Glycoqueuing: Isomer-Specific Quantification for Sialylation-Focused

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Glycoqueuing: Isomer-Specific Quantification for Sialylation-Focused Glycomics Wanjun Jin, Chengjian Wang, Meifang Yang, Ming Wei, Linjuan Huang, and Zhongfu Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01393 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Analytical Chemistry

Glycoqueuing: Isomer-Specific Quantification for SialylationFocused Glycomics Wanjun Jin,1,† Chengjian Wang,1,‡ Meifang Yang,† Ming Wei,† Linjuan Huang,*,†,‡ and Zhongfu Wang*,†,‡ College of Life Science, Northwest University, Xi’an 710069, China Research Center for Glycobiology and Glycotechnology, College of Food Science and Technology, Northwest University, Xi’an 710069, China † ‡

ABSTRACT: Changes of α2,3-/α2,6-linked sialic acids (SAs) in sialylglycans have been found to be closely related with some diseases. However, accurate quantification of sialylglycans at the isomeric level remains challenging due to their instability, structural complexity and low mass spectrometry (MS) detection sensitivity. Herein, we propose an analytical strategy named “glycoqueuing”, which allows sequential chromatographic elution and high-sensitivity MS quantification of various sialylglycan isomers based on isotopic labeling followed by analysis via online reversed-phase high performance liquid chromatography coupling with MS (RPHPLC-MS). The new method was validated by detailed structural identification and quantification of fetal bovine serum (FBS) Nlinked sialylglycan isomers, during which many branching isomers were successfully differentiated and twenty-eight sialylglycan compositions with Neu5Gc residues were analyzed. The method was successfully applied to isomer-specific, quantitative comparison of sialylated N-glycans between bovine and rabbit immunoglobulin G (IgG) and the search for serum sialylated N-glycan biomarker candidates of hepatocellular carcinoma, during which a 55% increase of α2,6-sialylated fucosylated N-glycans was revealed, demonstrating the great applicability and potential clinical usage of the method.

Sialic acids (SAs), a family of acidic nonose, usually occur as terminal sugars of free oligosaccharides, N- and O-glycans of glycoproteins and glycolipids.1 In mammalian cells, the most common SAs are Neu5Ac and Neu5Gc, and only the former is present in normal human cells.2 SAs are normally attached to terminal galactose residues via α2,3- or α2,6-linkages or Nacetylgalactosamine residues via α2,6-linkages, resulting in different biological activities.3 It has been reported that α2,6linked SAs are the sites binding to human influenza virus, whereas α2,3-linked SAs are the structures binding to avian influenza virus.4 Moreover, changes in linkages of SAs are closely related with development of cancer, which often involves an increase in α2,6-sialylation.5 As α2,3-sialylated antigens, Slea and Slex have also been demonstrated to be highly expressed in many malignant cancers.1,3,5 Therefore, quantification of sialylglycans at the isomeric level is essential and significant for the studies on their biofunctions as well as the search for sialylglycan biomarkers of many diseases. Mass spectrometry (MS) has emerged as a powerful tool for qualitative and quantitative analysis of glycans. However, it is still a challenge to quantify sialylglycans due to the low ionization efficiency and complicated isomers. Moreover, the SA residues are usually lost during MS ionization and sample preparation.6 Several methods have been reported to quantify sialylglycans by modifying the carboxyl groups of SAs in combination with isotopic labeling. Permethylation has been widely used to stabilize sialylglycans,7,8 while variable mass shifts were introduced during isotopic labeling due to the different number of labeling sites of different glycans. Recently, amidation of SAs was utilized for quantification of sialylglycans due to mild reaction conditions, good stability of

