Simplified Quantitative Glycomics Using the Stable Isotope Label

Nov 25, 2013 - Xuefei Zhong , Zhengwei Chen , Sergei Snovida , Yan Liu , John C. Rogers , and Lingjun Li. Analytical Chemistry 2015 87 (13), 6527-6534...
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Simplified Quantitative Glycomics Using the Stable Isotope Label Girard’s Reagent P by Electrospray Ionization Mass Spectrometry Chengjian Wang,† Zhiyu Wu,† Jiangbei Yuan,† Bo Wang,† Ping Zhang,‡ Ying Zhang,† Zhongfu Wang,*,† and Linjuan Huang*,† †

Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, College of Life Science, Northwest University, Xi’an 710069, China ‡ Chemistry and Chemical Engineering School, Xianyang Normal University, Xianyang 712000, China S Supporting Information *

ABSTRACT: Fast, sensitive, and simple methods for quantitative analysis of disparities in glycan expression between different biological samples are essential for studies of protein glycosylation patterns (glycomics) and the search for disease glycan biomarkers. Relative quantitation of glycans based on stable isotope labeling combined with mass spectrometric detection represents an emerging and promising technique. However, this technique is undermined by the complexity of mass spectra of isotope-labeled glycans caused by the presence of multiple metal ion adduct signals, which result in a decrease of detection sensitivity and an increase of difficulties in data interpretation. Herein we report a simplified quantitative glycomics strategy, which features nonreductive isotopic labeling of reducing glycans with either nondeuterated (d0-) or deuterated (d5-) Girard’s reagent P (GP) without salts introduced and simplified mass spectrometric profiles of d0- and d5-GP derivatives of neutral glycans as molecular ions without complex metal ion adducts, allowing rapid and sensitive quantitative comparison between different glycan samples. We have obtained optimized GP-labeling conditions and good quantitation linearity, reproducibility, and accuracy of data by the method. Its excellent applicability was validated by comparatively quantitative analysis of the neutral N-glycans released from bovine and porcine immunoglobulin G as well as of those from mouse and rat sera. Additionally, we have revealed the potential of this strategy for the high-sensitivity analysis of sialylated glycans as GP derivatives, which involves neutralization of the carboxyl group of sialic acid by chemical derivatization. KEYWORDS: glycomics, quantitation, stable isotope labeling, Girard’s reagent P, ESI-MS

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INTRODUCTION Glycosylation, one of the most common post-translational modifications of proteins, is receiving more and more attention from scientists. It is estimated that more than half of all proteins are glycoproteins.1 The glycan moieties of glycoproteins play pivotal roles in many biological processes, such as cell recognition, cell adhesion, signal transduction, immune response, and cell differentiation.2 Moreover, a series of recent studies have demonstrated conspicuous correlations between the alterations of glycans attached to proteins and many diseases.3−10 For example, serum glycoproteins of the patients suffering inflammation or cancer exhibit an increase in the amount of biantennary, core-fucosylated asparagine-linked glycans (N-glycans);9 the expression of the α-2,6-sialyltransferase ST6GALNAC5 has proved to facilitate metastasis of breast cancer cells to the brain by enhancing adhesion of tumor cells to brain tissues.10 Therefore, qualitative and quantitative determination of the changes in glycan expression between different cells, tissues, or biological fluids is essential for the study of glycosylation patterns of proteins (glycomics) and the search for glycan biomarkers of diseases, which promises great advances in medicine. © 2013 American Chemical Society

High-throughput qualitative and quantitative comparison of structurally complex glycans between different microamounts of biological samples, a current challenge in the field of glycomics, requires fast, sensitive, and simple analytical methods. Traditional chromatographic and electrophoretic techniques, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), gel electrophoresis (GE), and capillary electrophoresis (CE), have been extensively exploited for quantitative analysis of various glycans.11 However, these techniques suffer from an inability to perform identification of structurally diverse glycans by themselves, owing to the lack of commercially available glycan standards.12 In the past two decades, dramatically, an array of newly developed modern biological mass spectrometry (bio-MS) techniques based on soft ionization methods, such as matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS), electrospray ionization mass spectrometry (ESI-MS), liquid chromatography coupling with mass spectrometry (LC−MS) and tandem mass Received: April 20, 2013 Published: November 25, 2013 372

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glycomics. Previously, some researchers25−28 have explored the derivatization of reducing saccharides with some cationic reagents, including carboxymethyl trimethylammonium hydrazide (Girard’s reagent T, GT), carboxymethyl tripropylammonium hydrazide, 1-(2-hydrazino-2-oxoethyl)-4-phenylpyridinium, and glycidyltrimethylammonium (GTMA), to impart a permanent cation charge to the reducing end of glycans for highly sensitive identification by MS. Moreover, isotopic Girard’s reagent P [1-(2-hydrazino-2-oxoethyl)pyridinium, GP], another cationic reagent, has been utilized for the identification of carbonylation sites in oxidized proteins.29 Now we introduce isotopic GP into quantitative glycomics. In this study, reducing glycans are efficiently labeled with either nondeuterated (d0-) or 2,3,4,5,6-pentadeuterated (d5-) GP based on the hydrazone formation reaction under nonreductive conditions without any metal salts introduced, followed by direct high-sensitivity detection by ESI-MS, LC−MS, and MS/ MS techniques without any postprocessing steps, such as removal of superfluous labeling reagents and desalting. The d0and d5-GP derivatives exhibit only [M]+ type molecular ion signals in MS profiles, eliminating the interference from various complex ion adduct peaks such as [M + H]+, [M + Na]+, and [M + K]+ and simplifying exceedingly MS data of glycans and their interpretation. The MS signal intensity ratio between the d0- and d5-GP derivatives of each glycan allows rapid and highthroughput relative quantitation analysis, and MS/MS analysis allows detailed structural identification. We have synthesized d5GP, optimized the reaction conditions for the labeling of reducing glycans with GP, and examined lactose and bovine fetuin N-glycans as GP derivatives in terms of MS detection sensitivity to validate the feasibility of the new procedure. The quantitation linearity, reproducibility, and accuracy of this new strategy have been verified using the N-glycans released from ribonuclease B (RNase B) and chicken ovalbumin. Additionally, we have also successfully applied this method to comparative analysis of various neutral N-glycans released from bovine and porcine immunoglobulin G (IgG) as well as of those from two complex biological samples, mouse and rat sera, demonstrating definitively the excellent applicability of the suggested method.

