Accepted Manuscript

Comparison of Two Approaches for Quantitative O-linked Glycan AnalysisUsed in Characterization of Recombinant Proteins

Iva Turyan, Xaoping Hronowski, Zoran Sosic, Yelena Lyubarskaya

PII: S0003-2697(13)00499-5DOI: http://dx.doi.org/10.1016/j.ab.2013.10.019Reference: YABIO 11532

To appear in: Analytical Biochemistry

Received Date: 7 June 2013Revised Date: 9 October 2013Accepted Date: 11 October 2013

Please cite this article as: I. Turyan, X. Hronowski, Z. Sosic, Y. Lyubarskaya, Comparison of Two Approaches forQuantitative O-linked Glycan Analysis Used in Characterization of Recombinant Proteins, AnalyticalBiochemistry (2013), doi: http://dx.doi.org/10.1016/j.ab.2013.10.019

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Comparison of Two Approaches for Quantitative O-linked Glycan Analysis Used in

Characterization of Recombinant Proteins

Iva Turyan, Xaoping Hronowski#, Zoran Sosic, Yelena Lyubarskaya

Analytical Development, Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142, USA #Analytical Biochemistry, Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142, USA

Short title: Quantitative O-linked Glycan Analysis

Corresponding author:

Iva Turyan

Analytical Development

Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142, USA

phone: 617-914-0846

E-mail address: iva.turyan@biogenidec.com

Subject categories: carbohydrates; protein structure and analysis

2

Abstract

The principal aim of this manuscript is to demonstrate the optimization and fine tuning of

quantitative and non-selective analysis of O-linked glycans released from therapeutic

glycoproteins. Two approaches for quantitative release of O-linked glycans have been examined.

These include ammonia-based β-elimination and hydrazinolysis deglycosylation strategies. A

significant discrepancy in deglycosylation activity has been observed between ammonia-based

and hydrazinolysis procedures. Specifically, the release of O-glycans from glycoproteins has

been about 20-30 times more efficient with hydrazine compared to ammonia-based β-elimination

reagent. In addition, the ammonia-based reagent has demonstrated bias in the release of

particular glycan species. A robust quantitative hydrazinolysis procedure has been developed for

characterization of O-glycans. The method performance parameters have been evaluated. It has

been shown that this procedure is superior for quantitative non-selective release of O-glycans.

Identity confirmation and structure elucidation of O-glycans from HILIC fractions has also been

demonstrated using Linear Ion Trap Fourier Transform Mass Spectrometry (LTQ FT MS) with

the mass accuracy below 1 ppm.

Key words: O-linked glycan analysis, HILIC, LTQ FT MS.

List of the abbreviations: Linear Ion Trap Fourier Transform Mass Spectrometry (LTQ FT MS);

ethylamine (ETA); dimethylamine (DMA); hydrophilic interaction chromatography (HILIC); 2-

aminobenzamide (2-AB); solid phase extraction (SPE); O-GlcU glycans (O-glycans composed of

glucuronic acid); glycosaminoglycan (GAG); epidermal growth factor (EGF), proteoglycans

(PGs).

3

Introduction

Glycosylation is one of the most common and often rather complex post-translational

modifications affecting pharmacokinetics, pharmacodynamics and/or efficacy of glycoprotein

therapeutics. Therefore, glycosylation analysis has been one of the most critical tools in the

characterization of therapeutic proteins [1].

Oligosaccharide profiling of released N and/or O-glycans is one of the most common

methods for monitoring glycosylation consistency in biopharmaceutical process development.

The lack of an enzyme that ubiquitously cleaves O-glycans creates a need for the development of

chemical methods for O-glycan cleavage. Classically, alkaline beta elimination has been used for

deglycosylation of proteins [2-9]. Because of the high pH, the released carbohydrate chains can

be destroyed (peeling reaction) and a reducing agent, such as sodium borohydride, is added to

stabilize them. The reducing agent converts O-glycans to alditols and prevents the reductive

amination needed for the attachment of a fluorophore or chromophore. Therefore, pulsed

amperometric detection (PAD) is usually used for downstream analysis [10-17]. Isolation of

released glycans from the peptide-derived material produced by β-elimination is critical due to

peptide interference during PAD detection [11]. The approach also suffers from the excessive

cleaning required to remove the high salt content, which results in significant sample loses.

To amplify a signal for oligosaccharide detection after β-elimination in alkaline

borohydride solution, a mild periodate oxidation of oligosaccharide-alditols has been proposed

[18]. Specifically, the oligosaccharide-alditols are oxidized with sodium meta-periodate, labeled

with 2-aminopyridine, and analyzed by reverse-phase chromatography with fluorescence

detection. Alternatively, NaB3H4 is used to label the reduced glycans [18].

Recently, some attractive strategies for non-reductive beta elimination have been

explored [19-30]. These include applications of ammonium hydroxide [19-21], 28% ammonium

hydroxide saturated with ammonium carbonate [22-23], borane-ammonia complex in aqueous

ammonia [24], 70% ethylamine (ETA) [25-27], 40% dimethylamine (DMA) [28], methylamine

vapor with partial acid hydrolysis [29] and ammonium carbamate [30]. Oligosaccharides are

released with an intact reducing end to which a chromophore or a fluorophore may be attached,

thereby increasing the sensitivity of the downstream analysis. Furthermore, peeling reactions

have been significantly reduced.

