glycoprotein characterization by hilic-ms

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MSc Chemistry

Track Analytical Sciences

Literature Thesis

Glycoprotein Characterization by HILIC-MS

An overview on HILIC separation methods for intact glycoproteins,

glycopeptides and released glycans

by

Ditte Bijlmakers

UvA: 11812060 VU: 2630062

January 2020

12 ECTs

November 2019 – January 2020

Supervisor/Examiner: Examiner:

dr. A.F.G. Gargano dr. R. Haselberg

Van ‘t Hof Institute for Molecular Sciences

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Abstract This study summarizes recent advances in the characterization of glycosylated amino acids, peptides and proteins. In this process, termed as glycosylation, glycans are covalently attached to the amino acid backbone of a protein, and is commonly studied in post-translation modifications (PTMs). Glycosylation has gained more interest in the clinical field due to various roles in structural and functional biological processes. Recently, hydrophilic interaction liquid chromatography (HILIC) is an interesting alternative for reversed-phase liquid chromatography (RPLC). Stationary phases in HILIC analysis are mostly derivatized silica containing neutral (diol, amide or cyano), basic (amino) or zwitterionic (sulfobetaine) groups, and are used for the separation of polar or ionic compounds. Various studies demonstrate the usefulness of amide and zwitterionic phases for the separation of glycoproteins, glycopeptides and released glycans. Glycans structures are very diverse, resulting in structural heterogeneity. Their structure can be closely related (e.g. isomers) and their detailed characterization is challenging. Glycoforms may not be in high quantity or suffer of low ionization performance. Hence mass spectrometry and fluorescence methods are often employed. In addition to allow the study of low abundance glycoforms, enrichment approaches are used. Detailed knowledge of glycosylated proteins helps to understand their specific function, providing novel insights in target analysis and biomarker development. This study describes recent advances in glycoprotein characterization at various levels using HILIC with special attention to technical aspects, including the effect of the mobile phase, stationary phase, column temperature and detection.

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Abbreviations BPC base peak chromatogram CE capillary electrophoresis DTT dithiothreitol ECD electron capture dissociation EndoH Endoglycosidase H ETD electron transfer dissociation EThcD electron-transfer/higher-energy collision dissociation GalNac N-acetylgalactosamine GlcNac N-acetylglucosamine HILIC hydrophilic interaction liquid chromatography IdeS immunoglobulin-degrading enzyme LC liquid chromatography mAb monoclonal antibody Man mannose MALDI matrix assisted laser desorption ionization MS mass spectrometry MS/MS tandem mass spectrometry MSn high order tandem mass spectrometry NPLC normal phase liquid chromatography PA 2-aminopyridine PIL polymer ionic layer PNGaseF peptide N-glycosidase F PTM post translational modification rhEPO recombinant human erythropoietin rhIFN-β – 1a interferon-beta-1a RPLC reversed phase liquid chromatography TFA trifluoracetic acid TOF time of flight tR retention time UV ultraviolet UVPD ultraviolet photodissociation XIC extracted ion chromatogram ZIC zwitterionic phase

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Table of Contents 1. Introduction ....................................................................................................................................... 4

2. Theoretical Background...................................................................................................................... 5

2.1 Analysis of Protein Glycosylation .................................................................................................. 5

2.1.1 Analysis of Released Glycan ................................................................................................... 5

2.1.2 Peptide Level Analysis and Middle-up ................................................................................... 7

2.1.3 Analysis of the Intact Protein ................................................................................................. 7

2.2 Hydrophilic Interaction Liquid Chromatography .......................................................................... 9

3. Results & Discussion ......................................................................................................................... 11

3.1 The Effects of Mobile Phase Solvents and Additives to the Retention in HILIC .......................... 11

3.2 Column Temperature ................................................................................................................. 13

3.4 Stationary Phases used in HILIC for Glycoform Analysis ............................................................. 14

3.4.1 Amide phases ...................................................................................................................... 15

3.4.2 Zwitterionic phases.............................................................................................................. 20

3.4.3 Overview of HILIC Characterization of Glycoforms .............................................................. 23

3.5 HILIC vs RPLC .............................................................................................................................. 28

3.6 HILIC for Sample Enrichment of Glycoforms............................................................................... 30

3.6.1 Glycoprotein Enrichment ..................................................................................................... 30

3.6.2 Glycopeptide Enrichment .................................................................................................... 31

3.6.3 Glycan Enrichment .............................................................................................................. 34

3.7 Detection of Glycoforms............................................................................................................. 37

3.8 Applications of HILIC to Glycoform Investigations ...................................................................... 38

4. Conclusion ........................................................................................................................................ 39

5. References ........................................................................................................................................ 40

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1. Introduction Glycoproteins are polar macromolecules with a molecular weight up to MDa (e.g. Mucin-16) [1]. Two-third of proteins in the human body are glycosylated into glycoproteins [2]. Glycosylation is an enzymatic process where glycans are covalently attached to the amino acid backbone of a protein [3,4]. Protein glycosylation is commonly studied in post-translation modifications (PTMs), and plays a various role in structural and functional biological processes as cellular activities and intercellular communication [3,5,6]. Glycosylation influences the cellular machinery and is a complex process and therefore an interesting target in proteomic research. Glycosylation has been extensively studied in diseases, and can be associated with cancer and neurological diseases [7]. Understanding protein glycosylation can give a broader insight into the progression and genesis of diseases, for example in prostate cancer, and will be discussed in section 3.8. Additionally, glycosylation patterns are important as the majority of biopharmaceuticals are glycosylated and variation in the glycosylation profile may influence the activity and/or stability of the product [8,9]. Therefore, sensitive and selective methods are fundamental for the structure sequencing of glycoproteins and understanding the functional significance of glycosylation. Recently, the use of hydrophilic interaction liquid chromatography (HILIC) analysis in glycoprotein research has gained more attention, because of the capacity of resolving glycoforms of glycoproteins at different levels (intact, middle-up, bottom-up and released glycans) [10]. Glycoprotein characterization can be performed at three different levels, namely: studying the intact glycoprotein, glycopeptide and released glycans (Table 1). The analysis of the intact protein can offer information on the glycoform or proteoform, as well as on the pairing of glycans at the protein backbone [11]. The analysis of glycopeptides typically involves protein digestion (trypsin or IDES), providing information on the composition of the glycan, the glycosylation site and the peptide sequence. Released glycan analysis can provide insights in the glycan structure glycosidic linkages [11]. These three levels of glycosylation analysis all provide different (structural) information. Table 1 – Glycosylation analysis at three levels using LC-MS – formatted from Zhang et al. (2016) [11]

Intact protein analysis

• Glycoforms

• Proteoforms

• Glycan “pairing"

Glycopeptide analysis

• Peptide sequence

• Glycan compositions

• Microheterogeneity

Released glycan analysis

• Quantitative glycan distribution

• Glycan linkage sites

The number of HILIC publications in glycoproteomic research has continuously grown since 2010 (Figure 1). The use of organic solvents makes HILIC compatible with mass spectrometry (MS). This detection method provides structural information on the intact protein, as well as glycopeptides and released glycans. Recent developments in MS instrumentation gained increased interest in HILIC-MS analysis. In this review the different HILIC approaches are discussed to characterize glycoproteins at various levels, looking at methods to study intact proteins, large fragments, peptides and glycans. First, we shortly discuss the relevance of glycoprotein analysis, followed by a discussion on the separation using HILIC and its relevance in glycoprotein analysis. Subsequently, we discuss the use of HILIC as enrichment technique, HILIC separation looking at the types of stationary phases used for the different levels of characterization of glycoproteins, and conditions that influence retention and selectivity. Next, the detection and applications of glycoproteomics are evaluated. Last, different studies using HILIC for glycoproteins characterization at various levels are compared, followed by concluding remarks on the use of HILIC for glycoprotein separation.

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Figure 1 – Number of publications per year of Scopus search results of “Hydrophilic Interaction Liquid Chromatography” and “Glycoprotein”

2. Theoretical Background

2.1 Analysis of Protein Glycosylation The total number of glycosylated proteins in the human body is enormous. As discussed in the introduction, glycosylation plays various roles in biological processes. The type of glycans exposed by a cell influence their functioning and play a role in the immune system [12]. Due to the large size of the protein backbone, glycans have multiple linking sites, resulting in a variety of structural variations. This can lead to multiple glycoforms, where different glycans are attached to identical proteins and play different cellular roles [12]. This makes the identification of glycosylation sites challenging. There are two main approaches for the determination of glycosylation sites. The first approach involves the release of the glycan from the peptide backbone, also termed as glycomics. However, this method does not provide site-specific information on the glycosylation site, since the information on the attachment site is lost in the process of glycan release from the protein [13]. Conversely, in the other approach termed as glycoproteomics, glycans are not released from the peptide and provide information on the glycosylation sites [13]. Now, we have three levels of analysis of glycoproteins, namely characterization of the released glycan, analysis of the glycopeptide and the intact glycoprotein. Next, these three levels will be discussed.

2.1.1 Analysis of Released Glycan The two most commonly types of glycosylation occurring are either N-linked and O-linked [3]. In N-glycosylation, glycans are covalently linked to the amide residue of the amino acid asparagine (Asp). The structure of N-glycans is composed of two N-acetylglucosamine (GlcNac) and three mannoses (Man) (Figure 2). These structures can be classified into three groups: high mannose, hybrid, or complex (Figure 3) [14]. The glycan structure of high mannose structures only contain mannose groups attached to the amino acid GlcNac. The hybrid glycan type contains both mannose, galactose (Gal) and GlcNac groups. The complex glycan structures contains beside mannose, Gal and GlcNac, also a sialic acid groups and is therefore more complex than the other two glycan structures.