spectrometry (MS/MS and MSn), provided a powerful platform for the qualitative and quantitative analysis of various naturally occurring glycans, due to their unique features of highsensitivity, high-throughput, and high-molecular-weight detectabilities.13 Relative quantitation of glycans based on stable isotope labeling combined with bio-MS detection represents an emerging and promising analytical strategy. This strategy generally consists of three major operation steps, including tagging of two groups of glycans derived from different samples with the light and heavy forms of stable-isotope labels, respectively, mixing of equal quantities of the two groups of isotope-labeled glycans, and glycan identification by MS/MS and quantitation by MS. Two glycans with the same monosaccharide composition but from different samples can be recognized as a pair of peaks with a certain mass difference (Δm) compared to each other in mass spectra, and their difference in quantity can be determined according to the MS signal intensity ratio between the light- and heavy-form derivatives, due to the same ionization efficiency of them. Thus, the relative quantitation strategy is an ideal analytical tool for the qualitative and quantitative comparison of different groups of microamounts of glycans derived from different biological samples. The study of relative quantitation methods of glycans has achieved many advances in recent years, mainly involving a series of isotopic labeling procedures based on permethylation, metabolic labeling, and reducing end tagging. The permethylation procedures using isotopic methyl iodide [12CD3I coupled with 12CH3I (12CD3I/12CH3I),14 12CH3I/ 13CH3I,15 or 13 CH3I/12CH2DI16,17] are quite suitable for high-sensitivity qualitative and quantitative analysis of trace-level glycan samples, especially structural identification by MSn, due to the stable ionization efficiency of permethylated glycans in mass spectrometer ion sources. However, the mass differences between the heavy and light forms of isotopically permethylated glycans vary with the number of methylation sites, complicating the mass spectra and increasing the difficulties in data interpretation. The methods of metabolic labeling,18,19 alternatively named enzymatic labeling, using some isotopic metabolic substrates, can incorporate isotopes into some glycans and perform relative quantitation of some certain types of glycan samples but are very complicated. The reducing end derivatization approaches with isotopic labels, such as reductive amination using 12C6/13C6-2-aminobenzoic acid (2AA) (Δm = 6 Da),20 nondeuterated (d0-) 2-aminopyridine (2AP) coupled with hexadeuterated (d6-) 2-AP (Δm = 4 Da)21 or 12 C6/13C6-aniline (Δm = 6 Da),22 Michael addition using nondeuterated 1-phenyl-3-methyl-5-pyrazolone (d0-PMP) coupled with pentadeuterated (d5-) PMP (Δm = 10 Da),23 and hydrazone formation using 12C6/13C6-4-phenethyl benzohydrazide (Δm = 6 Da),24 can provide invariable Δm between the heavy and light forms of isotopically labeled glycans and allow qualitative and quantitative analysis of various reducing glycans by different techniques, such as MS, LC−MS, and MS/ MS. However, these methods are usually undermined by the complexity of mass spectra of isotope-labeled glycans caused by the presence of multiple ion adduct signals, such as [M + H]+, [M + Na]+, and [M + K]+, resulting in a decrease of detection sensitivity and an increase of difficulties in data interpretation. Here, we describe a conspicuously improved relative quantitation procedure based on reducing end tagging of glycans, namely, simplified quantitative glycomics strategy, to solve these problems and facilitate studies of comparative

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EXPERIMENTAL SECTION

Material

1-(2-Hydrazino-2-oxoethyl)pyridinium chloride (Girard’s reagent P, d0-GP) was purchased from TCI Development Co., Ltd. (Japan). Ethyl chloroacetate was purchased from AlfaAesar (Ward Hill, MA, USA). Deuterated pyridine (d5pyridine), carboxymethyl trimethylammonium chloride hydrazide (GT), 2-aminobenzoic acid (2-AA), 2-aminopyradine (2AP), aniline, 1-phenyl-3-methyl-5-pyrazolone (PMP), β-cyclodextrin, ovalbumin (chicken egg white albumin), ribonuclease B (RNase B), porcine IgG (reagent grade), bovine IgG (technical grade), and microcrystalline cellulose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine fetuin was purchased from Merck (Darmstadt, Germany). Peptide Nglycosidase F (PNGase F) was obtained from New England BioLabs (Ipswich, MA, USA). Lactose was from Beijing Aoboxing Biotech Co. Ltd. (Beijing, China). Hydrazine hydrate was a product of the Third Chemical Company of Tianjin (Tianjin, China). The MD34 (MW 8,000−14,000) dialysis membrane was from Union Carbide (Danbury, CT, USA). DLDithiothreitol (DTT), sodium dodecyl sulfate (SDS), and Nonidet P-40 (NP-40) were purchased from Aladdin Industrial 373

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onto a hand-packed microcrystalline cellulose column (1 mL) prewashed with 200 mL of water and equilibrated with 10 mL of 4:1:1 (v/v/v) butanol/methanol/water. After a wash of the column with 50 mL of 4:1:1 (v/v/v) butanol/methanol/water to remove peptides, the glycans were eluted with 2 mL of water. The eluates were completely dried, redissolved in 3 mL of water, and then loaded onto a graphitized carbon SPE column prewashed with 3 mL of ACN and equilibrated with 5 mL of water. After a wash of the column with 40 mL of water to remove salts, the glycans were eluted with 3 mL of 50% ACN containing 0.1% TFA. The obtained glycan sample was dried under a stream of nitrogen gas for further use.

