Sialic Acid Derivatization of Fluorescently Labeled N-Glycans Allows Linkage Differentiation by Reversed-Phase Liquid Chromatography–Fluorescence Detection–Mass Spectrometry

Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS. Despite this, these approaches are unable to simultaneously provide unambiguous assignment of sialic acid linkages and assess further isomeric glycan features within a single measurement. Thus, for the first time, we present the combination of procainamide fluorescent labeling with sialic acid linkage-specific derivatization via ethyl esterification and amidation for the analysis of released plasma N-glycans using reversed-phase (RP)LC-fluorescence detection (FD)-MS. As a result, α2,3- and α2,6-sialylated N-glycans, with the same mass prior to derivatization, are differentiated based on retention time, precursor mass, and fragmentation spectra, and additional sialylated isomers were also separated. Furthermore, improved glycan coverage and protocol precision were found via the novel application using a combined FD-MS quantification approach. Overall, this platform achieved unambiguous assignment of N-glycan sialic acid linkages within a single RPLC-FD-MS measurement, and by improving their retention on RPLC, this technique can be used for future investigations of released N-glycans as an additional or orthogonal method to current analytical approaches.


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S3 S1. Supporting Information -Experimental Section S1.1 PNGase F release Plasma (5 µL) and H2O (4 µL) were added to a 120 µL 96-well PCR plate. In addition, 10X denaturing solution (1 µL), consisting of 5% sodium dodecyl sulfate with 400 mM dithiothreitol [LZ-rPNGASEF-96], was added to the samples and the plate was sealed with a foil pierce seal. The plate was briefly vortexed and centrifuged prior to a 10 min incubation at 100 °C. The plate was left to cool to room temperature and the foil pierce seal was removed. Following this, 6 µL of H2O, 2 µL of concentrated reaction buffer (500 mM sodium phosphate, pH 7.5), and 2 µL 10% NP-40 were added to each sample. The PNGase F solution (150 µL) from the kit was diluted with 150 µL PNGase F storage buffer and 2 µL was added to each sample. The plate was sealed using a foil pierce seal and incubated overnight at 37 °C. Following the incubation, the foil pierce seal was removed and 5 µL of a 5% FA/H2O solution (v/v) was added to each sample. The plate was sealed again, vortexed to mix, briefly centrifuged and incubated at room temperature for 40 min. In order to remove the enzyme and other protein contaminants, a PBM plate was set up on the vacuum manifold on the Hamilton robot. The plate was prepared by washing each well first with 100 µL of methanol, followed by 300 µL of H2O. The solutions were removed by applying a vacuum prior to adding the next solution. The samples in the PCR plate (27 µL) were added to the PBM plate and the PCR plate was washed with 90 µL H2O, which was added to the corresponding sample well. Following this, the PBM plate was placed on top of a 2 mL collection plate and centrifuged using a Heraeus Labofuge 400 R (800 rcf for 3 min, 22°C). Finally, 100 µL H2O was added to the PBM plate and elution via centrifugation was repeated. The samples were transferred to a 300 µL 96-well PCR plate and dried down using a Thermo Savant vacuum centrifuge.

S1.2 N-glycan fluorescent labeling
Plasma samples were procainamide-labeled in the PCR plate using the Hamilton robot whereas the Nglycan and human milk oligosaccharide standards were prepared manually, as previously described. 1 In order to prepare the procainamide labeling kit for the Hamilton robot-based protocol, 1.2 mL of 30% acetic acid in dimethyl sulfoxide from the kit and 1.2 mL H2O were added to the procainamide dye followed by vortexing to ensure the dye was dissolved. This procainamide solution was added to the sodium cyanoborohydride reductant and vortexed again to mix and dissolve. The solution was added to two 2 mL Sarstedt vials that were placed on the robot deck, which added 20 µL of the labeling solution to each sample. Following this, the 300 µL 96-well PCR plate was manually sealed with a pierce-foil seal. The plate was briefly vortexed and centrifuged followed by incubating the samples for 60 min at 65 °C.
A HILIC clean-up plate was used to remove an excess of the labeling reagent. The clean-up plate was placed on the vacuum manifold on the Hamilton robot and successive washes with 200 µL of 70% EtOH/ H2O (v/v), 200 µL of H2O and 200 µL of CH3CN were performed. To each sample CH3CN (230 µL) was added, aspirated and dispensed five times to mix after being transferred to the clean-up plate. The samples were eluted under gravity for 10 min before a vacuum was applied. The samples were then washed with 200 µL of CH3CN and elution was performed under gravity for 5 min before a vacuum was applied. This step was performed two more times. The clean-up plate was blotted briefly onto a paper towel in order to remove excess CH3CN before being placed back on the vacuum manifold. Following this, a 96-well 2 mL collection plate was placed inside the vacuum manifold and 100 µL H2O was added to the samples. To start the sample elution a vacuum was used for about 5 sec, followed by elution under gravity for 15 min before a vacuum was applied again to elute any remaining sample. This step was repeated in order to elute the released and labeled N-glycans in a final volume of 200 µL. Following this, 120 µL of sample was transferred to a 300 µL 96-well PCR plate and reduced to dryness using a vacuum centrifuge. The 96-well 2 mL collection plate which contained the remaining 80 µL of sample was stored at -20 °C prior to HILIC-MS analysis.