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Figure 2 – N- and O-linked glycosylation of a protein backbone using monosaccharides [12]

Deglycosylation techniques are used to enzymatically remove the glycan from the peptide. Peptide N-glycosidase F (PNGaseF) is commonly used to enzymatically remove N-glycans from the peptide backbone [13,15]. This amidase cleaves between the glycan and the Asp residue, removes the intact N-linked glycan by hydrolyzation, and keeps the peptide backbone intact (Figure 3) [2,16]. Consequently, the Asp residue is converted into aspartic acid [17]. Another way to release the N-glycosylation site is by using the enzyme endoglycosidase H (EndoH). This enzyme releases the N-glycan from the glycopeptide. Differing from PNGaseF, EndoH leaves a GlcNac core at the peptide (Figure 3), which can provide information on the composition and location of the glycan [13,15].

Figure 3 – Glycosidase of N-linked glycan structures [18]

In contrast, glycosylation in O-linked proteins occurs at the hydroxyl group of serine (Ser) or threonine (Thr) [2–4]. Usually, O-linked glycans are removed after the removal of N-linked glycans by PNGaseF. O-linked glycans can be released from the peptide using β-elimination in the presence of dithiothreitol (DTT) [13,19]. This removal is carried out using a reducing agent, where the glycan is cleaved and released from the attached N-acetylgalactosamine (GalNAc) [20]. Structure determination of O-linked glycans is more challenging due to different possibilities of core sugar structures [17]. Due to the complex and intact structure of glycoproteins, characterization can be a great analytical challenge [3]. No methodology on its own can provide all necessary information in glycan analysis. Hence, the need of orthogonal techniques (e.g. HILIC-MS) to obtain a full structural analysis of glycans [15].

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2.1.2 Peptide Level Analysis and Middle-up Glycopeptide analysis and identification of the glycosylation site, can also be termed as site mapping [13]. Site mapping provides information on the peptide sequence of the glycosylation sites. The analysis of glycopeptides typically involves digestion. In glycoprotein studies for the analysis of immunoglobulins the protein digestion is usually performed using trypsin or immunoglobulin-degrading enzyme (IdeS) [19]. Here we focus on monoclonal antibodies (mAbs), because this category presents a major group of therapeutic drugs based on glycoproteins [11]. IdeS cleaves at the upper and lower region of mAbs and specifically digests below the hinge, resulting in the release of F(ab)’2 and Fc/2 fragments (Figure 4) [19,21]. This approach is named middle-up digestion. Furthermore, the sample is typically reduced using DTT, generating the Fd’ fragment and the LC subunits from F(ab)’2. The residual glycans or glycopeptides are analyzed to obtain detailed information of the glycosylation site [19]. Analysis of the resulting protein fragments can provide structural information, including multiple modifications (protein sequence and glycans). However, glycosylation analysis is challenging, due to the higher molecular weight of the species analyzed and specific glycoprotein combinations may not be revealed (e.g. correlation between glycosylation on Fd’ and Fc/2) [2,4].

Figure 4 – Middle-up approach using IdeS and DTT to get information of the glycosylation side [19] (IdeS = immunoglobulin-degrading enzyme; DTT = dithiothreitol)

A more common and general approach to study glycoproteins is by proteolytic digestion using trypsin. Trypsin cleaves at the carboxyl site of the amino acids lysine and arginine (with exception of proline), resulting in a mixture of peptide fragments. This technique provides an indirect measurement of peptide fragments derived from digested proteins [22]. In this analysis, the protein tryptic digest is subjected to LC combined with tandem mass spectrometry (MS/MS) for peptide identification. This identification is performed by comparing the obtained MS/MS spectra with a peptide database [23]. The protein is digested into small and variable peptide fractions. Therefore, a low percentage can be recovered of the total protein sequence. However, these peptide fractions can provide a significant amount of information on PTMs [22,23].

2.1.3 Analysis of the Intact Protein In contrast to bottom-up analysis, top-down proteomics is used to characterize the intact protein. With this approach, the proteins can be analyzed with LC-MS/MS or fractionated by LC and analyzed by offline electrospray ionization mass spectrometry (ESI-MS). The combination of intact mass measurements with fragmentation methods such as electron transfer dissociation (ETD), ultraviolet photodissociation (UVPD), electron-transfer/higher-energy collision dissociation (EThcD) allows in some cases to obtain nearly full sequence coverage [24]. When analyzing intact proteins, PTMs can be localized in the protein sequence and characterized. However, top-down analysis has some limitations compared to bottom-up analysis mainly arising from the higher molecular weight of the molecules analyzed. For example, peptide fragments are more easily separated by LC than peptides as well as their ionization and fragmentation is more complicated and less effective [22]. Figure 5 displays a schematic comparison between bottom-up and top-down proteomics.

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Figure 5 – Strategies for bottom-up and top-down analysis [23]

There are two MS approaches to analyze the intact (glyco)protein, namely denaturing and non-denaturing MS [25]. Denaturing conditions (e.g. low pH) disrupt the tertiary and quaternary protein structures. In contrast, native MS avoids specific loss of the protein organization in higher-order structures. In native MS analysis, proteins are kept in their original structure. This technique reveals functional information on glycoproteins, including structural information, protein-protein interactions and ligand binding [25]. As the folded structure of proteins is kept, the charge state distribution is narrower during ionization compared to denaturing MS analysis. This allows for a large space between different charge states, allowing for better separation of the masses of the glycoforms of highly heterogeneous proteins (Figure 6). However, the downside is that native MS analysis requires specialized expertise, because this is a low-throughput technique, resulting in challenging data interpretation due to possible isomers [25].

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Figure 6 – Structural protein heterogeneity in denaturing and native MS [25]

2.2 Hydrophilic Interaction Liquid Chromatography For the analysis of intact glycoproteins (native separation), glycopeptides and released glycans (denaturing separation), a variety of analytical methods have been reported. These analysis are mostly performed using capillary electrophoresis (CE) or liquid chromatography (LC) coupled to a mass spectrometer (MS) for mass determination and characterization of the intact protein. More recently, HILIC is considered as an attractive technique for the characterization of glycoforms [26]. The complex structural heterogeneity of intact proteins requires characterization with enhanced analytical methods [9]. HILIC has been widely used for the separation of release glycans, glycopeptides and more recently glycoproteins and glycoprotein fragments from middle-up digestion. The interaction (polar and/or electrostatic) between different glycan structures (e.g. number of OH) and conformation (linkage type or different order in the glycan structure) drives the selectivity of this technique. This allows reduced sample complexity prior to detection (e.g. MS) including in some cases the resolution of isobaric species [27]. Next we describe the general principles of the separation by HILIC. In HILIC, the separation is performed using a polar stationary phase with relatively polar mobile phases (ACN to water). The retention mechanism of HILIC arises from the partitioning of the analytes between the mobile phase and the water enriched stationary phase, as well as adsorption to the stationary phase and/or ion exchange interactions [28]. The partitioning of hydrophilic analytes is performed between the hydrophilic stationary phase and hydrophobic elution buffer, creating an aqueous layer. The

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separation mechanism of HILIC relies on the hydrophilic interactions between the analyte and the stationary phase. Compound elution is obtained increasing the water content in the mobile phase [29]. In the analysis of small molecules, the octanol-water distribution index (logDO/W) can be used as an indicator of the hydrophilic character of a compound. Compounds with a low hydrophilic character (high logDO/W), have typically low chromatographic retention [30]. Depending on the stationary phase, different interaction mechanisms take place, such as dipole-dipole interaction, hydrogen bonding, and electrostatic interactions [31]. The stationary phases used in HILIC are mostly based on silica (modified or non-modified) and having a polar surface chemistry, such as amide, diol, cyano, or zwitterionic sulfoalkylbetaine/phosphocholine [31]. With HILIC, both polar and ionized solutes can be separated [28]. This is an advantage compared with reversed-phase liquid chromatography (RPLC), where polar and ionic solutes have almost no retention. Additionally, lower back-pressure is observed in HILIC, because of the lower viscosity generated by mobile phases rich in acetonitrile. The use of volatile organic mobile phases provides desolvation in ESI, increasing sensitivity in MS detection [31]. Generally, buffers are used to increase the ionic strength and control the pH in the mobile phase. Depending on their concentration, the addition of buffer salts in the mobile phase, disrupt the electrostatic interactions. The choice of buffer salts is however limited, because salts are difficult to dissolve in highly organic HILIC buffers [29,32]. Buffer salts such as ammonium acetate or ammonium formate are mostly used in HILIC since they are highly soluble in acetonitrile and compatible with MS. The addition of buffer salts affects the separation in HILIC, and the addition of an acid (e.g. formic acid, acetic acid, or trifluoracetic acid (TFA)) to the mobile phase, can improve peak shapes, especially when basic analytes or analytes with basic groups are analyzed (ion-pair) [32]. Moreover, salts can enrich the aqueous layer (increasing the “polarity” of water), increasing the hydrophilicity of the stationary phase, and results in increased retention [29]. Another factor that influences the separation mechanism in HILIC, is the water-absorbing capacity of the stationary phase. Dinh (2013) and coworkers [33] evaluated the role of water-enriched layers in polar stationary phases in HILIC retention mechanisms. Lower water uptake in the stationary phase corresponds to lower retention, because of competition for polar interaction sites. Acetonitrile is an aprotic solvent and therefore a more suitable mobile phase in HILIC than methanol. Methanol is both a proton donor and acceptor which discourages hydrophilic partitioning, resulting in reduced retention [33,34]. Changing the pH of the buffer in the mobile phase can manipulate the retention characteristics of ionizable compounds, which can modify the selectivity of the stationary phase [29,35]. In HILIC, retention of peptides and proteins is mostly based on hydrogen bonding and electrostatic interactions [31]. Glycoproteins are molecules with a relatively large molecular weight, resulting in complex fragmentations derived from cleavage of the peptide and glycan. However, HILIC is commonly applied for the characterization of glycans or peptides, however rarely for the analysis of intact proteins. It is believed that the main reason is the number of hydrogen donor and acceptor groups, resulting in possible side chain charges. This makes the elution of proteins hard, since they intent to stick at the stationary phase [31]. Moreover, the use of high organic solvent concentration in HILIC can result in protein precipitation [36]. Furthermore, the characterization of glycosylation is difficult, because of the heterogeneity of glycans. As a result, low signal intensity is observed for glycopeptides. Glycopeptides are not chemically or enzymatically modified for HILIC enrichment/ separations, making HILIC applicable to in-depth glycoproteomic studies. Therefore, deglycosylation or enrichment steps are often required [29]. Practically, HILIC is an interesting alternative for normal phase liquid chromatography (NPLC), because in HILIC a relatively high percentage organic solvent (e.g. acetonitrile) is necessary for compound elution, which makes HILIC more compatible with MS due to increased ionization efficiency. Furthermore, sample preparation can be simplified due to direct column injection, and mobile phases