Inc. (Shanghai, China). Nonporous graphitized carbon (Carbograph) solid phase extraction (SPE) columns (150 mg/4 mL) were purchased from Alltech Associates (Deerfield, IL, USA). Analytical grade glacial acetic acid and anhydrous ethanol were purchased from Xi’an Sanpu Chemical Reagent Co. Ltd. (Xi’an, Shaanxi, China), and analytical grade ammonium acetate and nbutanol were from Tianli Chemical Reagent Co. Ltd. (Tianjin, China). Chemical grade trifluoroacetic acid (TFA) was from Shanghai Kefeng Chemical Reagent Co., Ltd. (Shanghai, China). Analytical grade sodium phosphate was from Shantou Xilong Chemical Factory (Guangdong, China). HPLC grade methanol and acetonitrile (ACN) were purchased from Fisher Scientific (Fairlawn, NJ, USA). The water used was purified through a Milli-Q purification system (Millipore, Milford, MA, USA).

Derivatization of Glycans with GP

The glycans released from 0.25 mg of glycoproteins were dissolved in 2 μL of 0.5 M GP in 9:1 (v/v) methanol/acetic acid mixture. The glycans derived from 1 mg of lyophilized total serum proteins were dissolved in 0.5 μL of 9:1 (v/v) methanol/acetic acid mixture containing 0.1 M GP, and then 1.5 μL of methanol was added. After incubation at 70 °C for 3 h, the samples were cooled, dried under a stream of nitrogen gas, and then redissolved in methanol for mass spectrometric analysis.

Synthesis and Characterization of d5-GP

d5-GP was synthesized according to a previously reported procedure.29 Briefly, a 100-mL round-bottom flask was filled with a mixture of ethanol (25 mL), ethyl chloroacetate (12.3 g, 0.1 mol), and anhydrous d5-pyridine (8.5 g, 0.1 mol), followed by heating at 70 °C for 12 h. The resulting mixture was cooled to room temperature, and then hydrazine hydrate (5 g, 0.1 mol) was slowly added with shaking. After the reaction stopped, a mass of crystalline 1-(2-hydrazino-2-oxoethyl)pyridinium chloride was precipitated. The solid target product was filtered out, washed with cold ethanol, and finally dried in air. The obtained product weighed 13.62 g (yield, 72.5%). Its ESI-MS spectrum in positive ion mode gave an m/z value of 157.08 ([M − Cl]+). Its 1H and 13C {1H} NMR spectra were recorded on a Varian INONA 400 instrument (Palo Alto, CA, USA) at 25 °C using d6-DMSO as a sample solvent and TMS as an internal reference standard. 1H NMR (400 MHz, ppm) δ: 5.48 (s, 2H, 1JCH = 146 Hz, CH2); the signal peak near 3.47 related to NH and NH2 was broad due to the presence of water. 13C NMR (100 MHz, ppm) δ: 163.76 (CO), 145.74, 127.20 (both 1:1:1 t, all 1JCD = 29.4 Hz, intensity ratio 3:2, p-, m-, and o-CD in d5-Py), 61.28 (CH2).

ESI-MS, Online LC−MS, and MS/MS Analysis

The analysis of GP-labeled glycans was performed on a linear quadrupole ion trap electrospray ionization mass spectrometer (LTQ XL, Thermo Scientific, USA) coupled with a highperformance liquid chromatography system (Thermo). To perform ESI-MS analysis, glycan samples were infused via a 2μL Rheodyne loop and brought into the electrospray ion source by a stream of 50% methanol at a flow rate of 200 μL/min. The spray voltage was set at 4.5 kV, with a sheath gas (nitrogen gas) flow rate of 30 arb, an auxiliary gas (nitrogen gas) flow rate of 10.0 arb, a capillary voltage of 48 V, a tube lens voltage of 240 V, and a capillary temperature of 350 °C. The generated ions from the electrospray ion source were transferred into the twodimensional linear ion trap and ion detection system (conversion dynode, −15 kV; electron multiplier 1, −1410 V; electron multiplier 2, −1430 V; multiplier gain, 300 000 counts) via an ion optics system, which consists of several tandem ion lens and multipole elements (multipole 00 offset, −4 V; lens 0, −4 V; multipole 0 offset, −4.5; lens 1, −20 V; gate lens, −58 V; multipole 1 offset, −12.5 V; multipole radio frequency, 405 Vp‑p; front lens, −5 V; front section offset, −9 V; center section offset, −12 V; back section offset, −7 V; back lens, 0 V). Data acquisitions were performed with normal scan rate (16,000 atomic mass unit per second), normal mass range (m/z 900−2000), full scan type, maximum injection time of 1000 ms, 3 microscans, and 10,000 automatic gain control target ions. The ESI-MS data were collected with LTQ Tune software (Thermo). To perform LC−MS analysis, GP-labeled glycans derived from ca. 1 mg of lyophilized total serum proteins were dissolved in 50 μL of 50% methanol, and 20-μL aliquots were injected by Surveyor automatic sampler in the partial loop injection mode. Glycan separation was achieved using a 2.0 mm ×150 mm TSKgel Amide-80 column (3 μm) (Tosoh Corporation, Tokyo, Japan) at ambient temperature (25 °C) with a gradient of mobile phase described as follows: solvent A, ACN; solvent B, 10 mM ammonium acetate (pH 6.0); time = 0 min (t = 0), 65% A, 35% B (200 μL/min); t = 60, 35% A, 65% B (200 μL/min). The parameters of electrospray ion source and LTQ used for LC−MS analysis were identical to those for ESI-MS analysis. The LC−MS data

