Site-specific glycan heterogeneity characterization by HILIC solid-phase extraction, RPLC fractionation, and capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry

Abstract

Reversed-phase chromatographic separation of glycopeptides tends to be dominated by the peptide composition. In contrast, capillary zone electrophoresis separation of glycopeptides is particularly sensitive to the sialic acid composition of the glycan. In this paper, we combine the two techniques to achieve superior N-glycopeptide analysis. Glycopeptides were first isolated from a tryptic digest using HILIC solid-phase extraction. The glycopeptides were separated using reversed-phase UHPLC to generate four fractions corresponding to different peptide backbones. CZE-ESI-MS/MS was used to analyze the fractions. We applied this method for the analysis of alpha-1-acid glycoprotein (AGP). A total of 268 site-specific N-glycopeptides were detected, representing eight different glycosylation sites from two isomers of AGP. Glycans included tetra-sialic acids with multi N-acetyllactosamine (LacNAc) repeats and unusual pentasialylated terminal sialic acids. Reversed-phase UHPLC coupled with CZE generated ~35% more N-glycopeptides than direct reversed-phase UHPLC-ESI-MS/MS analysis and ~70% more N-glycopeptides than direct CZE-ESI-MS/MS analysis. This approach is a promising tool for global, site-specific glycosylation analysis of highly heterogeneous glycoproteins with mass-limited samples.

Graphical Abstract

Glycosylation is one of the most common protein post-translational modifications, with at least 50% of the cellular proteome predicted to be glycosylated.^1,2 Aberrations in protein glycosylation have been implicated in a variety of diseases, such as immunological disorders,³ neurodegenerative diseases,⁴ and cancers.^5,6 Characterization of these aberrations, especially in the distribution of N-glycoforms at specific sites on proteins, should offer diagnostic and prognostic information, and the characterization of the site-specific microheterogeneity of protein N-glycosylation has drawn recent interest.^7–10

Reversed-phase liquid chromatography (RPLC) is commonly coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) for analysis of site-specific heterogeneous structures of glycopeptides. RPLC primarily separates N-glycopeptides based on the peptide’s hydrophobicity, and the glycan has relatively little effect on the separation. As a result, the retention time for a glycopeptide will be similar to the retention time of the deglycosylated peptide, and N-glycopeptides that share the same peptide backbone will elute with similar retention times.¹¹ In this narrow elution window, both the hydrophilicity and the number of terminal sialic acids on glycans contribute to the glycopeptide elution order on RPLC. The retention time is increased with an increase in sialic acid content and decreased by an increase in the number of monosaccharide units.^11,12

Capillary zone electrophoresis (CZE) coupled with ESI-MS/MS is emerging as a useful tool in proteomic analysis.^13–15 CZE is an orthogonal separation mode to RPLC, producing remarkably efficient separations based on differences in the charge-to-size ratio of peptides. We have shown that CZE coupled with an electrokinetically-pumped nanoelectrospray interface generated much higher peak intensities than reversed-phase UHPLC-ESI-MS for a wide range of peptides.¹⁶

CZE has also been shown to be a powerful tool for resolving glycans.^17,18 In particular, the electrophoretic migration order of glycans is strongly influenced by the number of sialic acids, each of which carries a negative charge.¹⁹ Glycans on the same peptide backbone migrate in the order of increasing numbers of sialic acids.²⁰

In this work, we take advantage of the complementary separations produced by RPLC and CZE for N-glycopeptides. Glycopeptides were first isolated from a tryptic digest using HILIC solid-phase extraction. The extracted glycopeptides were then fractionated with reversed-phase UHPLC, in which glycopeptides with the same peptide backbone were isolated. Each fraction was subsequently subjected to CZE-ESI-MS/MS for the second dimension separation and MS analysis. We also explored this approach for glycan structure characterization with manual precursor mass matching, because reversed-phase separation offers a retention time criterion for confirming peptide sequence, while CZE allows the estimation of the number of sialic acids in the glycan.

CZE produces extremely high sensitivity, favoring a second dimension analysis of fractions collected from single reversed-phase UHPLC run. As an alternative to the conventional two dimension high/low-pH RPLC-ESI-MS/MS method employed for intact N-glycopeptide analysis,¹² this system has much lower sample consumption, which is essential for the analysis of mass limited samples.

