Human Serum N-Glycome Profiling via the Newly Developed Asparagine Immobilized Cellulose/Polymer Nanohybrid

Abstract

Human serum N-linked glycans expression levels change during the disease progression. The low-abundance, structural diversity and coexisting matrices hinder their detection in MS analysis. Considering the hydrophilic nature of N-glycans, cellulose/polymer (1,2-Epoxy-5-hexene) nanohybrid is fabricated with oxirane groups functionalized of asparagine to develop solid phase extraction based hydrophilic interaction liquid chromatography sorbent (cellulose/1,2-Epoxy-5-hexene/asparagine). The morphology, elemental analysis and surface properties are studied through scanning electron microscopy, energy dispersive x-ray spectroscopy, and Fourier-transform infrared spectroscopy. Large surface area of cellulose/polymer nanohybrid (2.09×10² m²/g) facilitates the high density of asparagine immobilization resulting in better hydrophilic interaction liquid chromatography enrichment under optimized conditions. The enrichment capability of nanohybrid/asparagine is assessed by the N-Linked glycans released from ovalbumin and immunoglobulin G where 23 and 13 N-glycans are detected respectively. The nanohybrid/asparagine shows selectivity of 1:1200 with spiked bovine serum albumin and sensitivity down to 100 attomole. Human serum profiling for N-glycans identifies 52 glycan structures. This new enrichment strategy enriches serum N-linked glycans in the presence of salts, proteins, and endogenous serum peptides, etc.

1. Introduction

Protein glycosylation is the most significant protein post-translational change having role in biological processes including immune response [1], molecular recognition [2], protein folding and receptor stimulation. In general, glycans in N-glycosylation are covalently bound to the amide group on protein residue of asparagine having Man₃GlcNAc₂’s core oligosaccharide. Based on the structure and sequence of oligosaccharides, N-glycans can be divided into three classes: high-mannose, complex, and hybrid [3, 4]. Most studies are focused on N-glycans as their configuration is linked to diseases like oncogenesis, progression, and cancer transformation [5, 6]. More than 50% cancer biomarker candidates such as carcinoembryonic antigen (CA), mucin epitopes (CA 15–3), alpha-fetoprotein (CA 125) [7], human epidermal growth factor receptor-2 (HER2) [8], carbohydrate antigen 19–9 (CA 19–9), prolactin and leptin etc. used in clinical diagnosis are glycoproteins. Glycomics via the glycan profiling of cancer patients has become popular for biomarkers discovery from biofluids [9, 10].

Mass spectrometry (MS) is used to compare the glycan patterns in healthy controls and cancer patients [11]. Glycans are hardly detected by direct MS because of the abundant residual proteins, salts, endogenous peptides and the low abundant glycans in biological samples [12]. Developing a selective, sensitive, and reproducible enrichment method is in demand of glycomics. Methodologies for enriching glycans from biofluids include organic solvent precipitation [13], lectin, and hydrophilic interaction (liquid) chromatography (HILIC) [14]. HILIC approach for N-linked glycans rely on physical interactions like partitioning, hydrogen bonding, dipole-dipole interactions, and ionic interactions between the media and glycans [15]. HILIC phases have the bonded functional groups. Various saccharide-based phases have been developed where hydroxyl groups of immobilized saccharides provide sufficient hydrogen bonding [16] for enriching glycopeptides and glycans. Sepharose has been used for its non-ionic and compatible elution conditions [17]. The modified polysaccharide materials have been in focus because of biocompatibility and non-fouling properties [18].

A dextran-bonded silica phase prepared by bonding dextran to activated silica via the carbonyl diimidazole selectively enriches glycopeptides and glycans [19]. Hybrid magnetic nanoparticles (MNPs) branched polyethylene glycol (PEG) brushes with immobilized maltose have high selectivity and sensitivity for glycopeptides [20]. The alternate deposition of hyaluronan (HA) and chitosan (CS) using layer-by-layer (LbL) method develops multilayer polysaccharide shells onto MNPs (MNPs-(HA/CS)10), showing high selectivity, detection sensitivity, and binding potential for glycopeptides enrichment [21]. LbL-assembled nanostructures may lack stability in aqueous environment [22]. Diverse kinds of supports are used for enriching N-linked glycans and glycopeptides, especially the hydrophilic ones like metal oxides, metal organic frameworks (MOFs), ionic liquids and inorganic and organic materials [23,24]. Cellulose is a hydrophilic polymer phase with characteristics like biocompatibility, stability, non-toxicity and well-known chemistry [25,26]. Cellulose modifications produce acetylated cellulose as reversed-phase, polyethyleneimine (PEI) and diethyl aminoethyl (DEAE) as anion exchanger and cellulose phosphate as cation exchanger [27,28]. These affinities are immobilized onto the support material in addition to the support characteristics like particle dimensions which can affect enrichment [29].