amide bond, and high reaction efficiency.9,10 Zhang H. et al. performed isotopic modification of sialylglycans by amidation with p-toluidine to determine the number of SA residues and quantify sialylglycans by MALDI-MS.11 A dual modification strategy was developed to simultaneously quantify neutral and sialylated N-glycans by methylamidation of SA residues and reductive amination of reducing ends with an isotopic form of 2-aminobenzoic acid.12 Simultaneous quantification and distinction of sialylated and neutral N-glycans have also been performed by isotopic labeling on the SA residues by methylamidation and reductive amination derivatization at the reducing ends using amino acids.13 Although those methods facilitated the quantitative analysis of sialylglycans, the glycan isomers have not been differentiated and quantified. To differentiate the different linkages of SA residues of glycans, many techniques can be used. Initially, sialylglycan isomers were often separated by hydrophilic interaction chromatography (HILIC),3,14,15 porous graphitized carbon (PGC) materials,16 or capillary electrophoresis (CE)17, and the identification of linkage types of SA residues was performed by linkage-specific exoglycosidase, MS/MS and different pKa values. However, those methods are not suitable for accurate quantification due to the instability of sialylglycans3,14,15,17 or the variable mass shifts of permethylated derivatives.16 Another strategy involves linkage-specific derivatization of SA residues, during which α2,6-linked SAs are converted to esters or amides, whereas α2,3-linked SAs form lactones, producing different masses during MS detection and enabling rapid differentiation of linkage isomers of SAs.18,19,20 Nevertheless, more detailed isomeric structures of sialylglycans with different branches cannot be clearly distinguished and accurately quantified.

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Herein, we propose a novel analytical strategy named “glycoqueuing”, which enables sensitive and accurate isomerspecific quantification of sialylglycans. In this method, reducing glycans are subjected to nonselective isotopic labeling of SA residues by amidation with either nondeuterated (d0-) or deuterated (d5-) aniline, removal of non-sialylated glycans through fractionation using a C18 solid phase extraction (SPE) column and reducing-end derivatization with 1-(2-hydrazino-2oxoethyl) pyridinium chloride (Girard’s reagent P, GP). The obtained sialylglycan derivatives are analyzed by reversedphase high performance liquid chromatography coupling with MS (RP-HPLC-MS), and isomer-specific quantification is performed based on MS signal intensity. The SA linkage types of each separated isomer is confirmed by RP-HPLC-MS/MS analysis of the mixture of d0-aniline nonselectively-labeled GP derivatives and SA-linkage-specific d5-aniline-labeled GP derivatives of the sialylglycans, and the isomeric branching structures are identified by online tandem mass spectrometry (MS/MS). This strategy enables simultaneous qualitative and quantitative analysis of sialylglycan isomers with significant advantages. First, amidation of SA residues with aniline not only stabilizes the structure of sialylglycans, but also introduces hydrophobic and fluorescent groups, which enable highresolution RP-HPLC separation of sialylglycan isomers based on their differences in the nonreducing-end structure. As a result, sialylglycan isomers can be differentiated and recognized according to a regular elution order (glycoqueuing) that is closely related with the number of SA residues and the ratio between α2,3- and α2,6-linked SAs. Second, GP labeling can simplify the MS signals of glycans and greatly improve their MS detection sensitivity.21 Third, nonselective isotopic labeling of SA residues with d0/d5-aniline allows accurate quantification of sialylglycan isomers by RP-HPLC-MS and rapid recognition of the SA residue number of each glycanaccording to the mass differences between the corresponding light- and heavy-isotope form derivatives. The method has been validated in detail using fetal bovine serum (FBS) N-glycans 22 and successfully applied to isomer-specific, quantitative comparison of sialylated N-glycans between bovine and rabbit immunoglobulin G (IgG) and the search for serum sialylated N-glycan biomarker candidates of hepatocellular carcinoma (HCC). Additionally, the applicability of the method to sialylated O-glycans was evaluated. EXPERIMENTAL AND METHODS Materials and Reagents. FBS and HPLC grade acetonitrile (ACN) were obtained from Thermo Fisher Scientific (Fairlawn, NJ, USA). Microcrystalline cellulose, d5-aniline, 2,5dihydroxybenzoic acid (DHB), rabbit and bovine IgG, were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-(3(Dimethylamino) propyl) 3-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole monohydrate (HOBt), dimethyl sulfoxideand (DMSO) and trifluoroacetic acid (TFA) were from Aladdin Chemicals (Shanghai, China). 3′sialyllactose (3′-SL, α2,3-linked SA), and 6′-sialyllactose (6′SL, α2,6-linked SA) were products of Carbosynth Limited (United Kingdom). GP was from TCI Development Co., Ltd. (Japan). Peptide N-glycosidase F (PNGase F) was from New England BioLabs (Ipswich, MA, USA). Sep-Pak C18 (200

