Duplex Stable Isotope Labeling (DuSIL) for Simultaneous Quantitation

Subscriber access provided by Kaohsiung Medical University

Article

Duplex Stable Isotope Labeling (DuSIL) for Simultaneous Quantitation and Distinction of Sialylated and Neutral N-Glycans by MALDI-MS Lei Wei, Yan Cai, Lijun Yang, Ying Zhang, and Haojie Lu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02353 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Duplex Stable Isotope Labeling (DuSIL) for Simultaneous Quantitation and Distinction of Sialylated and Neutral N-Glycans by MALDI-MS Lei Weiab, Yan Caia,b, Lijun Yanga, Ying Zhang*b and Haojie Lu*ab a

Shanghai Cancer Center and Department of Chemistry, Fudan University, Shanghai, 200032, P. R. China Institutes of Biomedical Sciences and Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032, P. R. China b

A BSTRA CT: Quantitative N-glycomics provides an effective tool for detecting glycosylation changes in cancer and other diseases. However, the lability of sialic acid and its lower ionization efficiency compared with neutral glycans make their analysis by mass spectrometry complicated, and prevent the simultaneous quantitation and distinction of sialylated and neutral N-glycans by MS. To address this problem, we developed a novel approach duplex stable isotope labeling (DuSIL), to relatively quantify neutral and sialylated glycans concurrently by MALDI-MS. The duplex labeling strategy includes isotopic methylamidation labeling on the sialic acids and amino acid reductive amination on the reducing ends of N-glycans. Using this method, the labeled N-glycans showed doublet peaks with (6+3*N) Da mass difference for relative quantitation and discrimination of the number of sialic acids (N). The DuSIL strategy is of high labeling efficiency, high reproducibility (CV < 20%) and good linearity (R2 > 0.99) within two orders of magnitude of dynamic range. The strategy is successfully applied to measure N-glycans changes of IgG from human serum with colorectal cancer, demonstrating its potential in relative quantitation of N-glycome in clinical sample. Glycosylation is one of the most common and complex post-translational modifications of proteins. Changes in protein glycosylation are associated with various human diseases, such as immune deficiencies, cancer and dementia.1-3 Several studies have shown that the N-glycans could be used as disease biomarker.4,5 Notably, the nonreducing end of these N-linked complex glycans is typically occupied by sialic acids. Sialylation influences biological functions like immunogenic properties of the respective glycoprotein and the degree of sialylation affects various diseases.6,7 For example, the serum glycome of lung cancer patients shows significant alterations in sialylation with increases in trisialylated glycans and decreases in disialylated glycans.8 Stable isotopic labeling combined with mass spectrometry (MS) analysis is a powerful technology for the quantitative glycome. Relative abundances of glycan can be obtained by directly comparing peak intensities of pair signals with distinguishable mass difference after isotopic labelling. Based on a mass shift generated by stable isotopes like 2H, 13C and 15N, different types of isotope labeling methods such as permethylation,9,10 reductive amination,11,12 and hydrazine formation13 were developed. Most labeling-based quantitative strategies are effective in various ways, but each appears to be inefficient when it comes to sialylated glycans. Permethylation is used for quantitation of glycans by isotopic labeling with heavy and light methyl iodide.10 However, variable mass shifts were created due to the numerous number of their methylation sites such as biantennary or sialylated oligosaccharides. The variable mass differences bring difficulties in locating the pairs of relevant glycans from two respective samples for quantitation analysis.14 Additionally, different glycans may have different reaction efficiencies during permethylation because of side reaction, such as oxidative degradation of glycans.15,16 Isotopic derivatized the reducing end of glycans by reductive amination or hydrazone formation could result in the loss of sialic acid residues in some labeling conditions.11,13 Furthermore, the loss of sialic acids was found during 2-aminobenzoic acid (2-AB) labeling and the phenomenon was even more obvious for highly sialylated glycan.17 Particularly for the MALDI-MS analysis, sialylated glycans have much lower ionization efficiency than neutral N-glycans and easily get lost during positive ion mode caused by in- and post-source decay,18 hindering the direct comparison of neutral and