Herein, the cellulose based polymeric nanohybrid is developed for high-throughput N-glycans profiling. 1,2-Epoxy-5-hexene is grafted onto nanocellulose (illustrated in Fig. 1) having high surface area and high density of immobilization sites (oxirane groups) for asparagine to induce high sample loading capacity and strong interactions between N-linked glycans and HILIC sorbent from complex biological sample like serum.

Figure 1.

Synthetic scheme of cellulose/1,2-Epoxy-5-hexene/asparagine: (a) free radicals’ generation on cellulose backbone, (b) grafting of 1,2-Epoxy-5-hexene, and (c) immobilization of asparagine

2. Materials and Methods

2.1. Chemical and Reagents

Chicken egg albumin (OVA), immunoglobulin G (IgG), bovine serum albumin (BSA), ammonium cerium (IV) nitrate (98%), ammonium bicarbonate (ABC, ≥ 99%), cellulose, sodium hydrogen sulfite (≥ 98%), 1,2-Epoxy-5-hexene (≥ 97%), dithiothreitol (DTT, ≥ 99%), iodoacetamide (IAA, ≥ 99.9%), acetonitrile (ACN, ≥ 99.9%), hydrogen bromide (HBr, ≥ 48%), nitric acid (70%) and trifluoroacetic acid (TFA, ≥ 99.8%) were purchased from Sigma Aldrich (St. Luis, MO, USA). PNGase-F was from New England Biolabs. 2,5-Dihydroxybenzoic acid [(Gentisic Acid) (DHB)] was from Bruker (Bruker Daltons, Bremen, Germany). Deionized water (18.25 MΩ cm) was purified using Milli-Q system (Millipore, Bedford, MA, USA).

2.2. Synthesis of Nanocellulose

The designed protocol for nanocellulose preparation is provided in Supporting Information.

2.3. Synthesis of Nanohybrid Polymer

The cerium induced grafting reaction was performed in dilute nanocellulose suspension, typically 0.1% (w/w) in 0.1 M HNO₃. Nitrogen was bubbled through the suspension for 15 min and temperature was increased to 35 °C with stirring before adding ammonium cerium (IV) nitrate to a total concentration of 2 mM. 40 μmol/mg of 1,2-Epoxy-5-hexene was gradually added to the reaction mixture in 30 min. The product was washed with excess of water followed by THF.

2.4. Immobilization of Asparagine

For the immobilization of asparagine onto the oxirane groups of nanohybrid, 0.1 g nanohybrid was added to 10 mL of 2 M sodium carbonate solution in 50 mL flask equipped with condenser followed by the dropwise addition of 5 mL saturated asparagine solution at 80 °C. After 4 h, the reaction mixture was cooled to room temperature followed by water and methanol wash.

2.5. N-Glycan Release from Model Glycoproteins and Serum

Chicken ovalbumin (OVA) and immunoglobulin G (IgG) were used as model glycoproteins; 1 mg/mL protein solutions were prepared in 25 mM ammonium bicarbonate and heated at 95 °C for 10 min to denature the proteins. After cooling to room temperature, PNGase-F (1 μL = 1000U) was added and incubated for 16 h at 37 °C.

For the preparation of serum sample, 5 μL serum was diluted with 45 μL ammonium bicarbonate and centrifuged at 4 °C for 10 min. The supernatant was further diluted with 200 μL of 25 mM ammonium bicarbonate solution and denatured by heating for 10 min at 95 °C. N-Glycan were released by the overnight treatment of PNGase-F at 37 °C. For further analysis, samples were stored at −80 °C.

