopore

Glycosaminoglycans are highly anionic functional polysaccharides with information content in their structure that plays a major role in the communication between the cell and the extracellular environment. The study presented here reports the label-free detection and analysis of glycosaminoglycan molecules at the single molecule level using sensing by biological nanopore, thus addressing the need to decipher structural information in oligo- and polysaccharide sequences, which remains a major challenge for glycoscience. We demonstrate that a wild-type aerolysin nanopore can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, osidic bonds and epimers of uronic acid residues. Size discrimination of tetra- to icosasaccharides from heparin, chondroitin sulfate and dermatan sulfate was investigated and we show that different contents and distributions of sulfate groups can be detected. Remarkably, differences in α/β anomerization and 1,4/1,3 osidic linkages can also be detected in heparosan and hyaluronic acid, as well as the subtle difference between the glucuronic/iduronic epimers in chondroitin and dermatan sulfate. Although, at this stage, discrimination of each of the constituent units of GAGs is not yet achieved at the single-molecule level, the resolution reached in this study is an essential step toward this ultimate goal.


Supplementary Note 1
Optimization of the voltage, and type and concentration of the electrolyte: experimental parameters such as the applied voltage and type and concentration of the electrolyte were evaluated before recording the data. At voltages below +60 mV, the signal-to-noise ratio was not high enough to unambiguously distinguish true translocation events from noise, and at voltages above +60 mV, not only was the resolving power lower, but the event populations were distorted at the shorter translocation time portion of the distribution as shown in Supplementary Fig. 1. So, all the experiments were carried out at an applied voltage of +60 mV. that longer and deeper current blockades lead to better molecular discriminations, we searched for experimental conditions that slow down the translocation of GAGs. Previously, it was shown that the translocation time of a DNA molecule (which is also negatively charged) strongly increases when the size of the counter ion decreases from K + to Na + and Li + and when the concentration of the electrolyte increases. 1,2 Therefore, 3 M and 4 KCl M, as well as 4 M and 6 M LiCl were tried (Supplementary Fig. 2).
Supplementary Fig. 2 Comparing the event population using KCl 3 M (dark blue), LiCl 4 M (red) and LiCl 6 M (green) for a) HA dp10 and b) HN dp10.
From a practical point of view, it was difficult to work at KCl 4 M with our homemade horizontal Teflon lipid bilayer device, whose cis and trans compartments have a small volume (100 µL). Indeed, during the recording time, KCl precipitated, and, as a result, the salt concentration of the buffer changed. Therefore, use of LiCl with an extremely high solubility (83.2 g/100 mL (19.6 M) at 20 °C) 3 was preferred to KCl. Using 6 M LiCl, the translocation time was maximal ( Supplementary Fig. 2); however, pore insertion into the bilayer was difficult. Using 3 M KCl and 4 M LiCl was practically feasible; however, slightly longer and a b deeper events were observed using LiCl 4 M ( Supplementary Fig. 2), and therefore we chose to work under these conditions. Analyses were performed using two coupled SEC columns (PL aquagel-OH 20 5 µm, 7.5 x 300 mm) in series and reflectometry detection (Agilent 1260 Infinity II). 100 mM Na2PO4, 300 mM NaCl, pH 7.0 was used as the eluent at a flow rate of 1 mL/min. Supplementary Fig. 4 Scatter plots of (a) mixture of HP oligosaccharides containing dp4, dp6, dp10, dp16 and dp20 oligosaccharides, and (b) enoxaparin. The two clouds of event broaden over similar translocation timescales except at shortest times. Thus the presence of dp6-dp20 HP oligosaccharides in the enoxaparin sample can be qualitatively deduced from the plot. The data were recorded in 4 M LiCl, 25 mM HEPES buffer and 1.0 mM EDTA at pH 7.5, 20.0 °C, and at the bias voltage of +60 mV.

Supplementary Note 2
The ability of the nanopore detection system to differentiate different levels of heparin sulfation shown on Figure S6 was exploited to monitor the enzymatic regioselective 6-O-desulfation of HP dp 10 and HP dp20 catalyzed by the sulfatase HSulf-2 (Supplementary Fig. 7). A shift of the translocation times to shorter times after enzymatic desulfation were observed (with a larger shift for HP dp20 compared to HP dp10), in accordance with the above observations on Figure S6 with regioselectively desulfated heparins.