acidic glycans simultaneously. As the number of sialic acid moieties varies on a particular glycan, it becomes inherently difficult to detect these glycans, which can lead to incomplete characterization of a given sample.19 Therefore, to quantify the sialylated glycans, it is necessary to protect the sialylated glycans before or after reducing end labeling.12,20-22 Previous reported methods effectively prevent the loss of sialic acids during MALDI ionization process, while the loss of sialic acid during the reductive amination process is not considered. Moreover, these methods are unable to discriminate the number of sialic acids easily and the identification of sialic acids remains difficult. In this study, a novel method named duplex stable isotope labeling (DuSIL) for concurrent relative quantitation of neutral and sialylated N-glycans by MALDI-MS was developed. Isotopic methylamidation was first conducted to label the sialic acids on the N-glycans, followed by the isotopic amino acid reductive amination with all the N-glycans reducing ends. The labeled glycans from two samples produced doublet peaks with (6+3*N) Da mass difference, “N” represents the number of sialic acids. Here, 6 Da is from the reducing end and 3*N Da is from the sialic acid labeling. The new method can improve N-glycans identification and quantification with the following advantages. First, the labeling efficiency of the DuSIL method is high (almost 100%) with improved signal-to-noise (S/N) ratios of glycans (almost 10 folds) for more sensitive detection and quantitation of the sialylated glycans. Second, as the sialic acids are protected before reductive amination and MS measurement, relative quantitation of neutral and sialylated glycans from individual sample can be achieved simultaneously without the degradation of acid residues. Third, the created mass difference resulting in a mass shift of 3 Da per sialic acid, making the distinction of neutral and sialylated glycans easy because the sialylation degree of the sample can be conveniently determined. Fourth, isotopic methylamine and arginine reagents are both commercially available and nontoxic with more convenience than home-made reagents.

MATERIALS AND EXPERIMENTAL PROCEDURE Materials and Chemicals. IgG from human serum, bovine fetuin, methylamine hydrochloride(CH3NH2·HCl), methan-d3-amine hydrochloride(CD3NH2·HCl), L-Arginine (12C6-Arg), L-Arginine (13C6-Arg), dimethyl sulfoxide (DMSO), (7-azabenzotriazol-1-yloxy) trispyrrolidinoph-

ACS Paragon Plus Environment

1

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

osphonium hexafluorophosphate (PyAOP), 4-methylmorpholine, sodium cyanoborohydride (NaBH3CN), trifluoroacetic acid (TFA), ammonium bicarbonate (ABC) and 2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma (St. Louis, MO, USA). A2 glycan was acquired from Ludger (Oxford, UK). Peptide N-glycosidase (PNGase F) was obtained from New England Biolabs (Ipswich, MA, USA). HyperSep Hypercarb SPE was purchased from Thermo Fisher Scientific (CA, USA). HPLC-grade acetonitrile (ACN) and methanol were purchased from Merck (Darmstadt, Germany). Analytical grade acetic acid (CH3COOH) and methyl alcohol (MeOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Distilled water was purified by a Milli-Q system (Milford, MA, USA). Serum Samples. Blood samples were collected from 20 colorectal cancer (CRC) patients and 20 healthy donors at Fudan University Shanghai Cancer Center. The research was handled under an institutional review board-approved protocol with ethical and legal standards. Each sample was split into aliquots and stored at -80 °C until use. Serum IgG Purification. To analyze N-glycans of immunoglobulin G (IgG), purification of serum total IgG was performed on Protein G+A Sepharose (Roche) according to the procedure reported previously.23 Briefly, 20 μL beads were first loaded on a 96-well filter plate (Multiscreen Solvinert, 0.45 μm pore size low-binding hydrophilic PTFE; Millipore, Billerica, MA). After washing three times with five volumes of binding buffer (20 mM sodium phosphate), 20 μL serum and 250 μL binding buffer were successively applied to each well. The plate was incubated on a shaker at 4 °C for 15 min. Next, the retained beads were washed three times with 200 μL binding buffer. IgGs were eluted using 200 μL 0.1% TFA and immediately neutralized to pH 7.0 with 1 M ABC buffer (resulting in approximately 1 mg/mL). The purified IgG was quantified by BCA method and then equal amounts of IgG (200 μg) from each sample were prepared for later analysis. N-glycans Preparation. Glycoproteins (fetuin or IgG from human serum) were dissolved in 25 mM ABC buffer (pH 8.0) with a concentration of 1 mg/mL. After denaturation, 5 mU PNGase F solution was added and the enzymatic deglycosylation reaction was carried out at 37 °C overnight. The released N-glycans were further purified over a HyperSep Hypercarb SPE, the N-glycans were eluted by 2 mL H2O/ACN (70/30, v/v). Then the purified N-glycans were dried for subsequent derivatization.