Preparation and Pretreatment of Murine Sera

The blood samples of rat and mouse were collected from three healthy 1-month-old rats and three healthy 1-month-old mice for experimental use, respectively. The blood samples were left static for 30 min, prior to centrifugation at 3000 rpm for 5 min. The upper layers of the samples were collected and exhaustively dialyzed against Milli-Q water at 4 °C for 48 h. The dialyzed murine serum samples were finally lyophilized and stored at −20 °C for further use. Enzymatic Release of N-Glycans from Glycoproteins

This procedure was based on a previously reported method.23 Briefly, 1 mg of purified glycoprotein or 3−5 mg of lyophilized serum sample was dissolved in 400 μL of protein denaturation solution containing 0.4 M DTT and 5% SDS and heated at 100 °C for 10 min. After the sample cooled, 50 μL of 1 M sodium phosphate buffer (pH 7.5), 50 μL of 10% aqueous NP-40 (v/ v), and 1 μL of PNGase F solution (500 unit) were added, followed by incubation at 37 °C for 24 h. When the reaction was completed, the sample was boiled at 100 °C for 5 min to inactivate the enzyme and condensed to dryness under a stream of nitrogen gas. Purification of Glycan Samples

The enzymatically treated sample was dissolved in 300 μL of 4:1:1 (v/v/v) butanol/methanol/water mixture and loaded 374

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were recorded using Xcalibur software (Thermo). Molecular ions of GP-labeled glycans were fragmented by collisioninduced dissociation (CID) in the LTQ using helium (He) as the collision gas for MS/MS analysis (3 microscans, 10,000 target ions, 100 ms maximum injection time, m/z 3.00 isotope width, 35.0−65.0 normalized collision energy, 0.250 activation Q, and 30 ms activation time). Glycan compositions and sequences were assigned manually and then checked either in literature references or in databases such as CFG, Carbbank, and Glycosciences with GlycoWorkbench.30

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RESULTS AND DISCUSSION

Principle of the Simplified Quantitative Glycomics Strategy

As described above, many previously reported relative quantitation methods are usually troubled with complex mass spectra of isotopically labeled glycans, due to the presence of diverse types of ion adduct peaks, such as those assigned to [M + H]+, [M + Na]+, and [M + K]+. In fact, the ionization manners and ion types of glycan derivatives in biological mass spectrometers (bio-MS) are closely related to their structural features. Generally, if glycans are derivatized with neutral or anionic reagents, the obtained derivatives will tend to bind to protons and metal cations in samples to form cationic molecular ion adducts, enabling their detection by bio-MS in the positive ion mode. However, the diversity of native cations in samples results in complex types of cationic molecular ion adducts. Instead, if glycans are labeled with cationic reagents, the resulting glycan derivatives will be stable cationic molecular ions, which allow direct high-sensitivity detection by bio-MS in the positive ion mode without interference from various complex ion adduct peaks, greatly simplifying the analysis of glycans. Thus, relative quantitation of glycans based on stable isotope labeling with cationic reagents represents an attractive strategy. On the basis of these considerations, we designed a new relative quantitation method, termed simplified quantitative glycomics strategy. This procedure is developed on the basis of the hydrazone formation reaction between reducing glycans and d0/d5-GP (shown in Figure 1A). Obviously, compared with the broadly used reductive amination procedure, which needs sodium cyanoborohydride (NaBH3CN) as a reducing reagent and desalting steps prior to detection by MS, as well as other glycan derivatization methods, the hydrazone formation reaction is much easier to be carried out. The operation steps are summarized as a workflow scheme, which is presented in Figure 1B. As a result, all of the d0- and d5-GP derivatives exhibit only [M]+-type molecular ion signals in mass spectra, without interference from various complex ion adduct peaks, simplifying greatly MS data of glycans and their interpretation. The structure of these d0- and d5-GP derivatives of glycans can be identified in detail by LC−MS and MS/MS. Therefore, this strategy represents a rapid, sensitive, and simple procedure allowing qualitative and quantitative comparison between different groups of glycans.

Figure 1. Schematic diagram of simplified quantitative glycomics strategy. (A) Reducing glycans either occurring freely or released from glycoproteins are labeled with either nondeuterated (d0-) or deuterated (d5-) Girard’s reagent P (GP) based on the formation of hydrazone, without salts introduced. (B) The new strategy follows a workflow in principle. Two different glycan samples are derivatized with d0- and d5-GP, respectively, followed by mixing of them and analysis by MS, without any desalting steps. Two glycans with the same monosaccharide composition but from different samples are identified as a pair of MS peaks, with a mass difference (Δm) of 5 Da between their light form (d0-GP derivative) and heavy form (d5-GP derivative), and their difference in quantity can be determined according to the intensity ratio between the light and heavy forms. In contrast, the glycans present in one sample but absent in the other give single peaks in mass spectra.

molar ratios for GP labeling were successively investigated. The ESI-MS profiles of the reaction products obtained in different conditions (Figure S1 in Supporting Information) indicate a set of optimal reaction conditions, including the reaction temperature of 70 °C, the reaction time of 3 h, and the molar ratio of glycan to GP of 1:2. In addition, we also found an excessive amount of GP in glycan samples would result in the decrease of signal-to-noise ratios of ESI-MS profiles. Therefore, different amounts of glycans should be derivatized with different amounts of GP. Under these recommended reaction conditions, very small amounts of reducing glycans such as 1 nmol of lactose can be successfully derivatized with GP and analyzed by ESI-MS. Obviously, these reaction conditions for GP labeling are much simpler than those of most of other glycan derivatization methods reported previously.20−23 Thus, this method allows rapid and high-throughput derivatization of reducing glycans.