EXPERIMENTAL SECTION

Materials and Reagents

Bovine pancreas TPCK-treated trypsin (T1426), α1-Acid Glycoprotein (AGP, human plasma), dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA), and formic acid (FA) were purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile (ACN) and hydrofluoric acid (HF) were purchased from Fisher Scientific (Pittsburgh, PA). Methanol and water were purchased from Honeywell Burdick & Jackson (Wicklow, Ireland).

Protein Digestion

AGP (200 μg) dissolved in 50 mM NH₄HCO₃ (pH 8.0) containing 8 M urea at a concentration of 1 μg/μL was denatured at room temperature for 10 min, reduced by the addition of 2 μL of 500 mM DTT at room temperature for 1 h and alkylated with 5 μL of 500 mM IAA in the dark at room temperature for 30 min. Alkylated proteins were then transferred to a Microcon® −10 centrifugal filter unit (Merck, Darmstadt, Germany) for sample cleanup.²¹ Excess reagents were first removed by centrifugation for 30 min at 18,000 g. The proteins on the membrane were then washed three times with 200 μL of 50 mM NH₄HCO₃ (pH 8.0) via centrifugation at 16,000 g for each 15 min wash. Subsequently, the proteins were redissolved in 100 μL of 50 mM NH₄HCO₃ (pH 8.0) and transferred to a new centrifuge tube. This step was repeated, and the protein solutions were pooled. Finally, trypsin was added into the protein solution at an enzyme/substrate ratio (m/m) of 1:50 and incubated for 16 h at 37 °C. After terminating the digestion with TFA, the protein digest was lyophilized with a vacuum concentrator (Thermo Fisher Scientific, Marietta, OH)

Glycopeptide Enrichment

Glycopeptide enrichment was performed with a locally-constructed hydrophilic interaction chromatography (HILIC) solid phase extraction (SPE) column packed with ZIC^® glycocapture resin (ProteoExtract^® Glycopeptide Enrichment Kit, EMD Millipore, Billerica, MA). The AGP digest (~200 μg) was redissolved in 200 μL of ZIC^® loading buffer and then loaded onto two equilibrated HILIC SPE with 100 μL for each. After centrifugation at 1500×g for 2 min, the SPE column was washed with 300 μL of ZIC^® loading buffer four times to remove the non-specifically adsorbed peptides and then eluted twice with 100 μL of ZIC^® eluting buffer. The eluted glycopeptide fraction from the two HILIC SPE columns was collected, combined, and lyophilized using a vacuum concentrator. The dried glycopeptide fraction was stored at −20 °C before further analysis.

Reversed-phase UHPLC-ESI-MS/MS analysis

Unfractionated AGP glycopeptides were analyzed by a reversed-phase UHPLC-ESI-MS/MS system consisting of a nanoACQUITY UltraPerformance LC^® (UPLC^®) M-Class system (Waters, Milford, MA, USA). Chromatography was performed using a commercial C18 reversed phase column (Waters, 100 μm ×100 mm, 1.7 μm particle BEH130C18). Solvents A (0.1% FA in H₂O) and B (0.1% FA in ACN) were used to establish a 80 min gradient, comprised of 14 min of 2% B, then 1 min of 2–8% B, 60 min of 8–28%, 1 min of 28–80% B, and finally maintained at 80% B for 4 min, with the flow rate at 0.7 μL/min. The column was equilibrated for 15 min with 2% B at 0.7 μL/min before the run. AGP glycopeptide sample was dissolved in 30 μL of 2% ACN with 0.1% FA. The injection volume is 1 μL (with glycopeptides from ~6.7 μg of initial digest).

A Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) was operated in positive mode with a 1.8 kV applied spray voltage. Full MS scans were acquired in the Orbitrap mass analyzer over m/z 700–2000 with a resolution of 60000. The top-ten HCD scans per full scan were acquired with a normalized collision energy of 28% and an isolation window of ±2.0 Da. The resolution of the MS/MS scan was set at 15000. One microscan was set for each MS and MS/MS scan. The target value was 3.00 × 10⁶ and the maximum injection time was 50 ms. The dynamic exclusion duration was 30s.

Reversed-phase UHPLC-CZE-ESI-MS/MS analysis

Offline reversed-phase UHPLC prefractionation was performed based on the same chromatography condition as the direct reversed-phase UHPLC-ESI-MS/MS analysis mentioned above, including both the commercial C18 reversed phase column and separation gradient. Briefly, 1 μL of AGP glycopeptides (from ~6.7 μg of initial digest) was loaded onto the C18 column at a flow rate of 0.7 μL/min. An 80 min mobile phase gradient was generated using Solvent A and B. Eluates from 6.0 to 15.0 min, 27.0 to 33.0 min, 33.1 to 40.0 min, and 40.1 to 50 min were collected and lyophilized, to generate four fractions for the second dimension CZE-ESI-MS/MS analysis.