Isotopic labeling the sialic acid with methylamine. The sialic acid residues of glycans were all labeled using methylamidation according previous report.24 The dried glycans were dissolved in 10 μL of DMSO containing 5 M methylamine hydrochloride or methan-d3-amine hydrochloride. Then 10 μL of PyAOP (250 mM in 30% 4-methylmorpholine/DMSO) was added. The reaction proceeded at room temperature with constant shaking for 30 min. The derivatized glycans were purified by HILIC SPE, 5 mg of cellulose microcrystalline particles (Merck) was applied to each well of a 96-well filter plate (Multiscreen Solvinert, 0.45 μm pore size low-binding hydrophilic PTFE; Millipore, Billerica, MA). The HILIC stationary phases were prewashed using 3 × 200 μL of water, followed by activation using 3 × 200 μL of 80% ACN containing 1% TFA. The samples were loaded to the wells, subsequently washed using 3 × 200 μL 80% ACN containing 1% TFA. Glycans were eluted using 3×100 μL of water, followed by lyophilization for further analysis.

Isotopic labeling the reducing ends with amino acid. The methylamidation glycans were further reductive aminated by Arginine.12 Briefly, Arg(12C6) and Arg(13C6) were separately dissolved in distilled water at a concentration of 100 mg/mL. The glycan samples were redissolved in 10 μL of methanol-acetic acid (98:2, v/v) containing 0.1 mg/mL NaBH3CN, followed by the addition of 1 μL of Arg(12C6) or Arg(13C6). Then the sample solution was incubated at 65 °C for 3.5 hours. After reactions, the light and heavy labeled analytes samples were mixed. The

Page 2 of 8

mixed light and heavy labeled analytes were then sent for the second HILIC SPE purification as described above. MALDI -TOF MS Analyses. One microliter of sample and the same volume of DHB matrix solution (10 mg/mL in 50% ACN containing 0.1% TFA) were spotted on the MALDI plate for MS analysis. The MALDI-TOF MS and MS/MS spectra were acquired using 5800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA) equipped with a Nd: YAG laser (355 nm), an acceleration voltage of 20 kV and a repetition rate of 400 Hz. MALDI-TOF MS and MS/MS experiments were performed in positive ion reflection mode on a 5800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA) with a Nd: YAG laser (355 nm), an acceleration voltage of 20 kV and a repetition rate of 400 Hz. The spectrometer was operated in reflectron mode accumulated by 1000 laser shots. Resulting spectra were interpreted manually with the assistance of GlycoWorkbench software (Euro-CarbDB).25 Data Analysis. The MS data processing was further performed by Data Explorer 4.0 (AB SCIEX, Concord, Canada). Monoisotopic peak intensity was used as the index to calculate the relative quantitation ratio. Moreover, considering the shift in relative isotopic distribution by the heavy atoms, the relative quantification ratio was corrected by multiplying a correction coefficient “X”. The correction process was performed as described in Supporting information, Figure S1 and Table S1.