4

The efficiency of the non-reductive β-elimination procedure has remained a very

challenging problem. Ammonium-based alkali-catalyzed β-elimination [19-21] often poses an

issue for quantitative release of O-glycans. Moreover, while the released β1,4-linkage of the N-

glycan type has been shown to be stable under ammonium-based alkaline conditions, β1,3-

linkage is labile towards alkaline treatment, resulting in considerable peeling [21]. Ammonium

hydroxide saturated with ammonium carbonate has also been used to release O-glycans [22-23].

However, the quantitative aspects and selectivity of the procedure have not been confirmed.

β-Elimination with 70% ethylamine (ETA) has been preferred to triethylamine, sodium

hydroxide, and hydrazine [26]. However, a low yield of 20% and 40-50% is reported for acidic

and neutral oligosaccharides, respectively. In a different study, it has been shown that

approximately 70% of the oligosaccharides could be cleaved with 70% ETA, and quantitative

cleavage was achieved with 40% methylamine, at 50oC [25].

Application of methylamine for β-elimination reaction has been shown to result in

considerably higher release efficiencies compared to ammonia [29]. However, the efficiency of

the elimination-addition reaction has been different for glycosylated Ser and Thr residues [29].

Dimethylamine (DMA) has also been tested as a mild base, and microwave radiation was

applied to accelerate the substitution [28]. Improved yield has been reported with both bovine

fetuin and porcine stomach mucin compared to the classical strong base method [28]. Highly

efficient O-glycan release has been recently demonstrated with ammonium carbamate [30].

However, quantitative aspects have not been addressed.

Essentially, quantitative, non-biased release and recovery of intact glycans has been

reported using hydrazinolysis procedure [31-36]. Hydrazine releases glycans with a free reducing

end. The exact mechanism of hydrazinolysis is unknown, but it is generally agreed that it

proceeds via an initial β-elimination reaction followed by a reaction with hydrazine to form

hydrazine derivatives. Re-N-acetylation of the free amino groups with acetic anhydride is then

required to attach a chromophore or fluorophore, as N-acetyl moieties of all amino sugars are

hydrolyzed under reaction with hydrazine. Mild hydrazinolysis conditions (60oC, 4-6 h),

optimum sample preparation, and efficient recovery procedure are reported to result in high yield

of O-glycans with negligible degradation, regardless of the primary protein sequence [31].

Combination of β-elimination and hydrazinolysis has been used as a promising tool for O-glycan

analysis [37].

5

Overall, as reported in the literature to date, the most common methods used for O-glycan

characterization and quantitation are ammonia-based β-elimination and hydrosynolsis. However,

there are significant discrepancies in the reported data on deglycosylation efficiency by

ammonia-based β-elimination, and there is no consensus on the preferred deglycosylation

reagent for this procedure. Hydrazinolysis is reported to be an efficient deglycosylation

procedure, but it is quite lengthy and labor intensive, and thus is not very appealing in

biopharmaceutical development environment. Our goal was to assess the two procedures side by

side, and establish an optimum and robust approach to O-glycosylation analysis of

biopharmaceuticals. In this paper we summarize our recent findings, where the selection of a

deglycosylation reagent and optimization of deglycosylation reaction conditions led to a

simplified, robust and quantitative method for O-glycan analysis. The efficiency of ammonia-

based β-elimination deglycosylation strategy was examined. This procedure was then compared

to hydrazinolysis, performed using Glycan Hydrazinolysis Kit for O-deglycosylation and re-N

acetylation [38]. In both procedures, the glycan isolation and desalting was significantly

simplified using a solid-phase extraction (SPE) with Hypercarb cartridges. The released glycans

were derivatized with 2-aminobenzamide (2-AB) and purified from the excess dye by SPE using

GlycoClean S cartridges. The fluorescence labeled glycans were analyzed via hydrophilic

interaction chromatography (HILIC) with fluorescence detection. The data revealed that

ammonia-based β-elimination is much less efficient for glycan release than hydrazinolysis. In

addition, the preferential release of O-GlcU glycans was found using ammonia-based β-

elimination strategy. As a result, a simplified hydrazinolysis procedure was selected and further

optimized as a preferred method for O-glycan analysis.

Structural characterization of the released O-glycans has been done using high resolution,

high mass accuracy mass spectrometry, which evidently is the most powerful tool for structural

analysis of carbohydrates to date. Structure elucidation of O-glycans by MS is complicated due

to the presence of at least 8 different core structures, unlike the trimannosyl-chitobiose core

common to all N-glycans [39]. This makes the analysis of O-glycans rather challenging.

Reversed-phase LC-MS with triple quadrupole MS (data not shown) and fragmentation MS of

the HILIC fractions with Linear Ion Trap Fourier Transform Mass Spectrometry (LTQ FT MS)

were used for O-glycans identification and structural assignments. The application of hybrid

LTQ FT MS, which combines high resolution and high mass accuracy analysis in the FT MS

6

with automatic gain control and high sensitivity MSn was shown to be critical for the analysis of

subpicomole quantities of O-glycans.