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used in HILIC are similar to mobile phases used in RPLC [37]. The development of robust methods using HILIC is often more challenging than typical RPLC. One of the things to keep in mind is to maintain long column equilibration (e.g. at least 10 column volumes) [38]. Yet, full column equilibration can be facilitated using columns with relatively large pore sizes or using column temperatures above ambient [38]. Moreover, the use of acetonitrile can be difficult to dissolve polar compounds into the mobile phase. In contrast, the injection of large amounts of water can lead to peak distortion. Using a smaller injection volume reduces this problem. However, using small injection volumes can cause problems in quantification studies/analysis of low abundance compounds [28]. This study covers the use of HILIC for the separation of intact glycoproteins, glycopeptides and released glycans. Also insights in selectivity, sensitivity, and enrichment of HILIC approaches are discussed.

3. Results & Discussion This study describes the mobile phase conditions, column temperature effects, and the use of stationary phases for the characterization of different glycoforms, including intact glycoproteins, glycopeptides and released glycans. These parameters give insight about the recent separation approaches used in glycomics and glycoproteomics. Different HILIC columns are used for the separation of glycoforms. However, optimization is required to provide optimal separation with reasonable retention times. In this chapter, we discuss the use of HILIC characterization of different glycoforms.

3.1 The Effects of Mobile Phase Solvents and Additives to the Retention in HILIC The composition on the mobile phase has an extreme effect on retention in HILIC. Mobile phases in HILIC contain organic and aqueous solvents. Organic solvents are weak eluents in HILIC and used to focus polar compounds onto the column. Acetonitrile is preferred in HILIC separation due to its aprotic properties. Gradients in HILIC normally start with a concentration of 95% acetonitrile and 5% of an aqueous buffer. The elution strength of the mobile phase can be adjusted by increasing acetonitrile concentrations to achieve desirable retention. To elute polar compounds, the water content is increased. In addition, pH differences in the mobile phase influence selectivity. However, high percentages of acetonitrile suppress sample ionization, meaning differences in retention time as a result of pH are not desirable [39]. As previously discussed, buffers increase the ionic strength and control the pH in the mobile phase. Retention is affected by the pH in the mobile phase when it changes the ionization state of the analyte (protonated or deprotonated) [40]. Whereas buffer salts influence the separation of polar compounds in HILIC. For example, retention is mostly increased with higher salt concentrations by enhanced hydrogen bonding between the analyte and the stationary phase. This can be termed as the ‘salting-out’ effect. Nevertheless, increased ionic strength can decrease retention on some stationary phases (e.g. amino phases). On the other hand, increased ionic strength by addition of, for example, formic acid or TFA, provides better peak shapes [39,41]. Thus, high buffer(salt) concentrations are not desirable in HILIC-MS, because of ionization suppression in electrospray ionization (ESI). The use of ammonium formate in the mobile phase affected the charge state distribution of peptides according to Guan et al. (2019) [42]. The use of additives in the mobile phase increased ionization efficiency, and therefore provided better peak shapes. The effect of additives in the mobile phase in HILIC separation of peptides was evaluated by Roca et al. (2019 – In Press, Corrected Proof) [43]. In this study, the effect of mobile phase additives (10 mM ammonium acetate pH 6, 10 mM ammonium formate pH 3, and 0.3% formic acid) was investigated on three different stationary phases (amide and two silica columns) for the separation of peptides. In the mobile phase, acetonitrile/water with one of

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the additives was used. The amide column showed best performance using formic acid as an additive in the mobile phase, due to the highest signal intensity and sufficient resolution [43]. For the separation of peptides using a Zorbax silica column, ammonium formate was considered to be the optimal additive. The use of ammonium acetate as additive provided the most optimal separation using the Waters silica column [43]. These studies provide valuable insight into the use of additives and buffers into the mobile phase using different stationary phases in HILIC separation. Subsequently, selectivity differences between stationary phases are widely studied in the field of HILIC separation. McCalley et al. (2017) studied the retention behavior of different HILIC phases. The different physico-chemical properties provide various interactions, resulting in different interactions in HILIC separation. However, polar adsorption is considered as the main mechanism in HILIC [28]. Figure 7 illustrates the influence of mobile phase conditions on chromatographic HILIC separation. The separation was performed under the same chromatographic conditions on different stationary phases (diol, amino and ZIC). Differences in chromatographic separation are observed at increased salt concentration. Salt increases the ionic strength in the mobile phase. Therefore, an increase in retention is observed for the neutral (diol) phase. Separation on a ZIC column (sulfobetaine) is more likely based on hydrophilic interactions than electrostatic interactions. Therefore, the increase of salt concentration (from 10 to 15 mM ammonium acetate) has less effect on zwitterionic material than neutral (diol) and charged (amine) phases [44]. In contrast, this means that an increased water content has more effect on the separation using a ZIC column. This shows that HILIC separation can be manipulated to create a satisfying separation of polar compounds. Therefore, no optimal mobile phase composition was found in literature for the analysis of the intact glycoprotein, glycopeptides and released glycans. Later in the discussion, an overview of mobile phase compositions will be given for the separation of all three glycoforms (Table 4).

Figure 7 – The influence of mobile phase conditions on chromatographic separation of acidic compounds [44]

Mostly, ammonium salts (e.g. acetate, formate or bicarbonate) are used in the mobile phase. These salts have different impact on the retention on different columns. This effect was best observed on

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the retention time of model compounds (salicylic acid, aspirin and cytosine) by Guo and Gaiki (2005) (Table 2) [40]. Ammonium acetate was replaced by ammonium formate, resulting in small changes in retention time of polar compounds on different HILIC columns (i.e. TSKgel Amide 80, HILIC Silica, and ZIC). However, this replacement led to increased retention on an amino column. This was explained due to competing ions (acetate and formate) and their elution strength. In addition, acids showed a drastic decrease in retention time on all columns when ammonium bicarbonate was used in the mobile phase. This led to deteriorated separation [40]. In this study, retention increase was observed on stationary phases with different functional groups, indicating that electrostatic repulsion was not the only factor that influences the retention time. Hydrophilic partitioning processes were also responsible for retention increase. Additionally, stronger retention of the solutes was created by higher salt concentrations, increasing the volume of hydrophilicity of the liquid layer. Table 2 – Effect of ammonium salts on the retention time of model compounds on different columns using acetonitrile with 10 mM ammonium salt solution (85/15, v/v) [40]

Retention models (e.g. Neue-Kuss, adsorption, mixed-mode, exponential, and quadratic models) and the retention factor (k) can be useful for retention prediction based on solvent strength [43,45]. These models include separation factors such as the compound elution time, the column hold-up time (elution of unretained compounds), and the solvent elution strength factor. Increased acetonitrile content shows longer retention for polar compounds in HILIC [39]. A small increase in water content in the mobile phase results in a significant decrease in retention, because water is a strong eluent in HILIC. In contrast, retention increases with decreased acetonitrile concentration. The use of retention prediction models always contain an error. Jin et al. (2008) [46] studied different retention models on varying stationary phases used in HILIC. They predicted retention times with a relative error lower than 5%. Using retention prediction models, can be helpful for the optimization on a given chromatographic system.

3.2 Column Temperature Variation in column temperature has less effect on the retention than the mobile phase composition. Separation selectivity is significantly affected by the temperature of the column. These changes are mostly a result of analyte-stationary phase interactions [47]. Positive effects were found on chromatographic efficiency using increased column temperature, resulting from lower mobile phase viscosity and higher diffusion [37,39]. Increased temperature in HILIC usually decreases retention as a result of reduced hydrogen bonding, polar interactions, and differences in cohesive energy. Additionally, temperature affects the transformation rate of isomers. Split peaks, as a result of isomers, can become a single peak when different isomer configurations of the same analyte cannot be distinguished [47]. Also, peak resolution can be improved using higher column temperatures, because increased temperatures make the analyte retain longer on the column, resulting in greater retention differences between analytes [47].

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Figure 8 – van ‘t Hoff plot for Aspirin: Column temperature effect upon retention using 90% ACN (v/v) with 10 mM

ammonium acetate (▪) YMC-Pack NH2 ,( ) HILIC Silica, ( ) TSK gel Amide-80, and (x) ZIC-HILIC columns [40]

Temperature changes affect the retention factor (k) and analyte transferring enthalpy [39]. The van ‘t Hoff equation describes the retention factor and can be used to predict changes in retention using different column temperatures [48]. Figure 8 illustrates the effect of the column temperature on the retention of aspirin using different stationary phases in HILIC. The van ‘t Hoff plot shows different effects of column temperatures on the retention using the amine column. Generally, decreased retention was observed with increasing column temperatures. A small increase in retention was shown on the silica, amide, and ZIC column with increasing temperatures. These differences can be explained due to the fact that the retention process on the amine column was endothermic opposed to the other columns (i.e. silica, amide, and ZIC) where the retention process was exothermic [40]. Due to the use of different stationary phases in HILIC, column stability under increased temperatures has not been systematically studied. However, bare silica columns are believed to be more stable than derivatized silica columns [47].