Results and Discussion Optimization of the duplex stable isotope labeling (DuSIL) condition. The duplex stable isotope labeling strategy has two derivatization steps including methylamidation on the non-reducing end and reductive amination on the reducing end. Because the loss of sialic acids has been observed not only in MS ionization, but also in sample preparation step, we first label the sialic acids and then label the reducing end to minimize the loss of sialic acids during the sample preparation.26 Here, methylamidation of sialic acid is chosen because it can derivatize all sialic acid residues completely under mild conditions no matter what kind of linkage type (α2,3 and α2,6 linkage) without side products.24 We chose two isotopic reagent methan-d0/d3-amine hydrochloride to label sialylated glycans. The signal pairs with 3 *N Da mass shift (N is integer) can represent the relative abundance of corresponding sialylated glycans between two samples and obtain the number of sialic acids as well (Scheme 1). It is worth mentioning that, if we only aimed at sialylated glycans quantification, the following isotope labeled step on the reducing end would be skipped. To quantify the neutral glycans simultaneously, we add one more derivatization step to incorporated another isotope tag on the reducing end ofcompletely modified glycans with Arg have been well established by our group and its stable isotopic regents are commercially available.12 After the dual isotope reactions, two samples are mixed together and subjected to analyses by MALDI-MS. With the (6+3*N) Da mass difference of corresponding MS doublets, neutral and acidic glycans could be quantitated concurrently in positive ion mode. The N represents the number of sialic acids in a single glycoform. If N is zero, that means the doublet signal is attributed to a neutral glycan. We first investigate the labeling efficiency with a standard glycan A2 (a bi-antennary complex-type N-glycan containing two sialic acids). Figure 1A presented the [M - 2H + 3Na]+ ion of A2 at m/z 2291.0 with a signal-to-noise ratio (S/N) of 73. Due to the in- and/or post source decay, the profile of nonderivatized glycan is dominated by the glycans with sialic acids loss (m/z 1662.8, 1976.9). After dual labeling, the derivative of A2 was detected with the predicted mass shift at m/z 2407.8 (S/N 695) in the mass spectrum (Figure 1B). Moreover, we found that A2 glycan showed better ionization with a cleaner background and a higher S/N ratio (~10 fold) than its pristine counterpart. No signals of desialylated glycans and side products were observed, demonstrating the nearly 100% labeling efficiency, as well as the chemical ability to stabilize the sialic acid moieties and to improve ionization


2

Page 3 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme1. Workflow of the duplex isotopic labeling strategy for relative quantitation of N-glycans. N in mass difference represents the number of sialic acids in a single glycoform. If N is zero, that means the doublet signal belongs to a neutral glycan. response of sialylated glycan. The minor signals are sodium adductions of product, not from side reaction. Similar results are obtained with a mass difference of 12 Da (6+3*2 Da) between light and heavy isotope labeled A2 glycans in molar ratios of 1:1 (Figure 1C). Two sialic acid residues in the A2 glycan structure were confirmed by the created mass difference (6 Da). We also investigated the chromatographic behavior of light and heavy DuSIL labeled glycan (Supporting Information, Figure S2). To further validate this strategy, bovine fetuin, a glycoprotein which carries a series of multi-antennary sialoglycans was selected. Many side products (m/z 2028.4, 2275.5, 2525.1, 2649.5, and 2962.1) are observed in the dissociation of nonderivatized glycans caused by in- and post source decay, further suggesting that underivatized sialoglycan was not sensitive in positive ion mode MALDI-MS analysis (Supporting Information, Figure S3A). After duplex derivatization, the mass spectrum is improved with a higher S/N ratio and a cleaner background, all the peaks (m/z 2103.7, 2407.8, 2772.9, 3077.0, 3381.1) were the derivatized products of corresponding sialylated glycans of Man3GlcNAc4Gal2Sia1, Man3GlcNAc4Gal2Sia2, Man3GlcNAc5Gal3Sia2, Man3GlcNAc5Gal3Sia3 and Man3GlcNAc5Gal3Sia4 respectively as shown in (Supporting Information, Figure S3B). Importantly, the methylamidated sialic acid kept stable during the following reductive amination process (Supporting Information, Figure S4). Furthermore, the negative mode spectrum of N-glycan from fetuin was conducted to confirm the labeling efficiency and sialic acid stability, particularly for tri- and tetrasialylated species (Supporting Information, Figure S5). The underivatized sialic acids and the N-glycans with sialic acid loss are not observed, suggesting the high labeling efficiency of the DuSIL method. However, the ionization efficiency and S/N in the negative mode are much lower than that in the positive mode, which is not suitable for the quantitation analysis, so we used the positive mode to quantitate the N-glycans in this study. All these results suggest that this method can be used in profiling of sialoglycans with high labeling efficiency.