Materials and methods

Materials

HPLC grade acetonitrile, HPLC grade water, and 1M sodium hydroxide were purchased

from Fisher Scientific (Fair Lawn, NJ, USA). Methyl sulfoxide (DMSO), 2-aminobenzamide (2-

AB), sodium cyanoborohydride (NaCNBH3), ethylamine (ETA), ammonium carbonate, and

trifluoroacetic acid (TFA) were acquired from Aldrich.

Formic acid, 25% ammonia solution, 30% acetic acid, and glacial acetic acid were

obtained from Sigma. Discover Labmate microwave was purchased from CEM Inc (Matthews,

NC). Glycan Hydrazinolysis kit, GlycoClean S (regular and miniaturized) cartridges, GlycoSep

N-Plus HPLC column (4.6 x 150 mm) was purchased from Prozyme. The Hypercarb cartridges

were acquired from Fisher Scientific. Bovine Fetuin was purchased from Prozyme.

Two model proteins were used in this study. Model Protein 1 is a recombinant fusion

protein comprised of a single molecule of human coagulation Factor IX (FIX, Thr 148 variant)

covalently linked to the Fc domain of a human antibody (IgG1 isotype) [40]. It was produced

from an embryonic kidney cell line (HEK293) in house. The protein has two N-glycosylation

sites located on the factor portion of the molecule and one N-glycosylation site located on the Fc

portion of the molecule. There are 4 potential O-glycan occupancy sites on the factor portion of

the protein. The protein was purified and formulated at 11.9 mg/mL in 25 mM Histidine

HCl, 0.01% Polysorbate 20, pH 7.2 formulation buffer. Model Protein 2 is a recombinant human

dimeric fusion protein that consists of two copies of the extracellular ligand-binding domain of

the human TNF-α receptor [41] linked to the Fc portion of human immunoglobulin IgG1. It was

produced in Chinese hamster ovary (CHO) cells in house. Each chain of model Protein 2

contains three N-glycosylation sites occupied with complex type carbohydrates. Two N-

glycosylation sites are located on the receptor portion of the molecule and one N-glycosylation

site is located on the Fc portion of the molecule. There are 13 potential O-glycan occupancy

sites, which are clustered at the hinge region of the molecule. The protein sample used for this

study was at 3.2 mg/mL in 50 mM sodium phosphate buffer, pH 7.0.

7

Ammonia-based β-elimination procedure

200 µg of protein were buffer exchanged into 0.1% TFA and dried. The dried samples

were pre-mixed with 200 µL 40% aqueous ETA, consisting of 20 mg/mL ammonium carbonate,

and subjected to chemical deglycosylation under microwave-assisted mode at 55oC for 1 h. For

comparison, the same amount of glycoprotein was also deglycosylated at 60oC for 18 h (normal

mode). In both modes, acidification with 100 µL of 50% acetic acid was performed at 4oC for 18

h (normal mode), and at 30oC for 30 min (microwave-assisted mode).

Hydrazinolysis procedure

200 µg of protein were buffer exchanged into 0.1% TFA and dried extensively (not less

than 24 hours). The release of O-glycans was achieved using hydrazine from the Glycan

Hydrazinolysis Kit [38]. Briefly, the dried samples were pre-mixed with 60 µL hydrazine and

incubated at 60oC for 6 hours. After incubation, the samples were cooled and dried to evaporate

unreacted hydrazine. Re-N-acetylation was achieved by incubating the samples with 10 µL of re-

N-acetylation reagent and 40 µL of re-N-acetylation buffer from the Glycan Hydrazinolysis Kit

for 10 min at 4oC, followed by 1h 30 min at room temperature with gentle shaking [38].

Isolation of the released O-glycans with hypercarb stationary phase

The isolation and desalting of released glycans was performed via SPE with Hypercarb

stationary phase. Briefly, Hypercarb cartridges were first prepared by washing with 3 mL of 1M

NaOH, 6 mL water, 3 mL of 30% acetic acid, 3 mL water followed by 3 mL of 50% acetonitrile

plus 0.1% TFA(v/v) in water (Solvent A), and 6 mL of 5% acetonitrile plus 0.1% TFA (v/v) in

water (Solvent B). This removes impurities and prepares the surface to absorb the glycans. The

glycans are then absorbed onto the membrane by leaving the samples for 15 min. To remove

residual salts and non-hydrophobic, non-glycan material off the column, the cartridges were then

washed with 3 mL water, followed by 3 mL of Solvent B. The glycans are collected by eluting

with 4 x 0.5 mL of Solvent A.

Derivatization and clean-up of 2-AB labeled O-glycans

8

The isolated and dried O-linked glycans were derivatized with 2-aminobenzamide (2-

AB). The 2-AB labeling-reaction took place by reductive amination at 65°C for 3 h. The samples

were cleaned-up from the excess of the dye using GlycoClean S cartridges. Briefly, GlycoClean

S cartridges were first prepared by washing with 1 mL of water, 2 x 2.5 mL of 30% acetic acid, 3

mL of acetonitrile, and 1 mL of acetonitrile. The glycans were let absorb onto the membrane for

15 min. The excess of the dye was then removed by washing the cartridge with 1 mL of

acetonitrile, followed by 5 x 1 mL of 96% acetonitrile solution, allowing each aliquot to drain

before the next was applied. The glycans were then collected by eluting with 3 x 0.5 mL of

water.