2.6. Glycans Enrichment

10 mg cellulose/1,2-Epoxy-5-hexene/asparagine was suspended in 1 mL of 0.1% TFA with the final concentration of 10 mg/mL. For each enrichment batch, 10 μL was taken in Eppendorf vial (1.5 mL) and washed twice with 200 μL loading buffer (92% ACN, 1% TFA). Sample was diluted with 100 μL loading buffer and 2 μL diluted sample was loaded onto the activated HILIC sorbent by 20 min incubation. Three cycles of washing with 500 μL washing buffer I (92% ACN, 1% TFA) and for the complex samples one washing cycle with washing buffer II (89% ACN, 0.1% H₃PO₄) was carried out. The enriched N-linked glycans were eluted with 30 μL elution buffer (30% ACN, 0.1% TFA). The eluted glycans were concentrated by speed vacuum before spotting on MALDI plate without any desalting.

2.7. Regeneration of HILIC Sorbent

To regenerate the HILIC sorbent, cellulose/1,2-Epoxy-5-hexene/asparagine was washed with TA-30 (30% ACN, 1.0% TFA) in three cycles with 500 μL each time and once with 250 μL of 0.1% TFA. To start another batch of enrichment, the same procedure of washing, equilibration, loading, and elution was performed. This cycle of regeneration and reuse of HILIC sorbent was repeated up to 4 times.

2.8. MALDI-MS Analysis

1 μL of eluted fraction was mixed with 1 μL matrix (15 mg DHB in 1 mL of 50% ACN, 0.1 % TFA and 10 mM NaCl), 1 μL was spotted on MALDI plate and dried at room temperature. 0.5 μL alcohol was added to each spot for re-crystallization. MALDI-MS analysis was made by Ultraflex Extreme mass spectrometer (Bruker Daltons, Bremen, Germany) in positive ion mode with resolution 15000–20000. The spectra in mass range 800–2500 m/z were processed using Flex Analysis Version 3.4 supplied by Bruker Daltonics. GlycoMod tool was used to predict the possible glycan structure based on experimentally determined masses with 0.5 Da mass tolerance.

3. Results and Discussion

3.1. Synthesis Scheme of Nanohybrid/Asparagine

For hydrophilic interaction liquid chromatography (HILIC), sorbent with the antifouling properties and high density of hydrophilic groups are essential as they eliminate the non-specific bindings and enhance selectivity particularly when applied to complex biological samples [30]. So, keeping in view, the cellulose/polymer nanohybrid HILIC sorbent is fabricated. The synthesis scheme is shown in Fig. 1. First step involves the reaction of ceric ion with hydroxyl groups resulting in the formation of chelate complex (Fig. 1a). This complex is not stable and decomposes to generate free radicals on the cellulose backbone. These active free radicals assist polymerization of 1,2-Epoxy-5-hexene monomer on the cellulose fibers (Fig. 1b). The oxirane groups are modified with asparagine to attain hydrophilic characteristics (Fig. 1c). Asparagine has similar functional group chemistry as that of glutamine and hence the nanohybrid is also immobilized with glutamine to observe similarity in enrichment performance of affinity material.

SEM micrographs at different microscale levels of nanohybrid are shown in Fig. 2 a–b. At 1 μm, the appearance seems fibrous with rough edges and surface. Increase in zoom level to 0.5 μm reveals nanosized fibers between 100 to 250 nm. The surface area is determined by nitrogen adsorption-desorption isotherm (Fig. 2c) where BET surface area is calculated as 220.81 m²g⁻¹. This high surface area accommodates better quantities of asparagine resulting in large adsorption capacity, low steric hinderance and thus better enrichment of N-glycans. The fabrication involves ceric induced reaction, and the presence of metal may inflict non-specific bindings. EDX spectrum reveals the atomic percentage of carbon C as 94.51% and oxygen O as 05.49% (Fig. 2d) with no other species, confirming the impurity free method of nanohybrid synthesis. FTIR analysis compliments the synthesis of desired product and confirms the immobilization of asparagine through two stretch bands at 1586 and 1608 cm⁻¹ due to the asymmetrical vibrations of C-N bonds of guanidine group. The bands at 1428, 1367, 1334, 1027 cm⁻¹ and 896 cm⁻¹ belong to stretching and bending vibrations of -CH₂ and -CH, and C-O bonds in cellulose (Fig. S1).