Dynamic range and accuracy of relative quantification. To evaluate the dynamic range and accuracy of this

Figure 1. MALDI-TOF mass spectra of sialylated glycan (A2 glycan) (A) underivatized, (B) derivatized with light DuSIL method and (C) derivatized with light/heavy DuSIL method and mixed in a molar ratio of 1:1. “※” denotes the Na adducts.

quantitative strategy, sialylated N-glycans from fetuin were derivatized with dual light or heavy isotope reagents in a series of molar ratios (10:1, 5:1, 3:1, 1:1, 1:3, 1:5, and 1:10) with triplicates. Figure 2A shows the mass spectra of the equimolar mixture of fetuin N-glycans labeled with either methylamine hydrochloride or methan-d3-amine hydrochloride, isotopic peaks yielded the ratio of 1:1 as the expected. After that, the light and heavy methylamidated samples were labeled with Arg(12C6) or Arg(13C6) respectively, then subjected to MALDI-MS analysis (m/z 2103.7 vs 2112.7, 2407.9 vs 2419.9, 2772.9 vs 2784.9, 3077.1 vs 3092.1 and 3381.2 vs 3399.2, Figure 2B). The dual logarithm plots of the theoretical molar ratios vs the experimental ratios of each fetuin N-glycan were displayed in Figure 2C. The results showed good linearity (R2 > 0.99) within two orders of magnitude, suggesting the good reliability of the strategy. Additionally, mass difference in these two spectra accurately demonstrate the number of sialic acids, suggesting this method could be used to predicate the ambiguous glycoforms with multiple sialic acids easily in MS1.


3


Page 4 of 8

Figure 2. MALDI-TOF mass spectra of N-glycans from bovine fetuin (A) methylamidation with light/heavy labeling regent and mixed in 1:1 molar ratio. (B) derivatized with light/heavy DuSIL method and mixed in a molar ratio of 1:1, SA represents the number of sialic acids. (C) dynamic range and accuracy of quantitation of the N-glycans from fetuin (n=3).

Simultaneous Quantitation of Sialylated and Neutral N-Glycans. N-glycans from IgG have been linked to pathogenesis of diseases and the therapeutic functions of antibody-based

drugs.23,27 We further used IgG to validate the feasibility of the strategy and established a robust approach for simultaneous quantitation of sialylated and neutral N-Glycans from IgG. Two equal aliquots of IgG were processed with


4

Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. MALDI-MS spectrum of N-glycans from human IgG derivatized with light/heavy DuSIL method and mixed in a molar ratio of 1:1.

Figure 4. MALDI-MS spectra of N-glycans from human IgG derivatized with light /heavy DuSIL method and mixed in different molar ratios: (A) 1:1 (B) 3:1 (C) 5:1 (D) 10:1.


5


Page 6 of 8

Figure 5. Dual peaks in MALDI-MS spectra of N-glycans from IgG after processing DuSIL method (A) neutral, (B) sialylated N-glycans and MALDI-TOF/TOF tandem mass spectra of N-glycans from IgG (C) neutral, (D) sialylated. PNGase F in parallel and dually labeled with the light or heavy isotope reagents. As shown in Figure 3, a total of 25 pairs of the N-glycans compositions from IgG were detected as a doublet with (6+3*N) Da mass difference. From the mass difference we can discriminate the neutral and acidic glycans directly in MS1. Moreover, the two steps derivatization remarkably improve the ionization efficiency of trisialylated glycans (m/z 3077.0 vs 3092.0, 3223.1 vs 3238.1), which is at low abundance in IgG. Similar quantitation dynamic range experiment was also conducted in N-glycans from IgG released by PNGase F, a series of molar ratios (10:1, 5:1, 3:1, 1:1, 1:3, 1:5, and 1:10) native glycans were derivatized with dual light or heavy isotope reagents respectively with triplicates (Figure 4). The dual logarithm plots of the theoretical molar ratios vs the experimental ratios of corresponding glycans were displayed in Supporting information, Figure S6. The results have shown good linearity (R2 > 0.99) within two orders of magnitude, suggesting the reliability of this method for simultaneous quantitation of sialylated and neutral N-glycans from glycoprotein. To further confirm the deduced structure of IgG glycosylation in MS1, the tandem mass spectrometry was used to find out the unique fragment ions of N-glycans in MS/MS spectra. The MALDI-TOF MS/MS spectra of neutral and sialylated glycans from IgG after dual derivatizations are shown in Figure 5. For neutral glycans, a series of consecutive Y-type ions was predominately observed. B1 and consecutive Y ions (Figure 5B) indicated the absence of sialic acids from this m/z 1621.5, which is a neutral glycan with core-fucosylation. For sialylated glycan, B1-B4 and consecutive Y3-Y6 ions in

