9


Glycoproteins have arisen as promising biopharmaceutical therapeutics and diagnostic agents in recent research endeavours. Monoclonal antibodies (mAbs), for example, are used as therapeutics for several diseases. The specific structure of the N-linked glycans on these glycoproteins, including the linkage and positional isomers, are proven to have a significant impact on the effectiveness of glycoprotein therapeutics [7,11]. In light of the alterations to the efficacy of therapeutics that structural changes can have, development of reliable glycan characterisation methods is key for quality control [11,16]. Advances in research such as these have served to bring into focus the need for more sophisticated methods of glycan analysis.


A general strategy employed when analysing the structures of N-linked glycans involves releasing the glycans from the protein(s) via enzymatic or chemical digestion protocols. After the glycans are released, there are several possible methods for analysis available. A common practice consists of labelling the detached glycans with a fluorescent tag [17,19], such as procainamide (ProA) or 2-aminobenzamide (2-AB). Each of these commercially available fluorescent tags provides a number of advantages when analysing N-linked glycans [5,12,14,17- 20]. Both allow for fluorescence and UV detection, straightforward quantification, and increased sample solubility in high organic systems. Since hydrophilic interaction liquid chromatography uses high concentrations of organic solvents, the ability to dissolve samples in these solvents is necessary for LC separation. The ProA fluorescent tag is shown to provide greater fluorescence signal intensity, better labelling efficiency for minor glycan species, and it also facilitates significantly enhanced electrospray ionisation efficiency. These characteristics result in an increased MS sensitivity due to the increase in ionisation and signal intensity, and an improvement in the ability to identify minor glycan species due to the increased labelling efficiency [18-19].


There are several instruments used for the MS analysis of N-linked glycans, and the one predominantly used in this research is a triple quadrupole mass spectrometer (QqQ). While the mass resolution and range is exceeded by sector instruments, the QqQ has the benefit of having superior detection sensitivity and quantification while being efficient, easy to operate, and cheaper than some other traditional MS instruments [21]. The instrument setup of the QqQ allows


Table 1: Coefficients and statistical data obtained via multivariable linear regression for each chromatographically influencing monosaccharide represented by the retention prediction equation.


Coefficient Standard Error


Intercept GlcNAc Man Gal


-0.328 0.706 0.997 1.036


Neu5GC (3)


Neu5GC (6)


Fuc ant Fuc core


Neu5Ac (3) 1.794 Neu5Ac (6) 2.373 2.052


2.651


0.651 0.594


0.27878 0.04231 0.04465 0.06880 0.05893 0.05893 0.07617


0.07617


0.08105 0.08116


for four types of scans to be performed, and each has a specific purpose [21,22]. The scan type used for the development of the retention model is called a selected reaction monitoring (SRM) scan. During an SRM scan, the m/z values of the precursor ions in quadrupole 1 (Q1


of fragment ions in quadrupole 3 (Q3) are


used. Using both of these values allows only specific fragments from specific precursor ions to be detected, which results in greater sensitivity and a lower detection limit than precursor ion scans alone. If retention information for the N-linked glycans of interest is known, it can be incorporated into the SRM method to produce a scheduled SRM (sSRM) experiment. The addition of retention information allows for fewer transitions to be observed by the instrument at any given time, thereby increasing dwell time and providing more data points across chromatographic peaks. The integration of retention information not only results in improved accuracy and reproducibility, it also allows more analytes to be included without compromising the integrity of the experiment. While the use of SRM experiments is still relatively uncommon in glycomics research, the successful use of this technique in medical and pharmaceutical industries for proteomics research and analysis is well established [4,7,12,14,23].


Using an MS instrument alone may provide the structural identification of N-linked glycans, but obtaining specific details such as linkage identification is a much more difficult prospect. Several isomers can have the same m/z but have differences in things like the linkage position, such as glycans with a 2,3-linked versus 2,6-linked sialic acid. The incorporation of an LC method using a hydrophilic interaction


t Stat


-1.1748 16.6926 22.3268 15.0579 30.4354 40.2727 26.9418


32.1733


8.0298 7.3228


P-value Lower 95% Upper 95%


0.243418 2.95E-28 7.71E-37 1.87E-25 8.59E-47 3.01E-56 8.49E-43


1.22E-48


1.34E-13 8.36E-12


-0.8820 0.6221 0.9081 0.8992 1.6764 2.2561 1.9006


2.2991


0.4909 0.4341


0.2270 0.7904 1.0857 1.1729 1.9108 2.4906 2.2036


2.6021


0.8108 0.7544


) and the m/z values


liquid chromatography (HILIC) column into an experiment can result in separation of isomers such as these so identification is significantly easier. As glycan profiles are experimentally obtained, a database can be constructed and used to assist in identification of glycans during subsequent experiments [24,25]. Databases like these require time to develop, can only be used once a glycan profile is known, and may not provide sufficient information for isomer identification. The prediction model described here is an effective tool designed to complement the identification of isomeric glycoforms via HILIC LC-MS analysis. The model calculates retention based upon the individual influences that individual monosaccharide species have during separation, with variations attributable to linkage and position. This predictive tool allows both knowns and unknowns to be identified more readily and with greater precision.


Experimental Materials


Acetic acid, acetonitrile, alpha-1-acid glycoprotein (bovine), alpha-1-acid glycoprotein (human), asialofetuin (bovine), dextran ladder, dimethyl sulphoxide (DMSO), fetuin (bovine), human serum (human male, AB plasma), methanol, ovalbumin, procainamide hydrochloride, ribonuclease B, and trypsin (TPCK treated) were purchased from Sigma Aldrich (St. Louis, MO, US). Ammonium bicarbonate (AMBIC), ammonium formate, formic acid, and sodium cyanoborohydride were purchased from Fluka. PNGase F (glycerol free) was purchased from New England Biolabs (Ipswich, MA, United States).


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