S1.3 HILIC-FD-MS
HILIC-MS was implemented to analyze the released procainamide-labeled N-glycans without derivatization. In this case, 25 µL sample and 75 µL CH3CN were added to a Waters HPLC vial followed by a 20 µL injection onto a BEH glycan 1.

S1.4 Intermediate precision and repeatability
The protocol repeatability was validated using full technical replicates prepared over three days (n = 3); intra-day 1 (n = 24), intra-day 2 (n = 13) and intra-day 3 (n = 13). Measurement of all replicates was performed in a single batch and the intermediate precision (n = 50) of the sample processing protocol could be determined.

S1.5 Fluorescence detection (FD) data processing
Fluorescent chromatograms were saved in .txt format using Chromeleon (version 7.2) and further processed using HappyTools (version 0.1-beta1, build 190115a). 2 Baseline correction was applied to all chromatograms and peak calibration was performed using the elution time of specific peaks (Supporting Information, Table S5). The following parameters were applied during data processing: general: datapoints: 100, baseline function order: 1, background window: 1; peak detection: peak edge type: sigma, peak edge value: 2; calibration: minimum number of calibrants: 4, minimum S/N: 27. Peak integration was performed using an integration window (70% FWHM) that allowed the largest peak area to be quantified while also minimizing overlap with closely eluting peaks. However, in order to support S6 automated quantification, a specific integration window (0.05 sec) was applied to some low abundant peaks in order to avoid integrating nearby and more abundant peaks (Supporting Information, Table   S5). Furthermore, overlapping peaks and peaks with large shoulders were quantified using an integration window of 0.15 sec as this covered the entire peak area of both closely eluting peaks (Supporting Information, Table S5). Additionally, peaks were only included for further analysis if the measured retention time (RT) did not deviate more than 3 sec than the expected RT, and with a S/N above 9. Finally, the average background was determined in the blank samples by calculating the average intensity at expected peak RTs. Therefore, peaks at expected RTs were required to be above the average blank intensity plus nine times the standard deviation (s.d.) of the average blank intensity.

S1.6 MS data processing
Raw RPLC-/HILIC-MS data was converted into .mzXML files prior to being processed by LaCyTools (version 2.0, build 200723). Prior to data extraction, chromatograms were aligned according to assigned m/z (Supporting Information, Table S6) that spanned the entire RT. The alignment time window and mass window were set to 20 s and 0.1 Th, respectively. A minimum of five data points with S/N > 27 were required for alignment. Sum spectra were created for nine 'clusters' containing assigned N-glycans. Each sum spectra was calibrated using a mass window of 0.1 Th and based on a minimum of three calibration points (Supporting Information, Table S6) with S/N > 9. Finally, a minimum of 95% of the isotopic pattern was extracted for [M+2H] 2+ and [M+3H] 3+ . In cases where charge states were above or below the measured mass range, no extraction was performed. Although some sodium adduct ion formation was observed (<10%), as demonstrated by Supporting Information, Figure S5, only fully protonated species were used for quantification.
Quality control criteria were determined and applied prior to further analysis. According to the instrument specifications, a m/z tolerance of ± 100 ppm was implemented. In addition, a S/N > 9 was required.
Furthermore, the average isotopic pattern quality was calculated for all glycan signals with S/N > 9 for each extracted charge state. Then, an average isotopic pattern quality for all charge states was S7 determined for each sample and, following this, the cut-off was set to the average of all charge states plus the s.d. This resulted in an isotopic pattern quality with a deviation no greater than 35% and 20% for RPLC-and HILIC-MS data, respectively. Finally, following curation, the [M+2H] 2+ and [M+3H] 3+ signals of passing N-glycan compositions were summed to provide a total area of all charge states and adjusted to represent the entire isotopic envelope.