Figure 5D presented sialic acids in the terminal residue of this glycoform (m/z 2553.8). Therefore, the results of tandem mass spectrum confirmed the structure type deduced in MS1.

Quantitative analysis of IgG glycome from colorectal cancer patient serum. Colorectal cancer (CRC) is one of the most common malignancies in the world, with about one million new cases occurring every year.28 Altered glycosylation has significant consequences on Ig G and consequently on CRC immune surveillance.29 To verify the quantitation ability of dual isotope labeling strategy in real biological samples, N-glycans released from healthy human IgG and CRC patient IgG were tested. IgG was effectively purified by protein A+G, the purity of IgG from normal/CRC serum was confirmed by SDS-PAGE (Supporting Information, Figure S7). Before labeling, equimolar native DP7 was spiked into both samples as an internal standard to correct the mixed ratio. Two samples of N-glycans released from the same amount IgG of healthy control (pooled, n=20) and individual CRC patient (n=20) were separately derivatized with light and heavy DuSIL method, according to the method described above. Then, DuSIL labeled N-glycans between equal amount of healthy control group (light) and each individual healthy donor (heavy) was compared to set the criterion for unambiguous changes of the N-glycan. The ratios of these glycan species between normal (heavy)/normal (light) ratios were ranging from 0.73 to 1.16, so CRC (heavy)/normal (light) ratio of 1.16 were regarded as downregulation or upregulation. The CRC/normal ratios and CVs of glycoforms were illustrated in Supporting


6

Page 7 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


information, Table S2. The quantitative results show good reproducibility as the CVs are less than 20% (varied from 4.28 % to 19.60%). In total, 25 pairs of the N-glycans compositions from IgG were detected as a doublet with (6+3*N) Da mass difference, and all peaks were single hydrogen adducts. The result covered glycan various species from a simple neutral composition to a large trisialylated triantennary N-glycans. From Figure 6, we observed that 4 glycans were up-regulated, while 9 glycans were down-regulated. Most of the down-regulated glycans contain galactosylation or multi sialylation structures, and most of the up-regulated glycans are the neutral glycans with core-fucose. Furthermore, the difference in the sialylation mainly coming from decreases in disialylated glycans. The results are in accordance with previous reports that colorectal cancer associates with decrease in IgG galactosylation, sialylation and increase in core-fucosyltion of neutral glycans.30 Therefore, the dual isotope labeling strategy is applicable in the quantitative analysis of the complex N-glycomics. Among these changed N-glycans in CRC patient, the ratios for Man5GlcNAc2, Man3GlcNAc4Fuc1 were significantly higher than 1.16 and the ratios for Man3GlcNAc4Gal2Fuc1, Man3GlcNAc4Gal2Fuc1Sia2 were markedly lower than 0.73 (Man refers to mannose, GlcNac refers to N-acetyl glucosamine, Gal refers to Galactose, Fuc refers to Fucose, Sia refers to sialic acid), indicating the significant difference of these four glycans between healthy control and CRC. Those compositions with distinguished quantitative ratios might provide a perspective for further validation in the discovery of glycan-based biomarkers. In addition, DuSIL method was also feasible to quantify the N-glycans of complex mixture glycoproteins from human serum (Supporting Information, Figure S8).

associated with CRC. To conclude, the DuSIL strategy provides a unique approach for relative quantitation of N-glycome and has potential in clinical biomarker discovery.