CZE separation of AGP glycopeptide fraction was performed using an uncoated fused silica capillary (20 i.d. × 150 μm o.d. × 46 cm length, Polymicro Technologies, Phoenix, AZ). CZE was coupled to ESI-MS/MS via a third generation electrokinetically pumped sheath-flow interface (Figure S-1).²² Briefly, an emitter was prepared from a borosilicate glass tube (0.75 mm i.d. × 1.0 mm o.d. × 10 cm length), which was pulled to a ~20 μm diameter tip using a P-1000 flaming/brown micropipet puller (Sutter Instruments, Novato, CA). One end of the separation capillary was etched to reduce the o.d. to ~50 μm using hydrofluoric acid (Caution: use appropriate safety procedures while handling HF solutions). The electrospray interface was constructed from a plastic cross. The emitter was placed in one arm of the cross. The separation capillary was threaded through the cross into the emitter. The other two arms of the cross were connected to a sheath buffer reservoir with plastic tubing and to a syringe filled with sheath buffer for system flushing. The sheath electrolyte was 10% (v/v) methanol and 0.5% (v/v) FA.

The dried AGP glycopeptide fraction was resuspended in 2 μL of 0.1% NH₃.H₂O (~pH 10) to adjust the pH to ~8. The injection end of the capillary was fixed in a block that allowed pumping fluids with either pressure or voltage.²³ Samples were injected hydrodynamically for 2.5 seconds at 15 psi, producing an injection volume of ~2.2 nL. The background electrolyte for the separation was 5% CH₃COOH. The capillary was rinsed at 30 psi for 15 min with 1 M NaOH, water, and separation electrolyte prior to injection.

Separation voltage was provided with a Spellman CZE 1000R high-voltage power supplies; 26 kV was applied at the injection end of capillary and 2.0 kV was applied at the sheath buffer reservoir for electrospray. The power supplies were controlled by LabView software.

The operating parameters for Q Exactive HF mass spectrometer were the same as that used in direct reversed-phase UHPLC-ESI-MS/MS analysis, except the dynamic exclusion duration was set as 5 s.

Data Analysis

Reversed-phase UHPLC-ESI-MS/MS and reversed-phase UHPLC-CZE-ESI-MS/MS data were analyzed with Xcalibur software (Thermo Fisher Scientific). The glycopeptide structures were manually characterized by matching experimental precursors with theoretical ones in MS and experimental fragment ions with theoretical ones in MS/MS. Four abbreviations are used to represent monosaccharide residues: HexNAc, N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc); Hex, galactose or mannose; Fuc, fucose; and Neu5Ac, N-acetylneuraminic acid.¹² Glycan compositions are represented by assigning the existing number of each monosaccharide with the order of HexNAc; Hex; Fuc; and NeuAc. For example, G6704 represents a glycan composition of 6 HexNAc, 7 Hex, 0 Fuc, and 4 Neu5Ac.

RESULTS AND DISCUSSION

Our workflow is shown in Figure 1. Glycopeptides were isolated from a tryptic digest using HILIC solid-phase extraction. The enriched fractions were then used for three experiments. First, the peptides were deglycosylated and subjected to reversed-phase UHPLC analysis; this procedure provided the peptide backbone structure. Second, the intact glycopeptides were analyzed by reversed-phase UHPLC-ESI-MS/MS to provide a benchmark for the number of glycoform identifications. Third, the intact glycopeptides were fractionated using reversed-phase UHPLC, and four fractions were analyzed using CZE-ESI-MS/MS.

Figure 1.

Workflow for the site-specific N-glycan characterization of AGP with a reversed-phase UHPLC-CZE-ESI-MS/MS approach.

We evaluated the system by analysis of human AGP, which is a plasma protein with molecular weight of 41–43 kDa. The protein is heterogeneously glycosylated on its five N-glycosylation sites.^24,25 The tryptic digest contains a variety of peptide backbone sequences combined with a complex set of N-glycans. The protein backbone has two isotypes, AGP1 and AGP2, which share two glycopeptide sequences after trypsin digestion, Sites II and III.^21,24

Reversed-phase UHPLC-ESI-MS/MS analysis of N-glycopeptides from AGP

The deglycosylated peptides were first analyzed by single-shot reversed-phase UHPLC-ESI-MS/MS. Nine peptide sequences covering the eight N-glycosylation sites in AGP1 and AGP2 were confirmed (Table 1, Figure 2A).