HILIC analysis

The dried samples were reconstituted with 250 µL of the mixture of Acetonitrile:H2O =

80:20 and transferred to HPLC vials. The fluorescence labeled glycans were analyzed via

hydrophilic interaction chromatography (HILIC) with fluorescence detection (λexcitation = 330 nm,

λemission = 420 nm) utilizing GlycoSep N-Plus HPLC column, 4.6 x 150 mm at 30oC. Acetonitrile

and 50 mM ammonium formate (pH 4.4) were used as mobile phases A and B, respectively. The

gradient started from 80% and reduced to 56.4 % mobile phase A for 34 min at 0.67 mL/min

followed by column wash at 100% mobile phase B for 5 min. Finally, the column was re-

equilibrated at 80% mobile phase A prior to the next injection. The HPLC-FL system was

controlled by Empower 2 software from Waters.

O-glycans identification

To elucidate O-glycan structures, HILIC peaks of 2-AB labeled O-glycans were collected

and analyzed using Thermo Scientific LTQ FT Ultra Hybrid mass spectrometer. 5x loading was

used for fractions collection. The collected fractions were dried and reconstituted in 10 µL of

water. 1 µL of each fraction was further diluted with a mixture of acetonitrile/water (1:1) and 1%

acetic acid. Approximately, 2 µL of the solution in a static nanospray needle (purchased from

New Objective) was infused into LTQ FT MS, which was equipped with a nanospray source.

The instrument was operated at the positive mode. FT MS and LTQ MS/MS spectra were

acquired.

9

Results and discussion

Comparison of O-glycan release by ammonia-based β-elimination and hydrazinolysis

procedures

Two deglycosylation approaches, ammonia-based β-elimination and hydrazinolysis, were

compared using model Protein 1 and Bovine Fetuin. The released and fluorescently labeled O-

glycans were analyzed by HILIC chromatography.

Fig. 1 depicts HILIC chromatograms of 2-AB labeled O-glycans released from model

Protein 1 and Fetuin under treatment with hydrazine and 40% ETA, 20 mg/mL ammonium

carbonate. Ammonia-based β-elimination strategy was tested under a microwave-assisted mode

at 55oC for 1 h. In addition, the same amount of glycoprotein was also deglycosylated at 60

oC for

18 h (normal mode, data not shown).

Different parameters governing the performance of both procedures have been studied in

details and optimized. A significant discrepancy in deglycosylation activity was observed

between hydrazinolysis and ammonia-based β-elimination procedures (Fig. 1). Specifically, the

release of O-glycans was about 20-30 times more efficient with hydrazine compared to

ammonia-based β-elimination reagent for both proteins. Ammonia-based β-elimination

procedure was tested using a traditional heating system (normal mode, 18 h) or employing

microwave irradiation (1 h). To determine the optimum reaction time upon hydrazinolysis,

kinetic studies were conducted using a traditional heating system, as microwave mode is not

compatible with the hydrazinolysis procedure. Model Protein 1 samples were subjected to

hydrazinolysis at 60oC for different time intervals. No significant differences were observed

among samples prepared by hydrazinolysis at 60oC within 4-6 hours (data not shown).

Hydrazinolysis at 60oC for 6 hours was chosen as the standard condition.

Fig. 2 shows HILIC chromatograms of 2-AB labeled O-glycans released from model

Protein 2 using hydrazinolysis and ammonia-based β-elimination procedures. Surprisingly,

ammonia-based β-elimination reagent showed a strong bias towards O-glycan peak at RT 22.4

min. The relative peak area of this species was 24.6% using ammonia-based β-elimination

procedure vs. 2.1 % by hydrazinolysis. Low level of O-glycan peak at RT 22.4 min using

hydrazinolysis was consistent with tryptic peptide mapping analysis (data not shown). Therefore,

10

in addition to an overall lower efficiency of O-glycan release by ammonia-based β-elimination

reagent, an undesirable selective release of a particular O-glycan type has been demonstrated.

It has been previously shown that hydrazinolysis allows for the non-specific release of O-

linked glycans (>90% O-linked and <10%N-linked) independent of the primary sequence of the

protein [31]. To evaluate the effectiveness of hydrazinolysis procedure, the remaining

deglycosylated model Protein 2 was subjected to hydrazinolysis once again. The efficiency of

hydrazinolysis was assessed through comparison the quantity of the O-glycan pool before and after

repeated deglycosylation using the peak areas from corresponding chromatograms. The peak

areas were normalized versus peak area of the internal standard, 2-AB labeled GalGalNAc,

spiked into each O-glycan sample pool. It was found that only 8.0% O-glycans was obtained

after repeated hydrazinolysis, indicating the high efficiency of glycan release. It should be

noticed that the relative peak areas were comparable after the first and the repeated

deglycosylation procedures. This confirms that under mild conditions (60oC, 6 hours),

hydrazinolysis allows for non-selective, quantitative release of O-linked glycans.

Solid Phase Extraction (SPE) with hypercarb stationary phase for glycan isolation and desalting

as a replacement of ion-exchange procedure

It has been shown that residual protein or peptide material should be removed to avoid

interferences with the downstream analysis, which uses fluorescence detection [39].