Optimization of Reaction Conditions for the Labeling of Reducing Glycans with GP

To achieve efficient and quantitative labeling of reducing glycans with GP, the reaction conditions were optimized, using lactose as a model glycan, methanol as a sample solvent, and 10% glacial acetic acid (v/v) as a catalyst. With reference to previously reported reaction conditions for GT labeling,25 reaction temperature, reaction time, and reaction substrate 375

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Figure 2. Validation of the new method using N-glycans released from model glycoproteins. (A) ESI-MS profile of the equimolar mixture of d0- and d5-GP derivatives of N-glycans released from RNase B. MS peaks of all glycans are assigned to [M]+ ions. “R” denotes the intensity ratio between d0GP and d5-GP derivatives of each glycan, which consists of mean value and standard deviation obtained from three repeated experiments. (B) ESIMS profiles of d0- and d5-GP derivatives of the RNase B N-glycan Man5GlcNAc2 mixed in different molar ratios (1:1, 1:3, 1:5, and 1:10). (C) Linear correlations between experimentally detected MS signal intensity ratios and theoretical molar ratios of d0-GP-labeled N-glycans released from RNase B to their corresponding d5-GP derivatives. The inset presents an expanded part of the linear graph. “Mann” represents the high-mannose type Nglycan with a mannose residue number of “n”. (D) ESI-MS profile of an equimolar mixture of d0- and d5-GP-labeled N-glycans released from chicken ovalbumin. Structural formulas: square, N-acetylglucosamine; gray circle, mannose.

Verification of Quantitation Features of the Method

Supporting Information), while GP derivatives of sialylated glycans exhibit good structural stability under the reaction conditions for GP labeling but poor MS detection sensitivity (shown in Figure S3 in Supporting Information), which indicates the incompatibility of GP-labeled sialylated glycans with MS detection to a great extent. However, when the carboxyl group of these sialylated glycans is neutralized via methyl esterification, the MS profiles of both their d0- and d5GP derivatives give strong MS signals and high signal-to-noise

The feasibility and stability of this strategy were investigated. First, both neutral and sialylated glycans as GP derivatives were investigated in terms of MS detection sensitivity using lactose and bovine fetuin N-glycans, respectively (the details are described in Supporting Information). As a result, the GPderivatized neutral glycans exhibit excellent MS detection sensitivity and MS signal simplicity (shown in Figure S2 in 376

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Figure 3. Quantitative comparison of N-glycans released from bovine and porcine IgG. (A) ESI-MS profile of an equimolar mixture of d0- and d5-GP derivatives of bovine IgG N-glycans. (B) ESI-MS profile of an equimolar mixture of d0- and d5-GP derivatives of porcine IgG N-glycans. (C) ESI-MS profile of an equimolar mixture of d0-GP derivatives of bovine IgG N-glycans and d5-GP derivatives of porcine IgG N-glycans. All of the m/z values are assigned to [M]+ type ions. The blue and red ion peaks are the signals of d0-GP and d5-GP derivatives, respectively. The intensity ratio of the d0GP derivative of each glycan to its corresponding d5-GP derivative is shown as R, which consists of mean value and standard deviation obtained from three repeated experiments. Structural formulas: square, N-acetylglucosamine; gray circle, mannose; bright circle, galactose; triangle, fucose.

ratios (presented in Supplementary Figure S4). These results demonstrate definitely the feasibility of the recommended strategy for the sensitive detection of both neutral and sialylated glycans. Owing to the neutralization process of carboxyl groups by chemical derivatization, the analysis of sialylated glycans becomes relatively time-consuming to a certain extent.

Therefore, to better elucidate the concept of simplified quantitative glycomics, this study is focused on the analysis of various neutral glycans. Second, the quantitation linearity, reproducibility, and accuracy of the method were examined using high-mannose type N-glycans released from RNase B. Two groups of RNase B 377

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Quantitative Comparison of N-Glycans Released from Bovine and Porcine IgG

N-glycans in equal quantities were labeled with d0- and d5-GP, respectively, and mixed in different molar ratios (10:1, 7:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:7, and 1:10 of d0- to d5-GP derivatives), followed by analysis by ESI-MS. As shown in Figure 2A, the mass spectrum of the equimolar mixture of the d0- and d5-GP derivatives of RNase B N-glycans exhibits four pairs of peaks, including those with m/z 1368.67/1373.67, 1530.75/1535.75, 1692.83/1697.83, and 1854.92/1859.92, which are assigned to [M]+-type ions of d0- and d5-GP derivatives of Man5GlcNAc2 (Man5), Man6GlcNAc2 (Man6), Man7GlcNAc2 (Man7), and Man8GlcNAc2 (Man8), respectively.20,31 Each peak pair features an invariable 5-Da Δm between the light and heavy forms, allowing rapid and convenient distinguishment of reducing glycans from nonglycan impurities. The MS signal intensity ratio of d0- to d5-GP derivatives of each glycan is shown as R, which consists of a mean value close to the theoretical value 1.00 and a standard deviation (SD) value below 7% (coefficient of variability, CV) of the mean value (n = 3), indicating the sufficient accuracy and reproducibility of this method for relative quantitation. Similarly, the MS data of the d0- and d5GP derivatives of RNase B N-glycans mixed in other molar ratios were also obtained. As shown in Figure 2B, zoomed MS profiles of d0- and d5-GP derivatives of Man5 mixed in different molar ratios (1:1, 1:3, 1:5, and 1:10) exhibit excellent resolution, which allows complete distinguishment between MS peaks of the light and heavy forms of GP derivatives of each glycan and accurate calculation of experimental MS signal intensity ratios. The obtained MS signal intensity ratio data are presented in Table S1 in Supporting Information and Figure 2C. Obviously, correlation analysis reveals an excellent linear relationship between the experimental MS signal intensity ratios of d0-GP derivative to d5-GP derivative of each glycan and their theoretical molar ratios (mixing ratios) ranging from 0.1 to 10 (correlation coefficient R ≥ 0.9989), demonstrating definitely the feasibility of the suggested strategy for relative quantitation of N-glycans. Moreover, the CV values of these experimental MS signal intensity ratios remain below 6.6%, showing the good reproducibility of the method. However, the standard error increases markedly when the theoretical molar ratio becomes greater or smaller than 1.0, as well as when the relative abundance of these MS peaks decreases. Especially, an expansion of the theoretical molar ratio to 1:15 or 15:1 will result in an increase of the standard error up to 30.0%. Therefore, to guarantee the quantitation accuracy of this procedure, a 100-fold linear dynamic range of the molar ratios of d0-GP derivatives to d5-GP derivatives of glycans from 0.1 to 10 is recommended. Third, the usability of this strategy was confirmed by relatively quantitative analysis of complex and hybrid-type Nglycans released from chicken ovalbumin. Two aliquots of ovalbumin were digested by PNGase F, and the obtained two groups of N-glycans were derivatized with d0- and d5-GP, respectively. The d0- and d5-GP derivatives were mixed and analyzed by ESI-MS. As a result, 11 pairs of peaks of isotopically labeled glycans were observed (shown in Figure 2D). The sequences of these glycans were assigned according to previous literature reports.32 The MS signal intensity ratio of d0- to d5-GP derivatives of each glycan is close to the theoretical value 1.00 of an equimolar mixture of them, with a CV value below 5.6% (n = 3), indicating further the good accuracy and reproducibility of this method.