ASSOCIATED CONTENT Supporting Information Additional information as noted in the text. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Phone: +86 21 54237618. Fax: +86 21 54237961. E-mail: [email protected]. *Phone: +86 21 54237618. Fax: +86 21 54237961. E-mail: [email protected]. ORCID Ying Zhang: 0000-0003-0509-1098 Haojie Lu: 0000-0003-3477-7662 N otes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT The work was supported by the National Key Research and Development Program (2016YFA0501303), NSF (Grants 21335002, 31670835 and 21675031) and Shanghai Projects (Eastern Scholar, Rising star 15QA1400600, 15JC1400700 and B109) and Open Fund of Key Laboratory of Glycoconjugates Research, Fudan University, Ministry of Public Health.

REFERENCES Figure 6. Relative quantitation of N-glycans from CRC and normal human IgG. It has been reported that measurements of total sialic acid in the human biological samples can predict the risk of various diseases.31,32 To quantify the change of total sialylation degree, a sialylation index was developed according to a previous reported fucosylation degree evaluation method.33 It is defined as total sialylation degree =(1 *glycan S1 +2 *glycan S2 +3* glycan S3)/∑glycan, where glycan S1 denotes the sum of abundances of singly sialylated glycans, glycan S2 denotes that of disialylated glycans, and Σ glycan represents the sum of abundances of all glycans. We used this index to characterize the total sialylation level of IgG and to illustrate the differences between normal and CRC. The CRC group has a lower sialylation degree with a mean of 7.15% decrease compared with healthy group (Supporting Information, Figure S9A). The ROC curve (Supporting Information, Figure S9B) analysis between two groups resulted in an AUC value of 0.915, showed a statistically significant decrease in the sialylation.

Conclusion. In summary, an effective N-glycan relative quantitation strategy (DuSIL) has been developed using dual stable isotope labeling. By isotopic methylamidation of the sialic acid residues and amino acid reductive amination of glycan reducing ends sequentially, simultaneous quantitation and discrimination of both neutral and sialylated glycans was achieved. The DuSIL strategy shows good linearity (R2 > 0.99) and high reproducibility (CV < 20 %) to within two orders of magnitude in the dynamic range. It has been successfully applied in the analysis of N-glycosylation of complex biological samples like human IgG and human serum. The feasibility as also been demonstrated to track the changes in sialylation of human IgG