SPE with Hypercarb stationary phase was implemented to isolate released O-glycans.

Hypercarb cartridges allow for purification of glycans from non-cabrohydrate material, including

salts, proteins, and detergents. To evaluate the effectiveness of desalting and glycan isolation

with Hypercarb SPE, the following experiments were performed. Two sets of model Protein 1

samples were prepared for O-glycan analysis in triplicates using two procedures: 1) the desalting

procedure with the anion-exchanged resin from hydrozynolysis kit [38] followed by glycan

isolation with Hypercarb SPE cartridges and 2) sample desalting and glycan isolation using

Hypercarb SPE cartridges alone. The desalting with the anion-exchanged resin from

hydrozynolysis kit [38] is a tedious procedure. Moreover, the desalted samples still contain

residual protein/peptide material, therefore, an additional purification step is needed.

Relative distribution of 2-AB O-glycans released from model Protein 1 by hydrazinolysis

under both procedures is reported in Table 1. Method performance was assessed based on five

11

representative peaks (Table 1). The relative distribution of O-glycans between the two

procedures was shown to be comparable (Table 1). The overlay of O-glycan profiles in Fig. 3

demonstrates that the total amount of released glycans in samples prepared by the two

procedures is also comparable. This indicates that SPE Hypercarb cartridges are effective and

suitable for both, desalting and glycan isolation.

Evaluation of the completeness of the β-acetohydrazide derivatives cleavage

Re-N-acetylation of released glycans results in cleavage of the β-acetohydrazide

derivatives [38]. However, a small amount of these adducts may remain after the re-N-

acetylation reaction. It has been demonstrated that complete regeneration of the reducing end of

glycans can be achieved by mild hydrolysis with mineral or Lewis acid (5% TFA or 1 mM Cu

acetate in 1 mM acetic acid) [38]. To evaluate the completeness of the beta-acetohydrazide

derivatives cleavage after re-N-acetylation, the effect of mild hydrolysis with 5% TFA was

tested. Three replicate samples of model Protein 1 were prepared with and without the acid

hydrolysis and analyzed after the clean up and isolation using Hypercarb cartridges. Relative

distribution of 2-AB O-glycans released from model Protein 1 by hydrazinolysis with and

without the additional acidification step is reported in Table 2.

2-AB O-glycan analysis demonstrates no significant differences among samples

prepared with and without the additional acidification step. The data confirms that re-N-

acetylation alone results in the complete cleavage of β-acetohydrazide derivatives, while the

acidic environment of Hypercarb SPE cartridges used for glycan isolation assures the

regeneration of the reducing end of glycans.

Assessment of method performance parameters

Several performance parameters of O-glycan determination procedure have been

evaluated. To assess assay variability, intermediate precision, and limit of quantitation, samples

of model Protein 1 were subjected to hydrazinolysis at 60oC for 6 h. Repeatability data from the

analysis of O-glycans released from six samples of model Protein 1 on a single day is presented

in Table 3A. The calculated values of intra-assay precision for relative distribution of O-glycans

12

were determined to be in the range of 0.3-5.6% RSD. Intermediate precision was assessed by

performing the analysis of O-glycans released from model Protein 1 on three different days. The

data for intermediate precision over three days (n=6) is summarized in Table 3B. The calculated

values of intermediate precision (%RSD) for relative distribution of O-glycans were determined

to be in the range of 0.7-7.8 %. The results indicate good precision for monitoring O-glycan

distribution on different days.

To determine the variability associated with different analysts and different HILIC

column lots, model Protein 1 was analyzed by two different analysts using two different HILIC

columns. The data for relative distribution of major O-glycans obtained by two analysts using

different HILIC columns were in good agreement. The limit of quantitation (LOQ) for 2AB

labeled O-glycan analysis of model Protein 1 was found to be 0.4% of relative peak area. The

criteria for LOQ determination was the following: the %RSD value for replicate runs ≤ 20% of

the relative peak area and signal to noise ratio ≥ 10 (this set of data was obtained from 6

independent sample preparations and HPLC analysis performed on the same day).

The above findings demonstrate the reliability of the O-glycan determination procedure.

O-glycans identification using nanospray hybrid LTQ FT MS

Identification of glycan structure is an important part of biopharmaceutical product

characterization. Typical options for structure elucidation and identification of the released O-

glycans, such as comparing HILIC profiles of 2-AB labeled O-glycans released from model

Proteins and Fetuin, Sialidase A treatment and MS analysis with rpLC-MS have been explored

by us (data not shown). Ultimately, identification of O-glycan structures has been performed

with a nanospray hybrid LTQ FT MS, which allowed for confirmation of the known structures,

and identification of unknown glycans. The chromatographic fractions containing O-glycan

peaks have been collected, and further analyzed using static nanospray, as described in

“Materials and Methods”. The high resolution, high mass accuracy FT MS allowed assignment

of the precursor ions with the mass errors below 2 ppm [42]. The assigned precursor ions were

then analyzed by high sensitivity LTQ MS/MS. The unique parallel detection strategy allowed

for distinguishing O-glycan ions from high background noise and provided detailed structural

elucidation with high confidence of structural assignments.