Table 1.

List of N-glycosylated sites detected from AGP

Glycosylation Site	Isotype	Peptide Sequence	Y1 ion m/z (charge)
Site I	AGP1	LVPVPITN15ATLDQITGK	992.09 (2+)
Site I	AGP2	LVPVPITN15ATLDR	806.46 (2+)
Site II	AGP1/2*	NEEYN38K	999.43 (1+)
Site III	AGP1/2*	YFTPN54KTEDTIFLR (68-81)	974.49 (2+)
		FYFTPN54KTEDTIFLR (67-81)	1048.02 (2+)
Site IV	AGP1	QDQCIYN75TTYLNVQR	1059.99 (2+)
Site IV	AGP2	QNQCFYN75SSYLNVQR	1062.49 (2+)
Site V	AGP1	EN85GTISR	979.47 (1+)
Site V	AGP2	EN85GTVSR	965.45 (1+)

^*AGP1/2 represents glycosylation sites shared in both isotypes

Figure 2.

Base peak chromatograms obtained for A) deglycosylated AGP peptides, B) intact AGP glycopeptides. C) MS spectra of Site III glycopeptides with YFTPN₅₄KTEDTIFLR (68-81) backbone at 35.16 min by reversed-phase UHPLC-ESI-MS/MS analysis. Symbols: :N-Acetylglucosamine, : Mannose, : Galactose, : N-acetylneuraminic acid, : Fucose.

The peptide backbone information facilitated the analysis of intact glycopeptides (Figure 2B). The glycosylated forms of the peptide generated a set of peaks with reversed-phase UHPLC-based separation, and intact glycopeptides with different peptide backbones tended to overlap, e.g. Site I (AGP2) coeluted with Site IV (AGP1 and AGP2) and Site III (67-61, AGP1/2) coeluted with Site I (AGP1). In addition, glycopeptides based on Site II and V from both AGP1 and AGP2 were too hydrophilic to be well resolved during the reversed-phase separation.

The reversed-phase UHPLC-ESI-MS/MS analysis of N-glycopeptides from AGP identified 197 N-glycopeptides (Table S-1 to S-5), which is more than reported in earlier studies of AGP site-specific glycan microheterogeneity.^21,24,26 We obtained a more comprehensive N-glycan characterization on Site III of AGP1/2 because of the dramatically increased MS intensity of the YFTPN₅₄KTEDTIFLR (68-81) and FYFTPN₅₄KTEDTIFLR (67-81) species, which were produced by the nonspecific trypsin digestion (commonly seen on Site I of AGP). ^21,24,26 The amino acid sequences of both YFTPN₅₄KTEDTIFLR and FYFTPN₅₄KTEDTIFLR were confirmed with MS/MS fragmentation, Figure S-2. Earlier work reported the peptide sequence of Site III as SVQEIQATFFYFTPN₅₄K, and a limited number of N-glycans had been assigned to that sequence.^21,24,26 The relative abundance of Site III glycopeptides was reported to be ~3%, the lowest of the glycosylation sites.²¹ As shown in Figure 2A, deglycosylated YFTPN₅₄KTEDTIFLR (Site III, 68-81) produced ~17% of the total abundance of the deglycosylated peptides. This improved intensity allowed identification of many bifucosylated N-glycans on Site III without lectin purification. Highly complex N-glycans with different numbers of sialic acid were also detected (Table S-3). Nevertheless, N-glycans on Site III with different numbers of N-acetyllactosamine (β-Gal(1,4)GlcNAc, LacNAc) and sialic acids tended to coelute using reversed-phase UHPLC under optimized conditions (Figure 2C).

CZE-ESI-MS/MS analysis of N-glycopeptides from AGP with reversed-phase UHPLC prefractionation

CZE separations are based on the analyte charge to size ratio, and CZE should be ideally suited for resolution of differentially sialylated glycans that co-elute in reversed-phase UHPLC. Four fractions were generated using reversed-phase UHPLC (Figure 2B). Fraction 1 contained glycopeptides from Site II (AGP1/2) and Site V (AGP1 and AGP2). Fraction 2 contained glycopeptides from Site I (AGP2) and Site IV (AGP1 and AGP2). Fraction 3 contained glycopeptides mostly from Site III (68-61, AGP1/2). Fraction 4 contained glycopeptides from Site III (67-61, AGP1/2) and Site I (AGP1). Each fraction was then subjected to the following CZE-ESI-MS/MS analysis.