Figure 2.

Cellulose/1,2-Epoxy-5-hexene, (a) SEM image at 1 μM, (b) SEM image at 0.5 μM, (c) N2-adsorption/desorption plot, and (d) EDX spectrum

3.2. Enrichment of N-Linked Glycans

Ovalbumin and human IgG are selected as standards to enrich glycans by cellulose/1,2-Epoxy-5-hexene/asparagine. In HILIC-based enrichment, optimization of buffers (loading and washing) is critical for the selective enrichment with minimum non-specific bindings. Keeping this in view, the ACN concentration is optimized in loading/washing buffer. ACN concentrations as 80, 85, 90, 92, and 95% with 1% TFA are used as loading buffer for nanohybrid immobilized asparagine with ovalbumin as sample. The comparative MS spectra show improvement in signal intensity and quality with increase in ACN concentration up to 92% (Figure S2). MS results are same with no significant change in enrichment. Thus 92% ACN in 1% TFA is applied as loading and washing buffer for enrichment studies. Ovalbumin is the best characterized glycoprotein glycosylated with high-mannose and hybrid glycan structures [31]. It is therefore the most suited model sample in glycomics. Before enrichment, 13 N-glycans with low intensities are observed along with high intensity background (Fig. 3a). Such detections are not useful to account for the sensitive studies at clinical level. After applying the sample to nanohybrid HILIC sorbent, both the number and signal intensity are improved with negligible background (Fig. 3b). Total of 23 N- glycans are detected with 10 new N-glycans at m/z 956, 1298, 1460, 1581, 1948, 2028, 2110, 2151, 2272, and 2314. The peaks at m/z 1339, 1419, 1501, 1704, 1905 and 2067 have improved signal intensity with no prominent background peak (Fig. 3b). The enhanced hydrophilicity is thus the key in attaining enrichment efficacy. Structural information and composition of enriched N-glycans are provided in Table S1. Nanohybrid HILIC sorbent immobilized with glutamine is also subjected to enrichment using ovalbumin under similar conditions. The obtained MS profile for detected N-glycans is like asparagine with no obvious variation in enrichment behavior of affinity material (Figure S3).

Figure 3.

MALDI-MS spectra recorded for N-linked glycans from oval albumin, (a) prior to enrichment, and (b) after enrichment with cellulose/1,2-Epoxy-5-hexene/asparagine. Detail of N-linked glycans is provided in Table S1

The compatibility of nanohybrid with sample subjected to different pretreatment conditions for N-glycan release is also investigated. Most used sample conditions are (i) trypsin digested followed by PNGase F treatment and SDS/NP-40 treated samples. Ovalbumin is treated under both conditions and samples are applied to nanohybrid immobilized with asparagine. The optimized enrichment protocol is followed and characteristic N-glycans are detected (Figure S4). The experiment shows that fabricated material remains selective for N-glycans and the nature of sample pretreatment has no effect on enrichment ability of nanohybrid.

There is a relationship between IgG glycosylation, inflammation, and its biological role. IgG glycosylation pattern changes are related to age, disease and lifestyle variables which signify its role as biomarker. N-glycans in human IgG are predominantly fucosylated with varying degrees of galactose, α2-6-linked sialic acid, and bisected N-acetylglucosamine. The health status of an individual is related to galactosylation and fucosylation considering structural integrity and conformation of Fc domain. MS analysis of enriched human IgG shows 13 N-linked glycans with high intensities and clear background (Fig. 4). There is a shift of 23 in m/z values of identified N-glycans due to sodium adduct formation. The nanohybrid has enriched fucosylated as well as N-acetylglucosamine N-glycans which are important in studying underlying health issues.

Figure 4.