13

Table 4 summarizes O-glycans identified from model Proteins 1 and 2. FT-MS and LTQ-

MS/MS fragmentation data confirmed the identities of mucin type O-glycans, i.e.,

monosialylated core 1 (peaks B, C) and disialylated core 1 (peaks D and E) [32].

The major structure of the O-linked sugar chain of Model Protein 1 was found to be

PentPentHex (peak elutes in HILIC run at 12.3 min, Table 4). Table 5A shows MS/MS

fragmentation data of the precursor ion m/z = 565.22412 (1+) in the linear ion trap mode. The

fragment ions of m/z = 433.18167 (1+) and m/z = 301.13943 (1+) were observed, revealing the

glycosidic bond cleavage with a loss of two pentoses. The remaining mass corresponds to Hex-

2AB. The loss of specific ions allowed for structural elucidation of O-glycan eluted at 12.3 min,

as PentPentHex-2AB (Table 4). XylXylGlc O-glycan was identified in the epidermal growth

factor (EGF)-like domains of human and bovine clotting factor VII (Ser-52), factor IX (Ser-53),

protein Z (Ser-53) and bovine platelet glycoprotein thrombospondin [43].

Two ions (m/z = 941.3731 (1+) and m/z = 650.27646 (1+)) elute in HILIC run at 19.4

min (Table 4, RT 19.4). The mass difference of 291.1 Da corresponds to the loss of NeuAc.

Table 5B presents MS/MS fragmentation data of O-glycan ion with m/z = 650.27646 (1+) in the

linear ion trap mode. Glycosidic bond cleavage follows by a sequential loss of Hex and HexNAc

from DeoxyHex-2AB. The fragmentation pattern indicates the structure of O-glycan peak eluted

at 19.4 min as NeuAcHexHexNAcDeoxyHex-2AB. This tetrasaccharide O-fucosidically linked

to Ser-61 was found in the first EGF-like domain of human factor IX [43].

The ammonia-based reagent, as described in Section 1, had a strong bias towards

releasing the O-glycan peak eluted in HILIC run at 22.4 min. (model Protein 2). The ion with

m/z =753.25588 (1+) was detected by FT-MS analysis under the HILIC peak at 22.4 min (Table

4). The observed fragment ions in the linear ion trap mode revealed a sequential loss of 162, 158

and 162 Da from the precursor mass (Fig. 4, Table 6A). The fragmentation data indicates the

presence of O-Pent glycan composed of dehydrated glucuronic acid: HexGlcUdehydratedHexPent-

2AB. MS/MS analysis of HILIC peak collected at RT 24.6 min (model Protein 2) revealed

another O-Pent glycan containing glucuronic acid: GlcU(Hex)2Pent (Fig. 5, Table 6B). GlcUβ(1-

3)Galβ(1-3)Galβ(1-4)Xyl was identified in recombinant human α-Thrombomodulin [44] as a

glycosaminoglycan (GAG)-protein linkage tetrasaccharide common to various proteoglycans

(PGs). It is considered as a biosynthetic intermediate of an immature GAG chain [44].

14

The levels of O-glycan containing glucuronic acid were found to be overestimated, when

ammonium-based chemical deglycosylation procedure was used. This strongly indicates that

choosing deglycosylation reagent is critical to provide non-selective analysis of O-linked glycans

released from therapeutic glycoproteins.

Conclusions

Although multiple means of O-glycan analysis have been described in literature to date, a

comprehensive quantitative analysis of O-glycans released from therapeutic proteins has shown

to be a challenge. Ammonia-based deglycosylation strategy, which is still widely used, has been

compared with the release of O-glycans by hydrazinolysis, and was shown to be suboptimal.

Specifically, significant discrepancy in deglycosylation activity and selectivity was found

between the ammonia-based and hydrazinolysis procedures. The method workflow for

hydrazinolysis has been significantly simplified by combining the tedious procedures for glycan

desalting and isolation. Thus, an overall improvement to the hydrazinolysis procedure has been

demonstrated. A robust quantitative method for Q-glycan analysis by hydrazinolysis has been

developed. HILIC chromatography with fluorescence detection has been used as an effective and

robust tool for determining the relative O-glycan distribution. Method performance parameters

have been evaluated and the method demonstrated excellent O-glycan release efficiency, good

robustness and reproducibility. The optimization and streamlining of hydrazinolysis is essential

for robust and quantitative analysis, which is currently a method of choice for O-glycan analysis.

The LTQ FT hybrid-type mass spectrometer with a nanospray source used in this study

enabled identification of O-glycans in HILIC fractions. As a result, the following O-glycan

structures: mucin type O-glycans, PentPentHex, NeuAcHexHexNAcDeoxyHex,

HexGlcUdehydratedHexPent, GlcU(Hex)2Pent, have been identified with the mass accuracy better

than 1 ppm.

The combination of hydrazinolysis, HILIC chromatography of fluorescence labeled

glycans, and FT-MS analysis allows for both, accurate quantitation and comprehensive O-glycan

characterization.