CZE separation of fractions

An uncoated fused capillary (20 μm i.d. × 150 μm o.d. × 46 cm) coupled to a Q-Exactive HF mass spectrometer was used for CZE-ESI-MS/MS analysis of the four fractions generated by reversed-phase UHPLC separation of the intact glycopeptides. These separations were completed in 10 min (Figure S-3).

Figure 3 presents the CZE separation of reversed-phase UHPLC Fraction 1. CZE separated differentially sialylated glycopeptides from the same glycosylation site with resolution of greater than 2, Figure 3A. In contrast, glycopeptides containing the same number of sialic acids were separated with much lower resolution; glycan hydrodynamic volume has a much weaker influence on the intact glycopeptide migration compared with the negative charge associated with sialic acid.

Figure 3.

CZE-ESI-MS/MS analysis of AGP glycopeptide fraction collected at 6.0–15.0 min from Fraction 1 from the reversed-phase UHPLC separation. A) Base peak electropherogram. B) Summed MS spectra of monosialylated peptides of Site V migrating at 5.42–5.48 min. C) Summed MS spectra of pentasialylated peptides of Site V migrating at 8.4–8.5 min. Symbols: :N-Acetylglucosamine (HexNAc), : Mannose (Hex), : Galactose (Hex), : N-acetylneuraminic acid (Neu5Ac), : Fucose (Fuc). Glycan compositions were represented as G (HexNAc; Hex; Fuc; and NeuAc)

Resolution of glycopeptides based on different peptide backbones could also be observed in CZE, marked as green for Site II and yellow for Site V in Figure 3A. Among all of the factors that play roles in CZE separation of intact glycopeptides, the negative charge on sialic acid contributes most, thus enabling an observation of rearranged glycan-based separation of N-glycopeptides on CZE, which is complementary to conventional RPLC analysis.

We identified 91 N-glycopeptides from Fraction 1 by CZE-ESI-MS/MS analysis, which is nearly double the 55 N-glycopeptides identified with direct reversed-phase UHPLC-ESI-MS/MS analysis (Table 2). The isolation of low intensity CZE peaks that migrated at ~3, ~5.3, and ~8.4 min (Figure 3A) facilitated identification of asialylated Site II, monosialylated Site V (Figure 3B) and pentasialylated Site V (Figure 3C).

Table 2.

Identified AGP N-glycopeptides with Site II and V by reversed-phase UHPLC-ESI-MS/MS and reversed-phase UHPLC-CZE-ESI-MS/MS