To validate the applicability of the newly developed method to the quantitative glycomic comparison between two homologous glycoproteins, relative quantitation of the N-glycans of bovine and porcine IgG was performed. The bovine and porcine IgG N-glycans were prepared and labeled with d0- and d5-GP with the methods described above, followed by the mixing of the isotopically labeled glycan samples in equal ratios in three different ways described as follows: (i) d0- and d5-GP derivatives of bovine IgG N-glycans; (ii) d0- and d5-GP derivatives of porcine IgG N-glycans; (iii) d0-GP derivatives of bovine IgG N-glycans and d5-GP derivatives of porcine IgG N-glycans. These mixed samples were then analyzed by ESIMS, and the sequences of glycan derivatives were identified by MS/MS. The obtained ESI-MS data and proposed main glycan structure are displayed in Figure 3, and MS/MS data are presented in Figure S5 and Figure S6 in Supporting Information. As shown in Figure 3A, the equimolar mixture of d0- and d5GP derivatives of bovine IgG N-glycans presents 14 pairs of peaks in the MS profile, which are all assigned to [M]+-type molecular ions of d0- or d5-GP-labeled neutral N-glycans. In these glycans, 2 are high-mannose type, 11 complex type, 1 hybrid type, and 7 core-fucosylated, consistent with previous studies.33 The MS signal intensity ratio of d0- to d5-GP derivatives of each glycan is close to the theoretical value 1.00, with a CV value below 8.5% (n = 3), indicating the good reliability of the quantitation method. In contrast, as shown in Figure 3B, the equimolar mixture of d0- and d5-GP derivatives of porcine IgG N-glycans produces only seven pairs of peaks of [M]+ type molecular ions of d0- or d5-GP-labeled neutral hybrid type N-glycans, including four fucosylated ones. The MS signal intensity ratio of d0- to d5-GP derivatives of each glycan is also close to the expected value 1.00, with a CV value below 6.5% (n = 3), demonstrating further the good reliability of the quantitation method. For the real quantitative glycomic comparison between bovine and porcine IgG, as shown in Figure 3C, the equal-ratio mixture of d0-GP derivatives of bovine IgG N-glycans and d5GP derivatives of porcine IgG N-glycans exhibits seven pairs of peaks (m/z 1393.58/1398.58, 1450.50/1455.58, 1596.67/ 1601.67, 1612.58/1617.58, 1758.75/1763.75, 1774.75/ 1779.58, and 1920.83/1925.83) and seven single peaks (m/z 1368.50, 1409.50, 1530.58, 1555.58, 1571.67, 1799.58, and 1961.83) in the ESI-MS profile. All of these signals are assigned to [M]+-type molecular ions. The peak pairs indicate clearly the coexisting of the corresponding N-glycans in both bovine and porcine IgG, and the MS signal intensity ratio of d0- to d5-GP derivatives of each glycan represents its molar ratio between bovine and porcine IgG. Meanwhile, the single peaks of d0- or d5-GP derivatives indicate the existence of the corresponding N-glycans only in bovine or porcine IgG, respectively. Obviously, seven N-glycans exist in both bovine and porcine IgG, of which Fuc 1 Hex 4 HexNAc 4 , Hex 5 HexNAc 4 , and Fuc1Hex5HexNAc4 are much more in bovine IgG than in porcine IgG (by 1.63, 1.58, and 5.87 times, respectively), while Hex3HexNAc4, Fuc1Hex3HexNAc4, and Hex4HexNAc4 are much fewer in bovine IgG than in porcine IgG (by 0.25, 0.39, and 0.76 times, respectively). The glycan Fuc1Hex3HexNAc3 has an approximately equal content in bovine and porcine IgG. Most of the CV values of the MS peak intensity ratios remain below 10% (n = 3), demonstrating good 378

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Figure 4. Quantitative comparison of N-glycans derived from mouse and rat sera. (A) ESI-MS profile of an equimolar mixture of d0- and d5-GP derivatives of mouse serum N-glycans (MSGs). (B) ESI-MS profile of an equimolar mixture of d0- and d5-GP derivatives of rat serum N-glycans (RSGs). (C) ESI-MS profile of an equimolar mixture of d0-GP derivatives of MSGs and d5-GP derivatives of RSGs. All of the m/z values are assigned to [M]+ type ions. The blue and red ion peaks are the signals of d0-GP and d5-GP derivatives, respectively. The intensity ratio of the d0-GP derivative of each glycan to its corresponding d5-GP derivative is shown as R, which consists of mean value and standard deviation obtained from three repeated experiments. Structural formulas: square, N-acetylglucosamine; gray circle, mannose; bright circle, galactose; triangle, fucose.