15

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20

Figure Captions

Fig. 1. HILIC chromatography of 2-AB-labeled O-glycans: (A) released from Fetuin and (B)

released from model Protein 1 using: (1) ammonia-based (40% ETA, 20 mg/mL ammonium

carbonate) and (2) hydrazinolysis procedures. Peak B: NeuAcα(2-3)Galβ(1-3)GalNAc; Peak C:

Galβ(1-3)[NeuAcα(2-6)]GalNAc; Peak D: NeuAcα(2-3)Galβ(1-3)[NeuAcα(2-6)]GalNAc; Peak

E: NeuAcα(2-3)Galβ(1-3)[NeuAcα(2-3)Galβ(1-4)GlcNAcβ(1-6)]GalNAc (O-glycans identified

in Fetuin [32]).

Fig. 2. HILIC chromatography of 2-AB-labeled O-glycans released from model Protein 2 using:

(1) ammonia-based (40% ETA, 20 mg/mL ammonium carbonate) and (2) hydrazinolysis

procedures.

Fig. 3. Overlay of chromatographic profiles of 2-AB O-glycans released from model Protein 1 by

hydrazinolysis: 1) glycans desalted via anion-exchanged resin [38] followed by glycan isolation

with Hypercarb SPE cartridges (n=3 chemical replicates) and 2) glycans isolated using

Hypercarb SPE cartridges alone (n=3 chemical replicates).

Fig. 4. MS/MS spectrum of the O-glycan parent ion: m/z = 753.25588 (1+), corresponding to

HexGlcU(dehydrated)HexPent structure.

Fig. 5. MS/MS spectrum of the O-glycan parent ion: m/z = 771.26691 (1+), corresponding to

GlcU(Hex)2Pent structure.

21

Table 1. Relative Distribution of 2-AB-labeled O-glycans Released from Model Protein 2 by

Hydrazinolysis using Two Procedures: Desalting Resin + Hypercarb SPE (A) and

Hypercarb SPE alone (B)

A.

O-glycan ID RT,

min

s1 s2 s3 AVERAGE STDEV %RSD

PentPentHex 12.3 82.9 85.5 82.4 83.6 1.6 2.0

NeuAcHexHexNAcPent 19.4 7.3 6.2 7.4 7.0 0.7 9.7

21.3 2.0 1.7 2.0 1.9 0.2 9.2

D 25.8 3.2 2.8 3.5 3.2 0.4 11.8

E 29.9 4.6 3.9 4.6 4.4 0.4 10.3

B.

O-glycan ID RT,

min

s1 s2 s3 AVERAGE STDEV %RSD

PentPentHex 12.3 79.2 80.2 83.8 82.3 2.4 2.9

NeuAcHexHexNAcPent 19.4 9.2 8.8 6.9 7.6 1.2 15.8

21.3 2.5 2.4 2.1 2.1 0.2 8.8

D 25.8 3.9 3.5 3.0 3.3 0.5 14.1

E 29.9 5.3 5.1 4.2 4.6 0.6 12.5

Table 2. Relative Distribution of 2-AB-labeled O-glycans Released from Model Protein 2 by

Hydrazinolysis (A) with and (B) without Acidification Step

A.

O-glycan ID RT,

min

s1 s2 s3 AVERAGE STDEV %RSD

PentPentHex 12.3 83.2 84.1 83.7 83.7 0.4 0.4

NeuAcHexHexNAcPent 19.4 6.2 6.2 6.5 6.3 0.1 2.1

21.3 1.9 2.1 2.4 2.1 0.2 9.5

D 25.8 4.0 3.3 3.2 3.5 0.3 9.8

E 29.9 4.7 4.3 4.3 4.4 0.2 4.7

22

B.

O-glycan ID RT,

min

s1 s2 s3 AVERAGE STDEV %RSD

PentPentHex 12.3 81.2 81.1 80.3 80.9 0.4 0.5

NeuAcHexHexNAcPent 19.4 7.1 7.3 7.2 7.2 0.1 1.3

21.3 2.0 2.2 2.3 2.2 0.1 5.1

D 25.8 4.3 4.0 4.4 4.2 0.2 4.3

E 29.9 5.5 5.3 5.8 5.5 0.2 3.6

Table 3. Precision Data: A- Intra-assay Repeatability; B- Intermediate Precision

A.

O-glycan ID RT

,

mi

n

s1 s2 s3 s4 s5 s6 AVERAG

E

STDE

V

%RS

D

PentPentHex 12.

3

82.

7

82.

6

83.

2

82.

6

82.

8

82.

9

82.8 0.2 0.3

NeuAcHexHexNAcP

ent

19.

4

6.2 6.0 5.8 6.0 6.0 6.5 6.1 0.2 3.6

21.

3

1.8 1.8 1.6 1.7 1.7 1.9 1.7 0.1 4.5

D 25.

8

3.6 3.5 3.7 3.6 3.9 3.5 3.6 0.1 3.5

E 29.

9

5.6 6.2 5.6 6.0 5.6 5.2 5.7 0.3 5.6

B.

O-glycan ID RT,

min

Day1

(n=6)

Day2

(n=6)

Day3

(n=6)

AVERAGE STDEV %RSD

PentPentHex 12.3 82.7 81.6 82.3 82.7 0.6 0.7

NeuAcHexHexNAcPent 19.4 6.6 7.2 6.5 6.6 0.4 5.7

21.3 2.0 2.2 1.9 2.0 0.2 7.8

D 25.8 3.7 3.6 3.6 3.6 0.1 1.6

E 29.9 5.0 5.4 5.7 5.1 0.4 6.9

23

Table 4. Summary of O-glycan Identification Results Obtained by Nanospray Hybrid LTQ

FT MS

Model

Protei

n

Assigned Glycan Structure Pea

k ID

RT,

min

Observed

(m/z)

Calculated

(m/z)

Mass

Accurac

y (ppm)

1, 2 NeuAcαααα(2-3)Galββββ(1-

3)GalNAc*

B 18.