Peptide Sequence	Glycoform		Theoretical m/z	Observed m/z
				Reversed-phase UHPLC-ESI-MS/MS	Reversed-phase UHPLC-CZE-ESI-MS/MS
NEEYN38K	Asialylated	G4500	806.9800 (3+)	806.9841 (3+)	806.9800 (3+)
		G4510	855.6660 (3+)	×	855.6675 (3+)
		G5600	928.6907 (3+)	×	928.6913 (3+)
	Monosialylated	G3401	1172.9476 (2+)	×	1172.9479 (2+)
		G4401	849.9942 (3+)	×	849.9919 (3+)
		G4501	904.0118 (3+)	904.0175 (3+)	904.0110 (3+)
		G4511	952.6978 (3+)	952.7016 (3+)	952.6961 (3+)
		G5601	1025.7225 (3+)	1025.7286 (3+)	1025.7202 (3+)
		G5611	1074.4085 (3+)	1074.4156 (3+)	1074.4064 (3+)
		G6701	1147.4333 (3+)	1147.4392 (3+)	1147.4376 (3+)
		G6711	1196.1192 (3+)	×	1196.1154 (3+)
	Bisialylated	G4502	1001.0436 (3+)	1001.0496 (3+)	1001.0468(3+)
		G4512	1049.7296 (3+)	1049.7356 (3+)	1049.7297(3+)
		G5502	1068.7367 (3+)	×	1068.7313(3+)
		G5602	1122.7543 (3+)	1122.7606 (3+)	1122.7540 (3+)
		G5612	1171.4403 (3+)	1171.4470 (3+)	1171.4415 (3+)
		G6702	1244.465 (3+)	1244.4731 (3+)	1244.4539 (3+)
	Trisialylated	G5603	1219.7861 (3+)	1219.7919 (3+)	1219.7837 (3+)
		G5613	1268.4721 (3+)	1268.4785 (3+)	1268.4686 (3+)
		G5623	1317.1581 (3+)	1317.1625 (3+)	1317.1570 (3+)
		G6703	1341.4969 (3+)	×	1341.4994 (3+)
		G6713	1390.1828 (3+)	×	1390.1760 (3+)
EN85GTISR	Asialylated	G4500	1199.9873 (2+)	×	1199.9850 (2+)
	Monosialylated	G4501	897.3593 (3+)	897.3635 (3+)	897.3583 (3+)
		G5601	1019.0700 (3+)	×	1019.0710 (3+)
		G5611	1067.7560 (3+)	×	1067.7578 (3+)
		G6701	1140.7808 (3+)	1140.7877 (3+)	1140.7837 (3+)
		G6711	1189.4667 (3+)	×	1189.4662 (3+)
		G6721	1238.1527 (3+)	×	1238.1527 (3+)
		G7801	1262.4915 (3+)	×	1262.4904 (3+)
		G7811	1311.1775 (3+)	×	1311.1776 (3+)
	Bisialylated	G4502	994.3911 (3+)	994.3967 (3+)	994.3925 (3+)
		G5602	1116.1018 (3+)	1116.1080 (3+)	1116.0985 (3+)
		G5612	1164.7878 (3+)	1164.7932 (3+)	1164.7898 (3+)
		G6702	1237.8234 (3+)	1237.8234 (3+)	1237.8124 (3+)
		G6712	1286.5132 (3+)	1286.5132 (3+)	1286.5021 (3+)
	Trisialylated	G5603	1213.1336 (3+)	1213.1404 (3+)	1213.1299 (3+)
		G5613	1261.8196 (3+)	1261.8257 (3+)	1261.8143 (3+)
		G5623	1310.5056 (3+)	1310.5090 (3+)	1310.5037 (3+)
		G6703	1334.8444 (3+)	1334.8511 (3+)	1334.8402 (3+)
		G6713	1383.5303 (3+)	1383.5378 (3+)	1383.5253 (3+)
		G6723	1432.2163 (3+)	×	1432.2021 (3+)
		G6733	1480.9023 (3+)	×	1480.8983 (3+)
		G7803	1456.5551 (3+)	1456.5598 (3+)	1456.5485 (3+)
		G7813	1505.2411 (3+)	×	1505.2319 (3+)
	Tetrasialylated	G6704	1431.8762 (3+)	1431.8826 (3+)	1431.8717 (3+)
		G6714	1480.5621 (3+)	1480.5699 (3+)	1480.5583 (3+)
		G6724	1529.2481 (3+)	1529.2537 (3+)	1529.2448 (3+)
		G6734	1577.9341 (3+)	1577.9352 (3+)	1577.9226 (3+)
		G7804	1553.5869 (3+)	1553.5935 (3+)	1553.5856 (3+)
		G7814	1201.9566 (4+)	1201.9644 (4+)	1201.9531 (4+)
		G7824	1238.4711 (4+)	×	1238.4658 (4+)
		G7834	1274.9856 (4+)	×	1274.9803 (4+)
		G8904	1256.7261 (4+)	1256.7307 (4+)	1256.7178 (4+)
		G8914	1293.2397 (4+)	1293.2472 (4+)	1293.2349 (4+)
		G8924	1329.7542 (4+)	×	1329.7559 (4+)
		G9(10)04	1348.0083 (4+)	×	1348.0022 (4+)
		G9(10)14	1384.5227 (4+)	×	1384.5321 (4+)
	Pentasialylated	G8905	1329.4990 (4+)	×	1329.5043 (4+)
		G8915	1366.0135 (4+)	×	1366.0149 (4+)
		G8925	1402.5280 (4+)	×	1402.5204 (4+)
		G9(10)05	1420.7821 (4+)	×	1420.7756 (4+)
		C9(10)15	1457.2966 (4+)	×	1457.2939 (4+)
EN85GTVSR	Monosialylated	G4501	892.6874 (3+)	×	892.6888 (3+)
		G5601	1014.3982 (3+)	1014.4027 (3+)	1014.3954 (3+)
		G5611	1063.0841 (3+)	1063.0928 (3+)	1063.0844 (3+)
		G6701	1136.1089 (3+)	1136.1135 (3+)	1136.1073 (3+)
		G6711	1184.7949 (3+)	1184.7996 (3+)	1184.7943 (3+)
	Bisialylated	G4502	989.7192 (3+)	989.7225 (3+)	989.7205 (3+)
		G5602	1111.4230 (3+)	1111.4332 (3+)	1111.4310 (3+)
		G5612	1160.1159 (3+)	1160.1206 (3+)	1160.1124 (3+)
		G6702	1233.1407 (3+)	1233.1456 (3+)	1233.1334 (3+)
		G6712	1281.8267 (3+)	1281.8317 (3+)	×
	Trisialylated	G5603	1208.4618 (3+)	1208.4672 (3+)	1208.4576 (3+)
		G5613	1257.1477 (3+)	1257.1548 (3+)	1257.1482 (3+)
		G6703	1330.1725 (3+)	1330.1768 (3+)	1330.1670 (3+)
		G6713	1378.8585 (3+)	1378.8638 (3+)	1378.8590 (3+)
		G6723	1427.5444 (3+)	×	1427.5387 (3+)
	Tetrasialylated	G6704	1427.2043 (3+)	1427.2097 (3+)	1427.1981 (3+)
		G6714	1475.8903 (3+)	1475.8972 (3+)	1475.8844 (3+)
		G6724	1524.5762 (3+)	1524.5873 (3+)	1524.5752 (3+)
		G7804	1548.9150 (3+)	1549.2635 (3+)	1548.9236 (3+)
		G7814	1198.4527 (4+)	1198.4531 (4+)	1198.4491 (4+)
		G7824	1234.9672 (4+)	×	1234.9587 (4+)
		G8904	1253.2222 (4+)	1253.2255 (4+)	1253.2178 (4+)
		G8914	1289.7358 (4+)	1269.7380 (4+)	1289.7334 (4+)
		G9(10)04	1344.5044 (4+)	×	1344.7554 (4+)
		G9(10)14	1381.0188(4+)	×	1381.2648 (4+)
	Pentasialylated	G8905	1325.9951 (4+)	×	1326.0023 (4+)
		G8915	1362.5096 (4+)	×	1362.5182 (4+)
		G8925	1399.0241 (4+)	×	1339.0211 (4+)
		G9(10)05	1417.2782 (4+)	×	1417.2744 (4+)