Supplementary Note 3
It is to be noted that the action of the sulfatase enzyme on HP oligosaccharides is not complete, as shown by mass spectrometry analysis of the heparin dp10 oligosaccharide that was partially 6-Odesulfated upon overnight incubation with the HSulf sulfatase (Supplementary Fig. 8). Indeed, HSulf sulfatase is a processive enzyme that starts its action at the non-reducing end of the oligosaccharide and then progresses towards the reducing end without, however, succeeding in hydrolyzing all the 6-Osulfate groups. 35 . Therefore, compared to the oligosaccharide in Supplementary Fig. 6b, the oligosaccharide in Supplementary Fig. 7b is only partially 6-O-desulfated. It, therefore, occupies about the same volume in the pore lumen but carries a lower negative charge, thus not affecting the magnitude  Table 1 Description of the commercially purchased GAGs studied in this work.

HP oligosaccharides (Iduron)
These oligosaccharides have been prepared by high resolution gel filtration of partial heparin lyase digestion of high quality heparin.
Although the main disaccharide unit in these products is IdoUA,2S -GlcNS,6S, (approx 75%) saccharides in each size class show some variation in degree and pattern of sulfation.

Desulphated Heparin Oligosaccharides (Iduron)
These products have been modified by standard chemical methods to selectively remove sulfate groups from C2 of Iduronate, C6 of glucosamine or the N-sulfate of Glucosamine. The N-desulfated heparin contains the free amino group (NH + 3); in N-desulfated re N-acetylated heparin the free amino group has been modified by acetylation.

HA oligosaccharides (Iduron)
These oligosaccharides have been prepared by high resolution gel filtration of partial endolyase digestion of purified Streptococcal HA.

CS oligosaccharides (Iduron)
These oligosaccharides have been prepared by high resolution gel filtration of partial chondroitin ABC lyase digestion of mixed isomer chondroitin sulfate. The main disaccharide repeat in the original chondroitin sulphate is GlcA -GalNAc sulphated at C-6 or C-4 of the GalNAc residue; the CSD disaccharide unit (GlcA,2S -GalNAc,6S) is a minor component comprising approx. 5% of total disaccharides.

DS oligosaccharides (Iduron)
These oligosaccharides have been prepared by high resolution gel filtration of partial chondroitin ABC lyase digestion of dermatan sulfate. The main disaccharide in the dermatan sulfate used to prepare these oligosaccharides is IdoA -GalNAc,4S (88% of total disaccharides). The remainder are composed of 7% of disulfated units (IdoA,2S -GalNAc,4S) and 5% non-sulfated disaccharides. The 10.98 kDa low molecular weight Heparin is made via partial digestion of heparin with heparinase I, and is purified by size exclusion chromatography.

Enoxaparin Lovenox® (Sanofi-Aventis)
The pharmaceutical grade heparin preparation Enoxaparin is prepared by benzylation followed by alkaline depolymerization Data analysis was performed using Igor Pro 8.04A software (WaveMetrics, OR, USA) with in-house routines. The approach is based on a statistical analysis of current blockades induced by saccharides entering the pore. It involves at least several thousand events. The detection of each current blockade recorded as a time-dependent nanopore current is based on a current-threshold (Th) method. A blockade event is detected when the current magnitude becomes smaller than Th (downward crossing) until it returns to a value greater than Th (upward crossing). It defines the range of points used to compute the characteristic quantities of the blockade, such as the event duration ! and the mean value " (see Fig.   2). The threshold value is chosen according to the depth of the event typically ℎ = 0.5 # , where # is the average open-pore current. Before detection of blockades, the current trace is smoothed using a median filter with a smoothing window of 11 points. The average currents # and " are measured on the non-filtered trace. The translocation time is defined as the duration between the two crossings of the threshold. Characteristic translocation times were extracted from the long tail of the time distribution by a single-exponential function.