3.4 Stationary Phases used in HILIC for Glycoform Analysis In HILIC the choice of the stationary phase has a significant effect on selectivity and retention of the resulting chromatographic method. As discussed in section 2.2, the retention of glycoproteins depends on the type of used stationary phase, hydrogen bonding, dipole-dipole or electrostatic interactions, as well on the glycan size and hydrophilic properties of the peptide backbone [34]. In HILIC separation, chromatographic conditions influence selectivity and retention, i.e. (i) column dimension (stationary phase), (ii) mobile phases, (iii) salt concentration, (iv) column temperature, (v) analyte choice, and (vi) octanol-water partition coefficient [30]. This section covers the commonly used stationary phases in HILIC for the separation of glycoforms (i.e. intact glycoproteins, glycopeptides and glycans) (Table 4). First, we describe the use of non-modified silica phases. Silica stationary phases are widely employed in HILIC to separate small polar compounds, because of the combination of hydrophilic interaction, ion-exchange, and reversed-phase retention. Silica phases operate in pH ranging from 2.0 to 8.0. Using mobile phases below pH 2.0 will result in hydrolysis, however operation levels above pH 8.0 will result in dissolution of the silica particles. Silica phases contain negatively charged silanol groups, providing partitioning between the polar analyte and the adsorbed water layer [35]. Therefore, silica phases have strong cation exchange selectivity, resulting in longer retention of protonated bases than neutral compounds of similar hydrophilicity. Hence, silica phases lack of anionic selectivity [28]. Bare silica and aminopropyl columns both have limited stability in aqueous phases used in HILIC, resulting in dissolution and hydrolysis. Additionally, these two columns can both lead to irreversible adsorption of the analyte to the stationary phase [49].

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Table 3 – Common stationary phases used in HILIC

Column name Bonding type

Silica

Amide (BEH or TSK-Amide)

Diol

Cyano

ZIC (sulfobetaine)

Neutral stationary phases are more common in HILIC, including amide and diol stationary phases. The functional groups of neutral phases are uncharged. However, silanol groups can be deprotonated at a pH above 4-5, resulting in negatively charged functional groups [37]. Neutral 2,3-dihydroxypropyl (diol phases) or cyano-propyl (cyano phases) ligands are chemically bonded to the silica gel surface. Diol phases are commonly used to separate low molecular phenolic compounds in HILIC and RPLC [39]. Nevertheless, diol phases are rarely used for the separation of glycoproteins. Applications of diol- or cyano phases in HILIC of glycoforms are rare. Probably because these columns have a reduced hydrophilicity and hydrogen bond donor capabilities [28]. In contrast, stationary phases modified with amide groups and zwitterionic groups are commonly applied in HILIC for glycoform analysis. In this section, the use of amide and zwitterionic stationary phases in HILIC for the separation of glycoforms is evaluated. Some studies that were found interesting are described in this section. An overview of all evaluated studies on stationary phases (amide and zwitterionic phases) are summarized at the end of this section in Table 4.

3.4.1 Amide phases Recently, new amide phases have been developed and applied in HILIC. In this type of stationary phase, amide or carbamoyl groups are attached to a silica surface and linked either through a proprietary or propyl linker [37]. Amide stationary phases have no basic functional groups, in contrast to amino phases. Therefore, the retention of ionizable analytes is not based on ion-exchange interaction. Hence, no ionic mobile phases are required using buffer salts, making this stationary phase highly useful to combine with MS. Ethylene bridged hybrid (BEH) and TSK-gel Amide 80 are amide columns specifically designed for HILIC separation [39]. As shown in Figure 9, amide columns are mainly applied for the separation of intact glycoproteins, thanks to their reduced ion-exchange character and strong hydrophilicity properties [39].

16

Figure 9 – Pie chart of amide stationary phases used in HILIC characterization of glycoproteins, glycopeptides and glycans –

Based on 18 studies (Table 4)

Amide stationary phases are less sensitive to pH changes, therefore less (buffer)salts are needed into the mobile phase [39,50]. The reduction of salts/buffers into the mobile phase, is a favorable characteristic as it allows for increased sensitivity of LC-MS methods. However, the use of additives (e.g. formic acid or TFA) can improve peak shapes. The use of TFA instead of ammonium salt buffers (e.g. ammonium formate) in the mobile phase shows anion exchange behavior on amide columns, resulting in increased retention of strongly acidic solutes (Figure 10) [28]. This increase in retention may be caused by interactions (e.g. hydrolysis) between acetonitrile and TFA, producing charged products that may provide retention of acidic solutes. Next we discuss the use of amide columns for the HILIC analysis of intact glycoproteins, glycopeptides and released glycans.

Figure 10 – Chromatograms of HILIC analysis of acidic (red), basic (blue) and neutral (green) compounds on an Aligent amide column using 95% acetonitrile with either 0.1% TFA or 5 mM ammonium formate (AF) pH 3.0 [28]

56%

17%

27%

Amide Phases

Glycoprotein Glycopeptide Glycan

17

Separation of Intact Glycoproteins and Large Protein Fragments HILIC is widely used for glycopeptide and released glycan analysis. However, the use of HILIC for analysis of the intact protein has recently proven to be an interesting alternative to RPLC [14]. In several cases it has been reported the use of separation methods to resolve proteins of a sequence and molecular weight, is based on the amount of neutral sugars present in the glycosylation portion of the glycoprotein. One relevant example is the study of Zhang et al. (2013) [27] used a strongly aqueous layer on silica particles created by a brush layer of polyacrylamide (PAAm) for the separation of intact glycoproteins. This brushed layer was attached to the silica particles and swell in particle size by increased water content, making this layer extremely aqueous. Due to this extreme aqueous layer, a PAAm column was effective for HILIC. The brushed layer excluded proteins and allowed the carbohydrate moiety (glycan) to enter the stationary phase [27]. Since glycoforms differ by single mannose groups (Figure 4), isomers of glycans were easily resolved as illustrated in Figure 11. The use of a PAAm column showed good absorptivity of the glycan. Furthermore, peaks due to isomer glycans were resolved using relatively small particles (dp ~700 nm). Zhang stated that separation of glycoforms of ribonuclease B was improved using TFA combined with formic acid in the mobile phase. Despite TFA can cause ion suppression, combining a small amount of TFA with formic acid creates the ability to couple HILIC with MS [27]. However, detection with MS showed less resolution compared with UV detection. Due to the use of ~700 nm non-porous particles, there was no diffusion of intra-particles that affected the C-term in the Van Deemter equation [51]. Optimization of the PAAm packing is a significant progress in the use of HILIC. Increasing packing homogeneity and capacity for PAAm, makes this amide column valuable for complex glycoproteins.

Figure 11 – Separation of glycoforms of ribonuclease B using the PAAm column in HILIC-MS and 0.02% TFA combined with 0.5% formic acid in the mobile phase (water with acetonitrile) (PAAm = polyacrylamide; TFA = trifluoracetic acid) – Adjusted figure from Zhang et al. 2013 [27]

The use of amide columns for the separation of intact proteins continues in other studies. For instance, Pedrali et al. (2014) [52] used an amide column coupled to an UV detector for the characterization of intact Neo-glycoproteins, using RNase A as model protein. Furthermore, Tengattini et al. (2017) [53] used different amide columns (TSKgel Amide-80, XBridge BEH and AdvanceBio Glycan Mapping) for the separation of RNase A and RNase B. Then, a study of Domínquez-Vega et al. (2018) [4] used a superficially-porous amide HILIC stationary phase for the analysis of intact glycoproteins. In this study, interferon-beta-1a (rhIFN-β – 1a) and recombinant human erythropoietin (rhEPO) were analyzed. These glycoproteins present complex glycosylation, because of their variety of glycans. rhIFN-β – 1a contains one N-glycosylation site, and rhEPO has three N-glycosylation sites and one O-glycosylation site. rhIFN-β – 1a was directly analyzed with HILIC. In the base-peak chromatogram (BPC), one intensive peak was observed for rhIFN-β – 1a, which was confirmed with a main peak in the mass spectrum at the same retention time and the corresponding mass. Various glycoforms of rhIFN-β – 1a were

18

observed in the extracted-ion chromatogram (XIC) as illustrated in Figure 12. This separation showed increased retention with glycan size [4]. Furthermore, PTMs of the most abundant glycoform (tR 5.5 minutes) were observed in the XIC. These PTMs included succinimide, loss of N-terminal, deamidation, and sulfation of the glycan. Retention in HILIC was relatively decreased or increased due to these modifications.

Figure 12 – (left) XIC of glycoforms for rhIFN-β – 1a analyzed with a superficially-porous amide HILIC stationary phase; and (right) summarized obtained HILIC-MS data (XIC = extracted ion chromatogram) [4]

Glycoform profiling of aqueous rhEPO was also performed by direct analysis with HILIC. In this analysis, peaks were partially resolved. The most intense peak could be assigned to rhEPO with a glycan composition of Hex22HexNAc19Fuc3SiA19. With the peak profiles obtained from the mass spectra, 51 glycoform compositions of rhEPO were measured with different HexHexNAc or SiA residues [4]. Figure 13 displays the separation of rhEPO-glycoforms varying in the number of HexHexNAc units. Increased retention of rhEPO was observed by addition of one HexHexNAc unit, increasing protein polarity. The use of difluoroacetic acid (DFA) instead of TFA allowed for higher sensitivity as it gives less ion suppression. However, DFA is a weaker acid respect to TFA and more ion exchange takes place [4]. This study revealed the usefulness of an amide column in HILIC for the detection of intact glycoproteins by the large charge-state distribution using DFA and provided protonation of the protein.