Pentasialylated glycopeptides have rarely been reported.²⁷ Their low mobility results in excellent resolution from other components. Based on the Y1 ions detected in tandem MS analysis, pentasialylated glycans were only assigned on Site V. A dramatically increased relative intensity of sialic acid oxonium ion in MS/MS was observed for glycopeptides migrating at ~8 min compared to those at ~5 min, which further supports our assignment (Figure S-4). We identified 69, 49, and 59 glycopeptides from Fractions 2, 3, and 4, respectively.

Intense peak

In addition to providing a complementary separation to reversed-phase UHPLC, CZE also generated more intense peaks. The fractions for CZE-ESI-MS/MS analysis were collected from single reversed-phase UHPLC separation of AGP glycopeptides (enriched from 0.16 nmol of initial digest). Roughly 0.1% of each fraction was injected onto the CZE column, corresponding to an initial digest amount of 0.18 fmol. Despite this small injection amount, the observed base peak intensity is comparable with that obtained by reversed-phase UHPLC-ESI-MS/MS analysis of much larger sample amounts (Figure 2B and Figure S-3).

We observed a series of novel glycoforms on Site III by direct reversed-phase UHPLC-ESI-MS/MS. These novel glycoforms were relatively easy to detect because Site III glycopeptides (68-81) produced high peak intensity and were only slightly overlapped with other glycopeptides. In contrast, the second dimensional CZE-ESI-MS/MS facilitated identification of additional glycoforms on Site III, including monosialylated and highly complex tetrasialylated species (Table S-3).

As shown in Figure 4A, there was a systematic decrease in peak intensity as the numbers of fucose and LacNAc repeat increased on the tetrasialylated glycans of Site III, for both reversed-phase UHPLC-ESI-MS/MS and CZE-ESI-MS/MS analysis. CZE-ESI-MS/MS generated half of the peak intensity as reversed-phase UHPLC-ESI-MS/MS for the highly abundant G6704 glycopeptide. However, the differences in peak intensities between these two approaches narrowed as the glycopeptides abundance decreased. CZE-ESI-MS/MS produced more intense peaks for the low abundant G7814, G7824, G8904 and G9(10)04 glycopeptides, which allowed the detection of highly complex, multi-fucosylated tetrasialylated glycans on AGP Site III (Table S-3, Figure S-5).