MALDI-MS spectra recorded for N-linked glycans from human IgG enriched with cellulose/1,2-Epoxy-5-hexene/asparagine. Each N-linked glycan peak has m/z value with 1 Na+, composition and structure

3.3. Material Validation

Validation parameters like selectivity, sensitivity, and intra-laboratory reproducibility (precision) are tested through enrichments by cellulose/1,2-Epoxy-5-hexene/asparagine. Selectivity is evaluated by using ovalbumin N-linked glycans with different amounts of BSA as interfering protein. When ratio is 1:100, 1:200, 1:300, 1:400, 1:500 and 1:600, total of 22, 21, 20, 19, 17, and 16 N-linked glycans are detected (Fig. S5 a–f). When ratios are increased to 1:1000, and 1:1100, still strong signal intensities of 21 and 17 N-linked glycans are detected (Fig. S6 a–b).

To test the sensitivity, series of OVA N-linked glycans with different concentrations (100 pM, 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, 0.1 fM) are enriched with nanohybrid/asparagine. At the concentrations of 100 pM, 10 pM, 1 pM, 100 fM, 10 fM and 1 fM, a total of 22, 23, 21, 21, 18 and 15 N-linked glycans are detected (Fig. S7 a–f). When concentration decreases to 0.1 fM, 11 N-linked glycans are traced (Fig. S8). The achieved sensitivity is down to fM levels. There are number of materials reported in literature for glycan/glycopeptide enrichment. For comparative view (Table S2), tabular data is collected for reported HILIC-based materials, and parameters such as selectivity, sensitivity, and serum sample results are provided. The comparison provides perspective that previous materials have been reported with low sensitivity which might result in lower number of N-glycans detected from serum samples.

For intra-laboratory reproducibility, three enrichment batches are conducted. MS analysis demonstrates similar peak patterns of same intensities (Fig. S9). The nanohybrid/asparagine shows three-time reusability for enrichment of N-linked glycans. Twenty N-linked glycans could be detected third time after which the mass spectrum shows minor changes (Fig. S10).

3.4. Serum N-Linked Glycome Profiling

There is strong link between human serum glycans and diseases [32]. Trend follows the use of single molecule biomarker for diagnosis, progression, or prognosis but complete glycome profiles may work as signatures for certain diseases. Up/down regulation of fucosylation, change in expression of β1,6-N-acetylglucosamine, structural variation in N-linked glycan profiles can be linked to cancer progression or long-term prognosis. Glycans released from human serum glycoproteins after PNGase F treatment are profiled after their enrichment by nanohybrid/asparagine. MS profile after the enrichment identifies 52 N-linked glycans (Fig. 5). In serum N-glycans profile, 36 (69.23%) detected N-glycans belong to complex type whereas 16 (30.07%) are high-mannose. A comparison with similar studies shows the highest number of N-linked glycans identified using SPE based HILIC enrichment by cellulose/polymer/asparagine. Table S4 lists the comparison where most materials are carbon based with compromised hydrophilicity. The provided MS profile can be referred as the complete healthy human serum N-linked glycans profile reported so far.

Figure 5.

MALDI-MS spectra recorded for N-linked glycans from human serum enriched with cellulose/1,2-Epoxy-5-hexene/asparagine. Each N-linked glycan peak has m/z value with 1 Na+, composition and structure

4. Conclusion

HILIC based SPE material, cellulose/1,2-Epoxy-5-hexene/asparagine has been developed, characterized, and optimized for glycome profiling. Asparagine induces greater hydrophilicity and nanohybrid fabrication gives high surface area resulting in the detection of N-linked glycans with lower detection limit, improved selectivity, good recyclability, and biological compatibility. Human serum profiling provides N-linked glycome profile explaining the structural diversity of N-linked glycans. Conclusively, cellulose based polymeric nanohybrid immobilized with asparagine is a potential HILIC material for enriching and identifying N-linked glycans from model glycoproteins and complex serum samples.

List of Abbreviation

BET

Brunauer–Emmett–Teller

CS

Chitosan (CS)

DEAE

Diethyl aminoethyl

EDX

Energy dispersive X-ray

fM

femtomole

HBr

Hydrogen bromide

HA

Hyaluronan

IgG

Immunoglobulin G

LbL

Layer-by-layer

MOFs

Metal organic frameworks

MNPs

Magnetic nanoparticles

OVA

Chicken egg albumin

pM

pM picomole

PEI

Polyethyleneimine

TFA

Trifluoroacetic acid