In 2018 and 2019, two studies of D’Atri et al. [54,55] used amide columns for the characterization of therapeutic mAbs and Etanercept (middle-up analysis). Detailed characterization of Etanercept was achieved at the middle-up level (Fc/2) using enzymatic digestions (glycosidase, sialidase and proteolytic) combined with HILIC. In this study, they assessed overall N- and O-glycan compositions, the main PTMs and the distribution of glycans [54]. The variety of intact protein studies using amide columns suggests that this column shows great potential for intact protein separation.

19

Figure 13 – XIC of rhEPO-glycan compositions analyzed with a superficially-porous amide HILIC stationary phase and detection by MS (XIC = extracted ion chromatogram) [4]

Glycopeptide Analysis For the separation of glycopeptides from Proteinase K digests (asialofetuin and fetuin), Zauner et al. (2010) [56] used a nanoscale Amide-80 column. The Proteinase K enzyme unspecifically cleaves peptide bonds, leading to varying peptide lengths (2-15 amino acids). Consequently, relatively small peptide moieties resulted in less often observed peptides with multiple glycan fractions. The lack of specificity of this enzyme has the advantage to allow glycan assignment [56]. The resulting glycopeptides from the digested proteins were fractionate on the nanoscale amide column to confirm the glycosylation sites. As shown in Figure 14, the O-glycopeptides elute before the N-glycopeptides, providing a comprehensive view of the glycosylated protein [56]. The peptides digests were relatively small, and therefore easy to analyze with high order tandem mass spectrometry (MS3). In the fragmentation pattern, decomposition of the glycan portion was clearly observed, providing assignment of the peptide moiety and their glycan composition. Zauner and co-workers were able to identify 11 glycopeptides of the glycoprotein (even with MS/MS) [56].

Figure 14 – Separation of O-glycosylated peptides and N-glycosylated peptides from (A) an asialofetuin Proteinase K digest and (B) a fetuin Proteinase K digest [56]

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Analysis of Released Glycans Subsequently, Largy et al. (2017) [57] used a BEH amide column for the identification of N-glycosylation sites of mAbs (adalimumab and etanercept). The obtained fragmentation data provided structural identification of glycans. N-glycans were enzymatically digested using PNGase F and the middle-up approach was used to yield the glycan subunits. To enhance optical and MS detection, released glycans were labeled by a fluorophore (2-AB or RapiFluor-MS). The choice of labeling impacts the chromatographic separation, since labels can affect the hydrophobicity and charge of the glycan. Detection of O-glycosylation sites was carried out after N-deglycosylation and a desialylation step. As well, small proteins (e.g. RNase B) could be analyzed at the intact level. Largy stated that their method was applicable for a wide range of glycoproteins [57]. The separation of N-glycan isomers was achieved after using RapiFluor-MS labeling and HILIC-MS (Figure 15). For the chemical release of the O-glycan, β-elimination was used. In this study, O-glycans of bovine fetuin and etanercept were successfully detected without drifting in retention times [57].

Figure 15 – Fluorescence chromatogram of HILIC-MS analysis of labeled N-glycans (A) adalimumab and (B) etanercept with RapiFluor-MS. (C) illustrates the corresponding XIC of G1F isomers [57]

3.4.2 Zwitterionic phases Zwitterionic phases, also termed as mixed-mode phases, are widely used in HILIC as charged stationary phases. These columns use, for example, sulfobetaine groups that strongly bind to water at the polymer surface. This phase has both positively charged and negatively charged groups covalently attached to porous silica. The positively charged groups consist of a quaternary ammonium that may

21

interact with acidic portion of analytes. In contrast, the negatively charged groups consist of sulfonic acid, which may introduce electrostatic interactions with positively charged solutes. The combination of the two interaction sites provides high selectivity especially for amino acids and peptides [37,58]. The retention of glycoforms depends on the charge status of the protein, and the number of interaction sites. Because of structural recognition for neutral and charged compounds, ZIC-HILIC has a high capability of glycopeptide and released glycan separation (Figure 16) [59]. Studies on the use of zwitterionic phases (e.g. sulfobetaine) for glycoprotein separation are rare.

Figure 16 – Pie chart of sulfobetaine zwitterionic stationary phases used in HILIC characterization of the intact glycoproteins,

glycopeptides and released glycans – Based on 6 studies (Table 4)

Separation of Intact Proteins As shown in Figure 16, the use of ZIC-HILIC phases are rare in intact protein analysis. However, Tetaz et al. (2011) [60] used five columns (i.e. diol, ZIC, amide and silica) for the separation of intact proteins (human apoA-I, human apoM and equine cytochrome c). The intact proteins had relatively long retention on the ZIC column, due to ion-exchange interactions. The highly basic protein cytochrome c was not eluted on the ZIC column (Figure 17), because of the cation-exchange properties of the ZIC column [60]. In addition, broad peaks were observed, resulting from relatively small pore sizes (200 Å) of the ZIC column. As we compare this ZIC-HILIC separation with the separation of intact proteins using an amide column, we observe more satisfying separation on an amide column because of its hydrophilicity properties.

Figure 17 – ZIC-HILIC separation of (red) human apoA-I, (black) human apoM and (blue) equine cytochrome c [60]

17%

33%

50%

Sulfobetaine Phases (ZIC)

Glycoprotein Glycopeptide Glycan

22

Glycopeptide Analysis ZIC-HILIC analysis is commonly performed after protease digestion. A ZIC-HILIC method was demonstrated by Takegawa et al. (2006) [59] using a ZIC-HILIC column for the separation of sialylated N-glycopeptides and sialylated N-glycan isomers from alpha-1-acid glycoprotein (AGP). Their method showed great selectivity for peptides containing sialylated and non-sialylated N-glycans. Additionally, for neutral N-glycans (e.g. human serum IgG). Takegawa stated that their method showed highly capable separation based on structural recognition, as well for large isomeric N-glycan structures and different sialic acid linkage types (α2-3, α2-6) [59]. Figure 18A displays the mass spectra of AGP and its major N-glycopeptides and 2-aminopyridine (PA) N-glycans. Amino acid sequences were assigned to the peptides with their glycosylation sites. Though isomers show very similar separation patterns, effective recognition was performed with their N-glycan moieties. Different retention times were observed, because of different peptide lengths. It was noticed that all mass spectra resulted in two to four (isomeric) peaks. These peaks were well separated, although their glycan structures could not be specified. Nevertheless, this study was performed in 2006, indicating recent developments in MS and enrichment of glycoproteins are probably more useful to identify glycans. Analysis of Released Glycans Next, we evaluate the characterization of glycans using a zwitterionic column. Another work of Takegawa et al. (2006) [61] evaluated the separation of derivatized (PA) N-glycans using a ZIC-HILIC column with sulfobetaine groups. In this work, separation of IgG glycopeptides on a ZIC-HILIC column, provided sufficient separation of isomeric N-glycans with the same peptide sequence [61]. It was noted that PA N-glycan isomers were completely separated using the ZIC-HILIC column (Figure 18B), meaning this column has the capability of high structural recognition. This structural recognition is most likely based on the retention mechanism of ZIC-HILIC separation, including the hydrophilicity of N-glycans and electrostatic interactions. These electrostatic interactions take place between the N-glycans and the sulfobetaine groups on the surface of the zwitterionic column [61].

23

Figure 18 – ZIC-HILIC separation of tryptic (A) N-glycopeptides and (B) PA N-glycans [59]

3.4.3 Overview of HILIC Characterization of Glycoforms Table 4 gives an overview of HILIC characterization of intact glycoproteins, glycopeptides and released glycans. This table summarizes the columns, mobile phases and detection that are used in recent studies. As previously discussed, studies using ZIC columns for intact proteins were rare. Therefore, we reported only amide columns for the separation of intact glycoproteins. Overall, amide columns are more commonly used for the separation of glycoforms than ZIC columns, due to the lack of ion-exchange interaction. Therefore, the separation is more likely based on hydrophilicity properties. Contrary, the separation performed on ZIC columns is rather based on ion-exchange interactions. Studies on HILIC separation of intact proteins are still rare, because of their relatively high retention in HILIC [31]. Hence the need for columns that can reduce the retention of intact protein in HILIC. This can be achieved using columns with less hydrophilicity properties and less ion-exchange character. Furthermore, larger pore sizes (300-1000 Å) should be more suitable for the separation of relatively large proteins (e.g. intact mAbs) [31].

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Table 4 – Overview of HILIC characterization of glycoproteins, glycopeptides, and glycans

Glycoform Column name

Column Dimension Mobile Phase Buffer (salts) Column Temp.

Injection volume

Detection Ref.

Intact Glycoproteins

Amide Non-porous silica (30 x 2.1 mm , 700 nm) Lab made HILIC polyacrylamide

A: ACN with 0.1% TFA B: water with 0.1% TFA

0.5% (v/v) formic acid and 0.05% (v/v) TFA

30°C 0 – 12 µg MS [27]

BEH (150 x 2.1 mm, 1.7 µm) Waters, Milford, MA

A: 0.1% TFA (v/v) in water B: 0.1% TFA (v/v) in ACN

0.1% (v/v) TFA 80°C 1 µL ESI-TOF-MS [62]

BEH (150 × 2.1mm, 2.7 µm) Agilent Technologies


0.1% (v/v) TFA 40°C, 50°C, and 60°C

2 µL ESI-qTOF-MS [4]


A: ammonia solution B: ACN

50 mM ammonium formate

60°C NR Fluorescence and qTOF-MS

[57]



0.1% (v/v) TFA 80°C 0.5 µL UV-DAD and fluorescence detector

[8]

TSKgel Amide 80 (2.0 x 50 mm, 3 µm) Tosoh Corporation, Japan

A: 0.1% formic acid (v/v) in water B: 0.1% formic acid (v/v) in ACN

5 mM or 10 mM ammonium formate

NR 4 µL ESI-MS/MS [63]

TSKgel Amide 80 (2.0 × 150 mm, 3 µm) Tosoh Bioscience Montgomeryville, PA, USA

A: ACN B: water

10 mM HClO4 50°C 2 µL ESI-MS [52]