2 795.31365

(1+)

795.31420 -0.69

1, 2 Galββββ(1-3)[NeuAcαααα(2-

6)]GalNAc*

C 20.

2 795.31365

(1+)

795.31420 -0.69

1, 2 NeuAcαααα(2-3)Galββββ(1-

3)[NeuAcαααα(2-6)]GalNAc*

D 26.

0 1068.3994

4 (1+)

1068.3990

5

0.37

1 NeuAcαααα (2-3)Galββββ(1-

4)GlcNAcββββ(1-6)

[NeuAcαααα (2-3)Galββββ(1-

3)]GalNAc*

E 30.

0 1451.5414

0 (1+)

726.27434

(2+)

1451.5418

1

-0.28

1 PentPentHex** RT

12.3

12.

3

565.22412

(1+)

565.22393 0.34

1 NeuAcHexHexNAcDeoxyHex*

*

HexHexNAcDeoxyHex

RT

19.4

19.

4

941.37295

(1+)

650.27646

(1+)

941.37211

650.27669

0.89

-0.35

2 HexGlcUdehydratedHexPent

RT

22.4

22.

4

753.25588

(1+)

753.25602 -0.18

2 GlcU(Hex)2Pent***

RT

24.6

24.

6

771.26691

(1+)

771.26658 0.43

*O-glycans identified in Fetuin [32]; **Nortch EGF-like O-glycan [43]; ***GAG O-glycan

identified in recombinant protein [44].

24

Table 5. Model Protein 1: MS/MS Fragmentation Data of the Precursor Ion: m/z =

565.22412 (1+) (A) and MS/MS Fragmentation Data of the Precursor Ion: m/z = 650.27646

(1+) (B).

A.

Observed (m/z) Tentative Assignment

Calculated (m/z) Mass Accuracy

(ppm)

433.18167 PentHex-2AB 433.18167 0.00

301.13943 Hex-2AB 301.13941 0.07

B.

Observed (m/z) Tentative Assignment

Calculated (m/z) Mass Accuracy

(ppm)

488.22378 HexNAcDeoxyHex-2AB 488.22387 -0.18

366.13935 (HexHexNAc)-H2O 366.13947 -0.33

285.14443 DeoxyHex-2AB 285.14450 -0.24

Table 6: Model Protein 2: MS/MS Fragmentation Data of the Precursor Ion: m/z

=753.25588 (1+) (A); MS/MS Fragmentation Data of the Precursor Ion: m/z =771.26691

(1+) (B).

A.

Observed (m/z) Tentative Assignment

Calculated (m/z) Mass Accuracy

(ppm)

591.20370 GlcU(dehydrated)HexPent-

2AB

591.20319 0.86

433.18202 HexPent-2AB 433.18167 0.81

271.12898 Pent-2AB 271.12885 0.48

B.

Observed (m/z) Tentative Assignment

Calculated (m/z) Mass Accuracy

(ppm)

609.21416 GlcUHexPent-2AB 609.21376 0.66

595.23459 (Hex)2Pent-2AB 595.23449 0.16

433.18144 HexPent-2AB 433.18167 -0.53

271.12902 Pent-2AB 271.12885 0.63

Fig. 1

0.00 1

2

B

E D

RT 12.3

RT 19.4

10 12 14 16 18 20 22 24 26 28 30 Minutes

RT 21.3

B

C

D

E

A

1 2

10

B

C

D

E

0.00

B

C

D

E

B

C

D

E

Minutes

B

C E

A

1

2

12 14 16 18 20 22 24 26 28

5.0x102

1.0x103

1.5x103

2.0x103

2.5x103

3.0x103

30

D

3.5x103

EU

1.25x103

EU

5.0x102

7.5x102

1.0x103

2.5x102

Fig. 2

Fig. 3

5.0x102

1.5x103

2.5x103

3.5x103

4.5x103

Minutes

8 10 12 14 16 18 20 22 24 26 28 30

1

2

5.5x103

New peak (RT 22.4)

C

B

D

PentPentHex

EU

5.0x102

7.5x10 2

Minutes

10 12 14 16 18 20 22 24 26 28 30

1.25x103

1.0x103

RT 12.3

RT 19.4

E D

2.5x102

RT 21.3

EU

Fig. 4

Fig. 5

- Hex - GlcU dehydrated - Hex

350 400 450 500 550 600

Relative Abundance

433.18202 591.20370 271.12898

250 300 650 700 750 m/z

90

100 753.25588

dehydrated Hex GlcU Hex Pent -2AB

Pent-2AB

80

70

60

50

40

30

20

10

- GlcU - Hex - Hex

10

20

30

50

60

70

80

90

100 433.18144

Pent - 2AB

40

Relative Abundance

771.26691 595.23459 271.12902

Hex

GlcU

Hex Pent - 2AB

609.21416 Hex -

300 250 350 400 450 500 550 600 650 700 750 m/z