S2.1 Method development
The investigated parameters are described in Supporting Information, Table S2, plasma N-glycans were used to perform method development, unless stated otherwise. To this end, the combination of the derivatization procedure with fluorescent labeling was examined by performing it before and after labeling. The relative abundance of the most abundant fluorescent peak was similar whether EEA was performed before or after procainamide labeling (Supporting Information, Table S2). In this regard, fluorescent labeling followed by EEA was selected for further processing as this format is more compatible with the current N-glycan analysis procedure. In this manner, N-glycans are initially labeled with procainamide and, as a result, there are several options for analysis as samples may be assessed using HILIC-FD-MS or further investigated using EEA and RPLC-FD-MS.
Improvements to the sensitivity and throughput of the protocol were also achieved. For example, the sensitivity of the protocol was enhanced by effectively increasing the amount of sample injected for measurement. This was performed by increasing the sample and associated reagent volumes used for the EEA reaction as well as concentrating the sample prior to EEA. Further gains in sensitivity were achieved by decreasing the run time on RPLC from 70 min to 35 min. This was obtained by increasing the gradient steepness, as well as the flow rate from 0.2 to 0.4 mL/min. Both gradients produced similar profiles (Supporting Information, Figure S6). However, using the 35 min gradient resulted in an increase in absolute peak areas and a decrease in peak width, whilst maintaining similar relative abundances for the ten most abundant peaks (Supporting Information, Table S7). A peak corresponding to HOBt was observed in the chromatogram (Supporting Information, Figure S6).
However, this was not detected later during method validation, possibly due to the increased precision of the sample preparation by transferring the method to the robot. Finally, sample throughput was further increased when the HILIC clean-up plate method following EEA was modified, which facilitated a platebased processing method for EEA on the robot.
As summarized in Supporting Information, Table S8, the derivatization efficiency was estimated using fluorescent peak areas, which allows quantification free from ionization bias. 3,4 Ethyl esterification efficiency (α2,6 sialic acids) following fluorescent labeling was quantified using an N-glycan (A2G2S2 (H5N4S2,62)) and a human milk oligosaccharide (LST-C (H3N1S2,61)) as standards. Overall, this showed an efficiency of 94% (A2G2S2) and 88% (LST-C) for the expected main product of derivatization.
Assigned by-products and some unassigned by-products were also detected. Assigned by-products likely arise due to some contamination with the α2,3-linkage variant or mis-derivatization (amidation) of α2,6linked sialic acids. For the remaining (unassigned) by-products, the lack of any consistent mass shift made the source of these by-products difficult to determine. The derivatization efficiency of amidated α2,3-linked sialic acids was investigated using only the human milk oligosaccharide standard, LST-A (H3N1S2,31), as a complex α2,3-sialylated N-glycan standard was lacking. In this case, a derivatization efficiency of 91% was obtained. Similarly, assigned by-products due to under-derivatization and contamination/mis-derivatization were determined, although no unassigned by-products were observed.
Despite this, no detectable amounts of expected derivatization by-products, such as under-and partialderivatization, were determined during the examination of plasma N-glycans, although further unknown by-product formation was observed (Supporting Information, Table S8). The formation of by-products are commonly observed when employing derivatization strategies. 5,6,7 However, as detailed in the Method Validation section, the formation of such by-products did not affect the performance of the protocol. In the case of unknown by-product formation, future research may focus on using negative-mode ionization or orthogonal techniques such as NMR 8 in order to perform a full identification.   In some cases, one platform detected two isomers of a structure whereas the other platform detected only a single signal, which represents the sum abundance of both isomers. Thus, this was determined as one unique and one overlapping structure for the former, and one overlapping structure for the latter, respectively.