25

TSKgel Amide 80 (2.0 x 150 mm, 3 µm) Tosoh Bioscience Montgomeryville, PA, USA

A: ACN B: water

0.05% (v/v) TFA

50°C 2 µL UV and ESI-qTOF-MS

[53]

BEH Amide (3.0 x 150 mm, 2.5 µm) Waters, Milford, MA

A: ACN B: water

0.05% (v/v) TFA

50°C 2 µL UV and ESI-qTOF-MS

[53]

BEH Amide(2.1 x 150 mm, 1.7μm) Waters Milford, MA, USA

A: water B: ACN

0.08% (v/v) TFA and 0.02% (v/v) FA

45°C 1 µL ESI-qTOF-MS [55]

BEH Amide(2.1 x 150 mm, 1.7μm) Waters Milford, MA, USA

A: ACN B: water

0.1% (v/v) TFA 45°C 1 µL ESI-qTOF-MS [54]

ZIC ZIC-HILIC (4.6 x 150 mm, 5.0 μm) SeQuant, Germany

A: ACN + isopropanol + HFIP B: isopropanol + HFIP + L-tryptophan

50 mM ammonium formate and 50 mM formic acid

24°C 2 µL nESI-qTOF-MS [60]

Glycopeptides Amide BEH Amide (150 x 2.1 mm, 1.7 µm) Waters, Milford, MA




[57]

TSKgel Amide 80 (2.0 x 50 mm, 3 µm) Tosoh Corporation, Japan

A: 0.1% formic acid (v/v) in water B: 0.1% formic acid (v/v) in ACN

5 mM or 10 mM ammonium formate

NR 4 µL ESI-MS/MS [64]

TSKgel Amide 80 (75 µm × 180 mm, 3 µm) Tosoh Bioscience, Stuttgart, Germany

A: 80% ACN with ammonium formate B: ammonium formate


NR 5-10 µL ESI-MS [56]

26

ZIC ZIC-HILIC (2.1 x 150 mm, 3.5 µm) SeQuant, Umea, Sweden

75% ACN 10 mM ammonium bicarbonate

40°C 20 µL ESI-MS [59]

ZIC-HILIC (2.1 x 150 mm, 3.5 µm) SeQuant, Umea, Sweden

A: 50% ACN B: ACN C: Ammonium acetate

100 mM ammonium acetate

40°C 20 µL ESI-MS [61]

Glycans Amide TSKgel Amide 80 (250 × 4.6 mm, 5 µm) Anachem, Luton, U.K.


50 mM formic acid

30°C NR Fluorescence detector

[65]





[57]



0.1% (v/v) TFA 80°C 0.5 µL UV-DAD and fluorescence detector

[8]

TSKgel Amide 80 (75 µm × 150 mm, 5 µm) Tosoh Bioscience, LLC Montgomeryville, PA

A: 10% ACN with formic acid B: 95% ACN with 5% solvent A

50 mM formic acid

NR 4 µL ESI-qTOF-MS [63]

TSKgel Amide 80 (75 µm × 180 mm, 3 µm) Tosoh Bioscience, Stuttgart, Germany

A: 80% ACN with ammonium formate B: ammonium formate


NR 5-10 µL ESI-MS [56]

ZIC µZIC-HILIC (0.3 x 150 mm, 3 mm) SeQuant, Umea, Sweden

A: Ammonium acetate B: ACN


RT 0.15-0.25 µL

ESI-TOF-MS [66]

µZIC-HILIC (0.3 x 150 mm, 3.5 µm)

A: Ammonium acetate B: ACN


RT 0.25 µL ESI-MS/MS [67]

27

SeQuant, Umea, Sweden

ZIC-HILIC (2.1 x 150 mm, 3.5 µm) SeQuant, Umea, Sweden

A: 50% ACN B: ACN C: Ammonium acetate


40°C 20 µL ESI-MS [61]

Abbreviations: ACN = acetonitrile; ESI = electrospray ionization; HFIP = hexafluoroisopropanol, MS = mass spectrometer; NR = not reported; RT = room temperature; TFA = trifluoroacetic acid; (q)TOF = (quadrupole) Time of Flight

28

3.5 HILIC vs RPLC In the previous section, we compared different stationary phases used in HILIC. However, here we compare HILIC selectivity respect to RPLC. Theoretically, because of the separation is based primarily on polarity (and in some cases on ion-exchange), HILIC should be more suitable for resolving polar compounds. Various studies suggest that HILIC is preferred in glycoproteomic research for glycoproteins, as well for glycopeptides and glycans. As previously discussed in section 2.2, HILIC has many advantages over RPLC. For example, HILIC is more suitable for analyzing complex structures eluting near the void in RPLC [41]. Since ion pair reagents are not required in HILIC and the high content of organic solvents used in the mobile phase, HILIC is can be more easily coupled to MS. Periat (2016) [8] and coworkers used a BEH amide column for the characterization of intact protein biopharmaceuticals including interferon α-2b, insulins, and trastuzumab. In addition, a comparison has been made between HILIC and RPLC analysis of these intact proteins under the same conditions (i.e. column dimension, particle size, isocratic mode) looking at the oxidated products of this protein. Figure 19 shows the comparison of RPLC and HILIC analysis of interferon α-2b. As we look at the chromatograms, the oxidized forms of interferon α-2b retain before (RPLC) or after (HILIC) the “native” (non-oxidized) form. This was expected, since polarity is increased after oxidation using hydrogen peroxide [8]. This chemical reaction transforms the thioether group of interferon α-2b into sulfoxide. Due to increased polarity, less retention is observed for the oxidized forms of interferon α-2b in RPLC analysis. Hence, increased retention in HILIC. This gives the expectation of the opposite retention mechanism for the reduced form of interferon α-2b. This is confirmed in the chromatograms of Figure 19, where a decreased retention is observed under HILIC conditions, resulting from the hydrophobic amino acid residues [8]. This comparison confirms the difference in retention mechanism, making RPLC and HILIC orthogonal techniques. Another comparison between RPLC and HILIC has been made by D’Atri et al. (2017) [62], where a humanized mAb named Trastuzumab (Herceptin) was analyzed using the middle-up approach. Trastuzumab is commonly used in breast cancer treatments and is responsible for interfering with the signaling pathway, resulting in cancer cell proliferation [62]. Trastuzumab B has a similar glycosylation pattern, however, differs one amino acid residue from Trastuzumab (Herceptin), involving two charged residues. Hence, no shift in retention time is expected in the chromatogram. Figure 20 illustrates total ion chromatograms from Trastuzumab (Herceptin) and Trastuzumab B both analyzed with RPLC-MS and HILIC-MS. HILIC-MS analysis shows a reversed elution order from RPLC-MS analysis, as expected due to their opposite retention mechanism. It is noticed that the HILIC separation shows multiple peaks for the Fc/2 fragments, corresponding to different glycoforms [62]. This indicates that HILIC-MS can be used as a more powerful tool than RPLC-MS for the separation of protein biopharmaceuticals as it can resolve glycoforms on the basis of the number of neutral sugar presents. In addition, HILIC-based analysis can provide detailed structural glycan information. It is worth noticing that relatively large particles sizes (e.g. 3 µm) are used in HILIC separation of glycostructures (see Table 4). Mostly, acetonitrile is used in HILIC analysis, meaning smaller particles can be used due to lower backpressure and the low viscosity of acetonitrile [28,68]. Separation efficiency may be improved using smaller particles in RPLC. Compared to RPLC, HILIC shows a lower C-term, as derived from the Van Deemter equation [51]. However, it should be noted that the separation efficiency differs for each HILIC column. Since HILIC columns (silica, amide, ZIC and diol) are not commercially available for RPLC no real comparison has been made yet between HILIC and RPLC columns [68]. Nevertheless, a rough estimation has led to the statement that particle sizes have less influence on the separation efficiency in HILIC than in RPLC, because the separation selectivity depends on the chemistry of the stationary phase.

29

Figure 19 – Comparison of RPLC and HILIC analysis of intact, reduced and oxidized interferon α-2b [8]

Figure 20 – Total ion chromatograms of (a) Trastuzumab (Herceptin) and (b) Trastuzumab B analyzed with (i) RPLC-MS and (ii) HILIC-MS [62]

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3.6 HILIC for Sample Enrichment of Glycoforms The analysis of glycoforms can be challenging due to their heterogeneity, resulting in low sensitivity. Therefore, there is an increased interest in glycoprotein/glycopeptide enrichment. Sample enrichment techniques (i.e. lectin affinity chromatography, hydrazide chemistry, boronic affinity chromatography, and HILIC) are mostly applied because of the low abundance of certain glycoproteins/glycopeptides and/or glycans. Moreover, glycan structures in MS analysis may ionize less efficiently respect to non-glycated structures (e.g. glycopeptides vs non-glycosylated peptides) [69]. In lectin affinity chromatography, carbohydrate absorbance are used which bind to specific glycan structures that are attached to the protein [70]. This technique can facilitate glycoprotein separation due to its glycan-binding specificities [69]. However, enrichment techniques such as lectin affinity chromatography suffer from low specificity [71]. Prior to MS analysis, enrichment of glycopeptides is strongly recommended to enhance sensitivity [71,72]. The binding capacity for glycopeptides to the stationary surface is affected by limited hydrophilic groups and binding surface. To provide strong retention for glycoproteins and glycopeptides in HILIC, novel enrichment methods have to be developed. Among stationary phases used in HILIC (silica, amide, diol, cyano and ZIC), ZIC-HILIC is considered best for glycoprotein/glycopeptide/glycan enrichment [71,73]. ZIC-HILIC material can be modified with multilayer functionalized groups due to its both positive and negative charge. This phase reinforces the concentration of glycoproteins in the aqueous layer, due to its zwitterionic and hydrophilic groups [73]. This section covers alternative enrichment techniques for glycoproteins, glycopeptides and glycans. Figure 21 illustrates a workflow chart, reflecting the integration of glycomic techniques. In the sections that follow, we revise recent approaches to enrich glycoforms.