Figure 4.

A) Extracted ion separations of tetrasialylated Site III glycopeptides with the YFTPN₅₄KTEDTIFLR (68-81) backbone by reversed-phase UHPLC-ESI-MS/MS and CZE-ESI-MS/MS analysis. B) Venn diagrams of identified N-glycopeptides by direct reversed-phase UHPLC-ESI-MS/MS, CZE-ESI-MS/MS, and two-dimensional reversed-phase UHPLC-CZE-ESI-MS/MS analysis of the glycopeptides enriched from the AGP digest.

AGP glycosylation characterization

Reversed-phased UHPLC-CZE-ESI-MS/MS analysis identified 268 N-glycopeptides from AGP, which is a 35% increase over our direct reversed-phase UHPLC-ESI-MS/MS analysis (Figure 4B), which was an improvement over previous reports.^21,24,26 A number of newly characterized N-glycans were primarily located on Site III (AGP1/2) and Site V (AGP1 and AGP2), which appear to have a more complicated glycan distribution compared with other sites of AGP. Besides the monosialylated and pentasialylated Site V, highly branched Site III with various numbers of sialic acid were found (Table 2 and Table S-3).

Six known glycopeptides were not detected on Site IV (AGP1 and AGP2) with our approach (Table S-4). The failure to observe these peptides is likely due to the very similar charge to size ratio of the peptide backbone for Site I (AGP2) and Site IV. Glycopeptides with the same number of sialic acids from these two sites in Fraction 2 co-migrated during CZE separation, causing some low abundant Site IV glycopeptides to be suppressed by glycopeptides from Site I (AGP2). However, since Site IV and Site I (AGP2) did not share tetrasialylated glycans, CZE produced an efficient separation and detection of multiple tetrasialylated glycopeptides based on Site IV (Table S-4), demonstrating the complementary identification of reversed-phase UHPLC-ESI-MS/MS and CZE-ESI-MS/MS for complex glycopeptide mixtures.

Comparison with single-shot CZE-ESI-MS/MS

Unfractionated AGP glycopeptides were also analyzed with single-shot CZE-ESI-MS/MS. An uncoated fused capillary (20 μm i.d. × 150 μm o.d. × 80 cm) was coupled to a Q-Exactive HF mass spectrometer for this experiment (see Supporting Information). A total of 159 site-specific N-glycopeptides representing eight different glycosylation sites from AGP1 and AGP1 were identified (Table S-1 to S-5). The electrophoretic migration order of N-glycopeptides was strongly influenced by the negative charges associated with sialic acids (Figure S-6), facilitating the detection of pentasialylated Site V and tetrasialylated Site IV, which is complementary to the reversed-phased UHPLC-ESI-MS/MS results (Figure 4B). The separation of different peptide backbones could also be observed, but with lower resolution than reversed-phase UHPLC. We attributed the identification of a modest number of AGP N-glycopeptides by single-shot CZE-ESI-MS/MS to its relatively narrow separation window for peptide backbone differentiation, especially when the sample was complex in both peptide backbone and glycan components. Finally, the reversed-phased UHPLC-CZE-ESI-MS/MS approach generated ~70% more N-glycopeptides from AGP over direct CZE-ESI-MS/MS analysis (Figure 4B), demonstrating the value of applying two dimensional separations of N-glycopeptides from highly glycosylated proteins for comprehensive site-specific glycopeptide characterization.

Migration time-assisted N-glycopeptide characterization

The use of CZE for analysis of reversed-phase UHPLC glycopeptide fraction facilitated glycan structural elucidation. The first dimension reversed-phase UHPLC separation of deglycosylated peptides enabled the identification of peptide backbones. Glycopeptides with known peptide sequences were generated by reversed-phase UHPLC fractionation of the intact glycosylated peptides. The second dimension separation by CZE of the glycopeptides produced high-resolution based on the number of sialic acids, which produced a migration time-based prediction of the number of sialic acids. The precursor mass matching of successively eluted precursors observed in a particular cluster become straightforward. Assignment could be processed following the N-glycan growing on the lowest-mass glycopeptide. With 10 ppm mass tolerance window and migration time correction, site-specific glycan structures were characterized with high confidence, even when MS/MS spectra had poor quality. Additionally, the glycopeptide MS spectra could be assigned using commercial software such as Byonic and others. Automated spectral interpretation would simplify analysis of a large number of reversed-phase UHPLC fractions to achieve a more comprehensive characterization of complex samples.