(1) Pinho, S. S.; Reis, C. A. Nat. Rev. Cancer 2015, 15, 540-555. (2) Xu, C.; Ng, D. T. Nat. Rev. Mol. Cell Biol. 2015, 16, 742-752. (3) Zhang, Y.; Peng, Y.; Yang, L.; Lu, H. TrAC, Trends Anal. Chem. 2018, 99, 34-46. (4) Mariño, K.; Bones, J.; Kattla, J. J.; Rudd, P. M. Nat. Chem. Biol. 2010, 6, 713-723. (5) Adamczyk, B.; Tharmalingam, T.; Rudd, P. M. Biochim. Biophys. Acta. 2012, 1820, 1347-1353. (6) Sinclair, A. M.; Elliott, S. J. Pharm. Sci. 2005, 94, 1626-1635. (7) Bork, K.; Horstkorte, R.; Weidemann, W. J. Pharm. Sci. 2009, 98, 3499-3508. (8) Arnold, J. N.; Saldova, R.; Galligan, M. C.; Murphy, T. B.; Mimurakimura, Y.; Telford, J. E.; Godwin, A. K.; Rudd, P. M. J. Proteome Res. 2011, 10, 1755-1764. (9) Kang, P.; Mechref, Y.; Kyselova, Z.; And, J. A. G.; Novotny, M. V. Anal. Chem. 2007, 79, 6064-6073. (10) Hu, Y.; Desantosgarcia, J. L.; Mechref, Y. Rapid Commun. Mass Spectrom. 2013, 27, 865-877. (11) Prien, J. M.; Prater, B. D.; Qin, Q.; Cockrill, S. L. Anal. Chem. 2010, 82, 1498-1508. (12) Cai, Y.; Jiao, J.; Bin, Z.; Zhang, Y.; Yang, P.; Lu, H. Chem. Commun. 2015, 51, 772-775. (13) Walker, S. H.; Budhathokiuprety, J.; Novak, B. M.; Muddiman, D. C. Anal. Chem. 2011, 83, 6738-6745. (14) Shah, P.; Yang, S.; Sun, S.; Aiyetan, P.; Yarema, K. J.; Zhang, H. Anal. Chem. 2013, 85, 3606-3613. (15) Ionel Ciucanu, Costello, C. E. J. Am. Chem. Soc. 2003, 125, 16213-16219. (16) Pilsoo Kang, Y. M., Iveta Klouckova, Milos V. Novotny. Rapid Commun. Mass Spectrom. 2005, 19, 3421–3428. (17) Aich, U.; Hurum, D. C.; Basumallick, L.; Rao, S.; Pohl, C.; Rohrer, J. S.; Kandzia, S. Anal. Biochem. 2014, 458, 27-36. (18) Harvey, D. J. Mass Spectrom. Rev. 2012, 31, 183-194. (19) Nie, H.; Li, Y.; Sun, X. L. J. Proteomics 2012, 75, 3098-3112. (20) Zhou, H.; Warren, P. G.; Froehlich, J. W.; Lee, R. S. Anal. Chem. 2014, 86, 6277-6284. (21) Yang, L.; Peng, Y.; Jiao, J.; Tao, T.; Yao, J.; Zhang, Y.; Lu, H. Anal. Chem. 2017, 89,


7


Page 8 of 8

7470-7476. (22) Wang, C.; Wu, Y.; Zhang, L.; Liu, B. F.; Lin, Y.; Liu, X. Anal. Chim. Acta 2018, 1002, 50-61. (23) Wang, J. R.; Gao, W. N.; Grimm, R.; Jiang, S.; Liang, Y.; Ye, H.; Li, Z. G.; Yau, L. F.; Huang, H.; Liu, J. Nat. Commun. 2017, 8, 631-644. (24) Liu, X.; Qiu, H.; Lee, R. K.; Chen, W.; Li, J. Anal. Chem. 2010, 82, 8300-8306. (25) Ceroni, A.; Kai, M.; Geyer, H.; Geyer, R.; Dell, A.; Haslam, S. M. J. Proteome Res. 2008, 7, 1650-1659. (26) Yang, S.; Zhang, L.; Tomas, S.; Hu, Y.; Li, S.; John,C.; Zhang, H. Anal. Chem. 2017, 89, 6330-6335. (27) Harre, U.; Lang, S. C.; Pfeifle, R.; Rombouts, Y.; Frühbeißer, S.; Amara, K.; Bang, H.; Lux, A.; Koeleman, C. A.; Baum, W. Nat. Commun. 2015, 6, 6651-6662. (28) Ferlay, J.; Shin, H. R.; Bray, F.; Forman, D.; Mathers, C.; Parkin, D. M. Int. J. Cancer 2010, 127, 2893-2917. (29) Vesely, M. D.; Kershaw, M. H.; Schreiber, R. D.; Smyth, M. J. Annu. Rev. Immunol. 2011, 29, 235-271. (30) Vuä Koviä, F.; Theodoratou, E.; Thaã§I, K.; Timofeeva, M.; Vojta, A.; J, Å t.; Puä Iä-Bakoviä, M.; Rudd, P. M.; L, Ä. e.; Servis, D. Clin. Cancer Res. 2016, 22, 3078-3086. (31) Varki, A. Trends Mol. Med. 2008, 14, 351-360. (32) Ghosh, S. Glycoconjugate J. 2015, 32, 79-85. (33) Zhu, J.; Lin, Z.; Wu, J.; Yin, H.; Dai, J.; Feng, Z.; Marrero, J.; Lubman, D. M. J. Proteome Res. 2014, 13, 2986-2997.


8

Duplex Stable Isotope Labeling (DuSIL) for Simultaneous Quantitation

Recommend Documents