Figure 21 – Workflow chart for the identification of glycoproteins, glycosylation sites, and structural characterization of the glycan [1]

3.6.1 Glycoprotein Enrichment Besides lectin-binding affinity, enrichment techniques for glycoproteins are rare. In this section, we focused on alternative enrichment techniques for glycoproteins. Nanoparticles and nanospheres supporting different chemistries have been reported as potential alternatives, thanks to their high surface areas and aptness for sample preparation. Guo et al. (2018) [73] described the use of magnetic nanospheres (Fe3O4@PCL-PILs) for the isolation of glycoproteins using a zwitterionic HILIC coating. The magnetic nanospheres were successfully synthesized by the growth of polymer brushes (polymer ε-caprolactone – PCL) on Fe3O4 nanoparticles. The nanoparticles were packed with a coating of polymer ionic liquid (PIL). This layer provided more binding sites for protein adsorption and can be selectively removed using magnets [73]. The nanospheres enhanced the binding of glycoproteins by zwitterionic properties, resulting in more selective concentration of intact proteins. Figure 22 displays the use of

31

core-corona magnetic nanospheres for the isolation of glycoproteins. First, the nanospheres were synthesized and the magnetic core was coated with a PCL layer via a polymerization reaction, providing the surface of magnetic Fe3O4 with hydroxyl groups. Furthermore, PILs were introduced onto the nanospheres by esterification. Finally, free radical polymerization with VimCOOHBr generated Fe3O4@PCL-PILs core−corona nanospheres [73]. The hydrophilic layer (PIL) provides more binding sites and stimulates protein adsorption to the stationary phase.

adjusted figure from [73]

Model proteins were used to investigate the adsorption performance of the nanospheres. Differences were observed between glycoproteins (IgG, Trf, and Ova) and non-glycoproteins (Hb, BSA, Cytc, and Mb), because glycoproteins have different interaction sides than non-glycoproteins. Strong interaction between the ZIC-HILIC nanospheres and glycoproteins was noticed, resulting from the hydrophilic glycan groups. High adsorption was achieved at varying pH values (5, 7, and 9), however best adsorption for IgG was obtained at pH 9. This binding was facilitated by increased salinity (0 – 500 mM NaCl), causing water breakage clusters around the protein and outspread of hydrophilic PIL chains around the nanospheres, and increased adsorption efficiency [73]. This illustrates that the hydrophilicity enhanced nanospheres provided a high binding capacity toward glycoproteins. Additionally, the use of Fe3O4@PCL-PILs core−corona nanospheres can be promising in glyco-proteomics research [73].

3.6.2 Glycopeptide Enrichment The characterization of glycopeptides has proven to be a difficult task, because of ion suppression resulting from unmodified high abundance peptides [69]. Due to its increased interest, a variety of studies have been found for the enrichment of glycopeptides. Improvements in glycoproteomic technologies can provide more detailed information on glycan structures and glycopeptide sequences [1]. The evaluated methods for glycopeptide enrichment are listed in Table 5. These enrichment methods mostly provided increased sensitivity, hydrophilicity and selectivity. Furthermore, it was found that enrichment material contained the use of nanomaterials, because of its non-fouling and controllable properties (e.g. mechanical, thermal, interfacial and biocompatible properties) [74]. More interestingly, zwitterionic material was frequently synthesized. As previously discussed, ZIC is an attractive material to enrich because of its multiple charge properties. Alagesan and co-workers (2017) [34] evaluated the enrichment of glycopeptides using different mobile phases, including methanol, ethanol, acetonitrile and isopropanol (Figure 23). Tryptic protein digests were dissolved in 80% of one of the organic solvents with 1% TFA. An ideal mobile phase may not have hydrogen acceptor or donor functionalities, and has to be miscible with water. Because of its aprotic properties, acetonitrile is mostly used in HILIC [34]. Hence, methanol is non-favorable in HILIC, due to

Figure 22 – Schematic illustration of synthesizing Core-corona nanospheres and application for glycoprotein enrichment –

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hydrogen bonding which discourages hydrophilic partitioning, resulting in reduced retention of glycopeptides. Synthetic glycopeptides were better enriched using ethanol compared to acetonitrile and isopropanol. Since not all (glyco)peptides were soluble at 80% organic solvent, isopropanol provided best compromise for hydrophilic and hydrophobic glycopeptide enrichment. So isopropanol co-enriched unmodified peptides. However, acetonitrile provided best retention for all synthetic glycopeptides. These results indicate that retention not only depends on the polar stationary phase, but also on the organic mobile phase [34].

Figure 23 – Relative percentage of enriched glycopeptides of solvent effect in HILIC from (a) depleted proteins (b) depleted human serum (c) non-depleted human serum (Abbreviations: ACN = acetonitrile; EtOH = ethanol; IPA = isopropanol) [34]

A study of Neue et al. (2011) [75] used ZIC-HILIC (ZIC-HILIC ProteaTip, 10-200 µL, Marl, Germany) combined with solid-phase extraction (SPE) for the separation of N-glycopeptides and detection with nanoESI-MS. Glycoproteins were dissolved, loaded onto the SPE-column, eluted and digested using an ammonium bicarbonate buffer. Finally, the glycopeptides were separated using ZIC-HILIC ProteaTips. This method allowed separation of glycopeptides using unspecific protease without further purification steps. In addition, this approach provided mass-based separation of glycopeptides without overlapping signals. However, signal intensities were frequently low in MS due to the low ionization efficiency and high heterogeneity of glycopeptides [75]. Separation of glycopeptides (i.e. haptoglobin (HG), human immunoglobulin gamma (IgG), ribonuclease B (RNase B), R-1-acidglycoprotein(AGP), and asialofetuin (AF)) was evaluated. ZIC-HILIC separation showed excellent separation of N-glycopeptides from haptoglobin without overlapping signals (Figure 24). In addition, ZIC-HILIC-SPE resulted in almost complete separation of N- and O-glycopeptides (Figure 25). O-glycopeptides were detected in the residual of the loading- and wash solution. This can be termed as carry-over. The detection of O-glycopeptides as a result of carry-over, occurs due to the short O-glycan attached to a relatively long peptide backbone [75]. Contrary to O-glycopeptides, N-glycopeptides consist of a relatively large N-glycan moiety attached to a short peptide backbone. The N-glycopeptides interact with the zwitterionic stationary phase, resulting in separation from O-glycopeptides.

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Figure 24 – Mass spectra (nanoESI Q-TOF) with ZIC-HILIC separation from haptoglobin digested with thermolysin [75]

Figure 25 – Mass spectra (nanoESI Q-TOF) from separation of N- and O-glycopeptides using ZIC-HILIC-SPE [75]

Differing from other enrichment methods, Mysling et al. (2010) [76] used a solid phase extraction (SPE) microcolumn for glycopeptide enrichment. In this study, Mysling increased differences in hydrophilicity between glycopeptides and non-glycopeptides by TFA ion pairing. They defined their method as enrichment method for glycopeptides, since the use of TFA in the mobile phase significantly improved the detection (MALDI-TOF/MS) of glycopeptides from complex glycoprotein mixtures. TFA can be used in relatively high concentrations (e.g. 2% TFA v/v) in the mobile phase without reducing MS sensitivity of MALDI-TOF. Using ZIC-HILIC-SPE enrichment, Mysling achieved almost complete isolation of glycopeptides and non-glycopeptides [76]. Using TFA as an ion-pairing reagent promoted hydrogen bonding under low-pH mobile phase conditions (Figure 26). These low-pH conditions were shortly applied to the column, meaning no degradation of silica particles was observed. In addition, the influence of the low-pH on column equilibration and sample loading was negligible [76]. Under these conditions, glycopeptides were neutralized due to ion-pair formation (both positive and negative

34

charges). Now, electrostatic interactions can be formed by the peptide moiety, reducing hydrophilicity as a result of a lower hydrogen bonding potential. Glycopeptides differ from non-glycopeptides in terms of hydrophilicity due to differences in hydroxyl groups. A relatively large hydrophilic difference between glycopeptides and non-glycosylated peptides was created. This means that there is less competition between glycopeptides and non-glycopeptides, resulting in fewer coenriched non-glycopeptides. Eventually, this difference can be observed in improved separation between glycopeptides and non-glycopeptides.

Figure 26 – Glycopeptide enrichment using TFA in the mobile phase (A) normal mobile phase conditions (80% ACN) and (B) low-pH mobile phase conditions (1% TFA v/v with pH 1.6) [76]

Table 5 – Overview of glycopeptide enrichment methods (Abbreviations: RAFT = reversible addition-fragmentation chain transfer; PMSA =

poly(2-(methacryloyloxy)ethyl)dimethyl(3-sulfopropyl)ammonium hydroxide; CS@PGMA@IDA = Chitosan@Poly(glycidyl methacrylate)@iminodiacetic acid; TFA = trifluoroacetic acid; SPE = solid phase extraction; MoS2 = Molybdenum disulfide)

Enrichment material Synthesized material Ref.