reproducibility of these data. In addition, seven N-glycans, including Hex5HexNAc2, Hex4HexNAc3, Hex6HexNAc2, Fuc1Hex4HexNAc3, Hex5HexNAc3, Fuc1Hex3HexNAc5, and Fuc1Hex4HexNAc5, exist only in bovine IgG, consistent with the results of Figure 3A and B.

isotopically labeled glycan samples in equal ratios in three different ways described as follows: (i) d0- and d5-GP derivatives of MSGs; (ii) d0- and d5-GP derivatives of RSGs; (iii) d0-GP derivatives of MSGs and d5-GP derivatives of RSGs. These mixed samples were analyzed by ESI-MS and MS/MS, and the obtained mass spectra and proposed main glycan structure are displayed in Figure 4. As shown in Figure 4A, the equimolar mixture of d0- and d5GP derivatives of MSGs gives seven pairs of MS peaks, which are all assigned to [M]+ type molecular ions of d0- or d5-GPlabeled neutral N-glycans. The sequences of Hex5HexNAc2, Hex6HexNAc2, Fuc1Hex3HexNAc4 and Fuc1Hex4HexNAc4 were identified by MS/MS (shown in Figure S7 in Supporting

Quantitative Glycomic Comparison between Mouse and Rat Sera

In order to elucidate the applicability of this method to complex biological samples, the quantitative glycomic comparison between mouse and rat sera was performed. The mouse serum N-glycans (MSGs) and rat serum N-glycans (RSGs) were separately prepared and labeled with d0- and d5-GP with the methods described above, followed by the mixing of the 379

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Figure 5. Qualitative and quantitative comparison of N-glycan isomers derived from mouse and rat sera using the newly developed method combined with online HILIC-MS. (A) Total ion chromatograms (TICs) of d0- and d5-GP-labeled N-glycans derived from mouse and rat sera. (B) Online MS spectra of an equimolar mixture of d0-GP derivatives of mouse serum N-glycans (MSG) and d5-GP derivatives of rat serum N-glycans (RSG). The blue and red ion peaks are the signals of d0-GP and d5-GP derivatives, respectively. All of the m/z values are assigned to [M]+ type ions.

either MS/MS data (shown in Figure S8 in Supporting Information) or previous literature reports.34,35,37,38 Seven of these glycans are the same as MSGs, excluding a corefucosylated complex-type Fuc1Hex3HexNAc5. The MS signal intensity ratio of d0- to d5-GP derivatives of each glycan is also close to the expected value 1.00, with a CV value below 5.2% (n = 3), demonstrating further the good reliability of the quantitation method. For the real quantitative glycomic comparison between mouse and rat sera, as shown in Figure 4C, the equal-ratio mixture of d0-GP derivatives of MSGs and d5-GP derivatives of RSGs exhibits seven pairs of peaks (m/z 1368.75/1373.75, 1530.75/1535.83, 1597.83/1601.92,

Information), and the others in low relative abundance were assigned according to previous literature reports34 due to the difficulty in performing MS/MS experiments. In these glycans, four are high-mannose type, and three are core-fucosylated complex type, consistent with previous studies.34−36 The MS signal intensity ratio of d0- to d5-GP derivatives of each glycan is close to the theoretical value 1.00, with a CV value below 5.3% (n = 3), indicating the good reliability of the quantitation method. In contrast, as shown in Figure 4B, the equimolar mixture of d0- and d5-GP derivatives of RSGs presents eight pairs of MS peaks of [M]+-type molecular ions of d0- or d5-GPlabeled neutral N-glycans, which are assigned according to 380

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Table 1. Qualitative and Quantitative Comparison of the N-Linked Glycans from Sera of Mouse and Rat

a

d0-GP-MSG, nondeuterated Girard’s reagent P labeled mouse serum N-glycans; d5-GP-RSG, deuterated Girard’s reagent P labeled rat serum Nglycans. bF, deoxyhexose; H, hexose; N, N-acetylhexosamine. cThe structure was assigned on the basis of online HILIC-MS/MS analysis and some literature reported data.34−38 Structural formulas: square, N-acetylglucosamine; gray circle, mannose; bright circle, galactose; triangle, fucose. dSD, standard deviation.

samples were also analyzed by online hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS) and online HILIC-MS/MS. As shown in Figure 5A, the obtained total ion chromatograms (TICs) of these samples exhibit many differentiable peaks, suggesting an efficient separation of these glycan derivatives and the compatibility of GP derivatives of glycans with HILIC-MS techniques. Their structure and composition are presented in Table 1. Totally, 12 different N-glycans have been successfully separated, including three groups of glycan isomers. For the detailed characterization of these glycans, both d0- and d5-GP derivatives of each TIC peak were identified using the online MS/MS technique. The d0- and d5-GP derivatives of Fuc1Hex4HexNAc4 (peak 7), for example, produce similar online MS/MS profiles (Figure 6A), indicating the high correspondence of the d0- with d5-GP derivatives of each TIC peak. For the distinguishment of the isomers of a glycan, their MS/MS profiles were comprehensively compared with each

1692.67/1697.83, 1758.92/1764.00, 1854.92/1860.00, and 1921.00/1925.92) and one single peak (m/z 1805.00) in the ESI-MS profile. All of these signals are assigned to [M]+-type molecular ions. Obviously, seven N-glycans corresponding to the peak pairs exist in both mouse and rat sera, of which Fuc1Hex5HexNAc4 is much more in mouse serum than in rat serum (by 1.68 times), while Hex5HexNAc2, Hex6HexNAc2, Fuc1Hex3HexNAc4, Hex7HexNAc2, Fuc1Hex4HexNAc4, and Hex8HexNAc2 are much fewer in mouse serum than in rat serum (by 0.64, 0.52, 0.21, 0.33, 0.84, and 0.40 times, respectively). All of the CV values of the MS peak intensity ratios remain below 10% (n = 3), demonstrating good reproducibility of these data. Meanwhile, the glycan Fuc1Hex3HexNAc5 corresponding to the single peak of d5-GP derivatives exists only in rat serum, consistent with the results of Figure 4A and B. For the quantitative comparison of glycan isomers between mouse and rat sera, the three differentially mixed equal-ratio 381