Poly (amidoamine) dendrimer Zwitterionic functionalized material [71]

RAFT on silica nanoparticles Zwitterionic polymer brushes PMSA [74]

Immobilized ultrathin gold nanowires Magnetic graphene oxide linked zwitterionic groups

[72]

CS@PGMA@IDA nanomaterial Zwitterionic coating [77]

Carbon microspheres

Phosphorylcholine or sulfobetaine type zwitterionic groups

[78]

Metal-organic framework Zwitterionic tripeptide – Glutathione (GSH) [79]

MoS2/gold nanoparticles−L-cysteine Zwitterionic material [80]

ZIC-magnetic beads Zwitterionic polymers [81]

Polyethyleneimine (PEI) and hyaluronic acid (HA)

Dendritic mesoporous silica nanoparticles and magnetic graphene oxide

[82]

3.6.3 Glycan Enrichment In studies of Mancera-Arteu et al. (2016 and 2017) [66,67], a ZIC-HILIC (sulfobetaine) coupled to high-resolution tandem mass spectrometry (MS/MS) was used for the characterization of glycan isomers. With this method, they obtained valuable information about glycan structures, using hAGP as model glycoprotein. Desialylation with α2-3,6,8 sialidase after PNGase F treatment, revealed information about sialic acid and fucose linkage-type glycan isomers. The detection of these isomers is important for glycan-based biomarkers, since alterations in specific sialic acid or fucose linkage isomers can correlate with a specific pathology, for instance in cancer [66]. The characterization of glycan isomers was carried out using glycan reductive isotope labelling (GRIL) (Figure 27). This methodology allowed glycan comparison before and after digestion, and provided characterization of glycan isomers [66,67].

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N-glycans were labelled with [12C6]/[13C6] coded aniline and analyzed with ZIC-HILIC-MS/MS. With this labelling, the characterization of glycan isomers was evaluated in order to find new glycoprotein biomarkers. Desialylated N-glycans (bi-, tri-, and tetraantenary) were obtained from hAGP after digestion and labelled with [12C6]-aniline. Differences in fragment ions were observed after MS-analysis. Multiple ions provided structural information on the glycan-forms. Moreover, D-ions were used to identify the antenna positions, and provided information on the composition of 6-antenna. These D-ions were only present in H5N4 and H6N5 glycans. In addition, some glycans were digested with α2-3 sialidase for the complete characterization of N-glycan isomers. However, this approach showed lack of specificity [67]. Although, isomer glycans showed all sialic acids linked α2-6 isomers. Similarities in MS/MS spectra were observed, and D-ions revealed structural information on the glycan isomers. Relative intensities of fragment ions differed between isomers, and were used to identify the composition of glycan isomers (Table 6).

Figure 27 – Workflow of the glycan reductive isotope labelling (GRIL) methodology [66]

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Table 6 – Observed fragment ions used for the characterization of N-glycan isomer (α2-3 desialylated hAGP and aniline-labelled native forms) [67]

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3.7 Detection of Glycoforms Next, detection methods for the characterization of glycoforms will be discussed. MS-based approaches are commonly applied as detection methods, however UV or fluorescence detection is also used. First, the application of MS to glycoforms will be evaluated, followed by UV and fluorescence detection. Continuing improvements in MS result in increased detection performance in terms of resolution, sensitivity and upper mass range [83]. These developments also include the use of tandem MS (MS/MS and MSn), providing more sensitive detection to define glycosylation sites. To facilitate tandem MS experiments, matrix assisted laser desorption ionization tandem time-of-flight (MALDI-TOF/TOF), ESI and ion-trap instrumentation are commonly used in isomeric glycan characterization. The complexity of glycoprotein structures requires highly sensitive techniques, since structural analysis of glycans is typically performed in femtomolar ranges [84]. With MS, complete mass spectra of complex glycoforms, including glycoproteins, glycopeptides and released glycans, can be obtained. These mass spectra provide information on peptide fragmentation, glycosylation sites and peptide sequences [85]. Defining the site of glycosylation can be facilitated by introducing alternative fragmentation methodologies such as electron transfer dissociation (ETD) or electron capture dissociation (ECD). These applications allow direct mapping of both N- and O-glycosylation sites [83]. MS analysis can be used to yield information on released glycan composition. However, to provide structural information, chromatographic separation of glycans is necessary due to its large number of structures. The same applies for protein associated to glycan analysis. Because of the large diversitiy of protein glycoforms, chromatographic separation is essential, because protein-specific glycosylation requires more detailed information. Therefore, protein enrichment and tandem MS detection are often needed [84]. Nevertheless, low ionization efficiency and the lack of reference spectra makes quantification of peptide glycoforms challenging [86]. Hence, targeted MS-based quantification is required, because this detection method has the advantage of using parallel reaction monitoring (PRM) and selected reaction monitoring (SRM). Kawahara et al (2018) [86] stated that PRM showed great results for the quantification of intact glycopeptides due to improved accuracy. Subsequently, UV detection is mostly used when the analytical method is not compatible with MS. UV detection can be used for intact proteins to monitor glycosylation reactions. In addition, glycoproteins and non-glycoproteins can be discriminated using UV detection [53]. Peak assignment is less common using UV detection, because of the multiple glycosylation forms. Therefore, MS detection is preferred over UV detection due to its sensitivity. However, both detection methods mostly show comparable quantification precisions [8,87]. Furthermore, fluorescence detection is mostly used for fluorescently labeled glycans. Fluorescent labeling can be alternatively used to improve MS sensitivity (10 – 100 fold increase) [57,65]. The chromatographic separation can be influence by the choice of label, since it can alter the hydrophilicity and charge of glycans. For instance, RapiFluor labels are likely to protonate at acidic pH, resulting in a different chromatographic separation. The use of fluorescence labels allows for more sensitive detection for low intensity glycans [57]. Nevertheless, structures of N- and O-glycans in glycoproteins are mostly defined by MS.

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3.8 Applications of HILIC to Glycoform Investigations Glycoproteomic studies are widely used in clinical research to identify PTMs of proteins (e.g. glycosylation) [88]. It is demonstrated that malignant processes in cancer are more frequently reflected in glycosylation processes [86]. This yields that glycosylation can be altered in cancer compared to healthy cells. In cancer, protein glycosylation is regulated as a result of differences in glycan-modifying enzyme expression. Therefore, these proteins are also termed as tumor-associated antigens [1]. This implies that quantitative analysis of intact glycoproteins is interesting and opens novel ways to provide information on development, progression, and metastasis of tumors in cancer. Detailed knowledge of glycosylated proteins helps to understand their specific function [86,88]. Glycans have a major influence on immune responses as well as carcinogenesis. Therefore, glycan isomer separation is needed in biomarker discovery [27]. It is believed that glycosylation can be associated with prostate cancer, because of the glycosylation of prostate-specific antigen (PSA). Prostate cancer is worldwide frequently diagnosed in male patients [89]. A common biomarker in prostate cancer is PSA, however, lacks of specificity when measured in blood. Characterization of N-glycans, peptide compositions and glycosylation sites, are proposed as biomarker candidates for prostate cancer [86]. Better understanding is needed of how glycans act at the cell surface and change the glycosylation process. Consequently, the properties of tumor cells can be affected [90]. The characterization of proteins with tumor glycans is essential to understand cellular processes in cancer progression. mAbs can be used as a biomarker for the identification of tumor-specific glycosylation in, for example, the progression of cancer or Hodgkin’s lymphoma [90,91]. Powlesland et al. (2009) [90] demonstrated that a galactose mannose-binding protein (GalMBP) was effectively used to identify tumor-specific glycans in breast cancer, where the protein binds to glycans containing terminal galactose binding. GalMBP is considered to be an effective tool for the identification of glycoproteins in tumor cell lines, because this protein binds two subsets of galactose glycans as present in tumor cells [90]. Further research on structural information and information on the glycan binding sites can provide new sets of glycans for target analysis. The main application in glycopeptide analysis is to determine the glycosylation sites. For example, the measurement of intact glycopeptides can be useful to discriminate prostate cancer from benign prostate hyperplasia (BPH). Kawahara et al. (2018) [86] found in their study that glycoprotein levels were not altered in prostate cancer, however, differed in glycosylation sites. In addition, they found that quantitative changes in glycosylation are a result of specific glycoforms of glycoproteins. Since PSA has about 40 glycoforms, the separation of glycoforms can facilitate biomarker discovery [27]. Glycoproteins are more likely to be used to monitor disease progression rather than for screening tests, because levels of glycoproteins in serum correlate with tumor masses [1]. New developments, especially in MS measurements, enable better characterization strategies, such as isotope-coded glycosylation-site-specific tagging. This strategy combines lectin-based methods, HILIC, isotope labeling, and peptide identification by LC-MS. This methodology provides the identification of circa 1,000 glycoproteins and their glycosylation sites. Progress in glycoproteomics will depend on new developments towards enrichment methods for intact glycoproteins and glycopeptides, MS instrumentation, software and high-throughput screening methods [1,92]. In addition, the development and use of glycan-specific biomarkers depend on glycoprotein standards used in laboratories, minimizing intra-variability in samples [1].

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4. Conclusion The characterization of glycoproteins using HILIC can be challenging for several reasons. However, it has been shown that HILIC is a valuable alternative to RPLC, enabling to chromatographically resolve glycan structures at various glycoform levels. Therefore, an increased interest was recently shown for HILIC. There are multiple columns that are commercially available for the separation of glycoprotein at various levels. However, method optimization is required in all cases. Among all HILIC stationary phases, we found that amide columns were favorable for the characterization of all glycoforms (glycoproteins, glycopeptides and released glycans). Additionally, ZIC-HILIC columns were also used in characterization of glycopeptides and glycans. The separation efficiency depends on the type of stationary phase. However, the use of buffer salts in the mobile phase also influence the chromatographic separation. Ammonium formate is mostly used as additive for amide stationary phases. In contrast, ammonium acetate or ammonium bicarbonate are commonly used for ZIC columns. These stationary phases are mostly coupled to a MS-detector. MS provides multiple advantages over UV or fluorescence detection. However, fluorescent labeling can improve selectivity in MS detection. Glycoproteomic research is extremely important in the clinical field. Understanding glycosylation patterns can give more insights into specific functions of glycoforms and, for instance, the progression of cancer. Especially the characterization of glycopeptides and glycan structures can be valuable in biomarker development. Furthermore, structural information of glycan binding sites provide novel insights in target analysis. This review covers the use of HILIC for the characterization of glycoforms. The main goal was to create an overview of the HILIC detection based on recent literature. This overview can provide more insights in the practical use of HILIC in glycoproteomic research.

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