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Figure 6. Sequence identification of d0- and d5-GP-labeled N-glycan isomers derived from mouse and rat sera by online HILIC-MS/MS. (A) Online MS/MS spectra of d0- and d5-GP derivatives of N-glycans. (B) Online MS/MS spectra of d5-GP derivatives of N-glycan isomers. The MS/MS fragment ions were assigned using GlycoWorkbench.30 Structural formulas: square, N-acetylglucosamine; gray circle, mannose; bright circle, galactose; triangle, fucose.

literature reference.38 The other peaks (peak 9, peak 11, and peak 12) in the TICs were assigned by reference to the literature reports34,35,37,38 mentioned above. Obviously, due to the immovable positive charge center on GP, the MS/MS profiles of all of these d0- and d5-GP derivatives of glycans exhibits X-, Y-, and Z-type molecular fragment ions, without A-, B-, or C-type fragments detected, simplifying greatly the structural identification of various reducing glycans. Meanwhile, quantitative comparison of these glycan isomers between the mouse and rat sera were also performed using this new method. As shown in Figure 5B, the corresponding online mass spectra of the TIC peaks of the mixture of d0-GP derivatives of MSGs

other. The three isomers of Fuc1Hex3HexNAc4 labeled with d5GP generate three different MS/MS profiles (Figure 6B). The glycan of peak 1 in the TIC is characterized with the fragments of m/z 1038, 1056, and 1074, peak 2 with the coexistence of m/ z 1380, 1398, and 1439, and peak 5 with the absence of m/z 1421 and 1439, enabling the recognition of their structure. Similarly, the structure of the isomers of Hex5HexNAc2 (peak 4 and peak 8) and Hex6HexNAc2 (peak 6 and peak 10) was assigned on the basis of online MS/MS data (shown in Figure S9 and Figure S10 in Supporting Information). Peak 3 was assigned according to the MS/MS data of Fuc1Hex3HexNAc5 (shown in Figure S7 in Supporting Information) and a 382

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and d5-GP derivatives of RSGs exhibit 11 pairs of MS peaks and a single peak. The MS signal intensity ratio data are presented in Table 1. Obviously, these MS signal intensity ratios are all close to those obtained by ESI-MS, indicating that the isomers of each glycan are similar to their monosaccharide composition in the quantitative difference between MSGs and RSGs. These MS signal intensity ratios are also consistent with the differences in TIC peak area between MSGs and RSGs (shown in Figure 5A), suggesting the reliability of these quantitative data. Additionally, some sialylated N-glycans reported previously22,34,35 are not observed in either ESI-MS spectra or HILIC-MS profiles. As described above, for the analysis of sialylated glycans using this method, the carboxyl groups of the glycans need to be neutralized via some derivatization reactions such as methyl esterification before isotopic labeling with d0- and d5-GP.39,40 Those experiments are in progress.

glycomics strategy. Therefore, this strategy represents a facile and versatile analytical method for comparative glycomics.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Natural Science Foundation of China (Nos. 31370804, 31071506, 31170773) and the Hi-Tech Research and Development Program of China (No. 2006AA02Z146).

CONCLUSIONS A new strategy has been developed, based on stable isotope labeling with d0- and d5-GP, for the rapid and sensitive relative quantitation of reducing glycans. To our knowledge, this is the first demonstration of isotopic cationic reagents as a glycan label for comparative glycomics studies. We have synthesized d5-GP, obtained the optimized reaction conditions for GP labeling, and validated the detection sensitivity of the method using lactose and bovine fetuin N-glycans as model glycans. The feasibility and stability of this strategy were investigated on the N-glycans released from RNase B and chicken ovalbumin, confirming good linearity, reproducibility, and accuracy of the method. The excellent applicability of the suggested procedure was validated via quantitative comparison of the neutral Nglycans released from bovine and porcine IgG by ESI-MS, as well as of those from mouse and rat sera by ESI-MS and online HILIC-MS. There are several conspicuous advantages of this new strategy over most of the other relative quantitation methods, including (i) nonreductive isotopic labeling of reducing glycans with d0- and d5-GP, without salts introduced, allowing direct and rapid detection by MS without any postprocessing steps; (ii) simplified and sensitive MS spectra resulting from the permanent [M]+-type molecular ions of GPderivatized neutral glycans, without any interference from diverse complex ion adducts, allowing rapid data interpretation; (iii) simplified MS/MS profiles arising from only X-, Y-, and Ztype molecular fragment ions, without any detectable A-, B-, or C-type fragments, enabling rapid structural identification of various neutral reducing glycans. Sialylated glycans have good structural stability under the reaction conditions for GP labeling but suffer low detection sensitivity when they are determined as GP derivatives by ESI-MS. For the analysis of sialylated glycans using this method, the carboxyl group of sialic acid needs to be neutralized via some derivatization reactions such as methyl esterification before isotopic labeling with d0- and d5-GP. Another study aimed at achieving sensitive and quantitative analysis of sialylated glycans based on this strategy is in progress now. Although this study is focused on the analysis of N-linked glycans, the suggested strategy is directly applicable to other types of reducing glycans, such as reducing O-linked glycans released from glycoproteins, free reducing oligosaccharides from various biological samples, and reducing glycans released from glycolipids. Additionally, in addition to d0- and d5-GP, some other types of isotopic cationic reagents are also compatible with the concept of simplified quantitative

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dx.doi.org/10.1021/pr4010647 | J. Proteome Res. 2014, 13, 372−384