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mg/3 mL) and PGC (150 mg/3 mL) SPE columns were from Simon Aldrich (Germany). Water was purified through a MilliQ purification system (Millipore, Milford, MA, USA). Preparation and Pretreatment of Human Sera. Human serum samples from 20 healthy donors and 20 HCC patients were provided by The Second Affiliated Hospital of Xi’an Jiaotong University (Xi’an, Shaanxi, China) under permission from the local legal departments and scientific-ethics institutes. The clinical information of HCC patients is shown in Table S1. The people involved in the study are voluntary and no reward has been paid for them or their families. These healthy and HCC sera were pooled separately, and 500 μL of pooled healthy serum sample (HS) and 500 μL of HCC serum sample (HCCS) were exhaustively dialyzed in a 10-kDa dialysis membrane against Milli-Q water at 4 °C for 48 h. The obtained samples were lyophilized and stored at −20 °C for further use. Extraction of FBS Protein. As described above, FBS was dialyzed, lyophilized, and stored at −20 °C for use. N-Glycan Preparation. N-Glycans were enzymatically released according to a previously reported procedure.21 Briefly, glycoprotein or serum samples were dissolved in 500 μL of protein denaturation solution containing 0.4 M DTT and 5% SDS with a concentration of 1 mg/100 μL. After denaturation at 100 °C for 10 min, 50 μL of sodium phosphate buffer (1 M, pH 7.5), 50 μL of aqueous NP-40 (10% vol/vol), and 2 μL of PNGase F solution (1000 units) were added, followed by incubation at 37 °C for 24 h. The obtained samples were further purified using C18 and PGC SPE column. Initially, the sample solution was loaded onto a C18 column pre-washed with 3 mL of ACN and equilibrated with 10 mL of water. The glycans were eluted with 10 mL of water, and the elutes were then loaded onto a PGC column pretreated in the same manner as C18. After washing with 10 mL of water to remove salts, the glycans were eluted with 5 mL of 25% ACN (vol/vol) containing 0.1% TFA and then dried for further derivatization. Nonselective Amidation Modification of SA Residues. Based on a previously reported method,11 reducing glycans were mixed with 450 μL of aniline solution (1 M) and 90 μL of EDC (2 M, pH 4.5 adjusted with HCl), followed by incubation at room temperature (25-30 °C) for 4 h. Then the mixture was concentrated using a centrifuge concentrator (Thermo) and loaded onto a hand-packed paper chromatography column. After drying at 45 °C for 6 h, the column was washed with 40 mL of ACN and eluted with 5 mL of water. After concentration, the obtained sample solution was loaded onto a C18 SPE column pretreated as described above and washed with 3 mL of water to remove salts and non-sialylated glycans. Finally, the sialylglycan derivatives were eluted with 5 mL of 25% ACN (vol/vol) and dried for further use. Linkage-Specific Derivatization of SA Residues. 1 μL of glycan solution derived from 0.2 mg of glycoprotein sample was incubated at 60 °C for 1 h after an addition of 20 μL of solution A (250 mM EDC and 500 mM HOBt in DMSO), followed by an addition of 20 μL of solution B (500 mM EDC, 1 M HOBt and 500 mM aniline in DMSO) and incubation at 60 °C for 1 h. After the sample was cooled, 500 μL of ACN was added, prior to storage at −20 °C for 30 min. Finally, the sample was centrifuged and the supernatant was removed. The obtained precipitate was collected and stored at −20 °C for further derivatization. Reducing-End Modification of Glycans with GP. Glycan


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Figure 1. Schematic of the isomer-specific quantification strategy of sialylglycans. (A) Isomer-specific quantification of nonselectively derivatized sialylglycans by RP-HPLC-MS. (B) Identification of SA residue linkages of each nonselectively derivatized glycan isomer by comparative analysis of nonselectively and linkage-specifically derivatized siaglylglycans via RP-HPLC-MS. Detailed structure of each glycan isomer is confirmed based on online MS and MS/MS data, including molecular weight, retention time, peak area ratio, peak area increased and peak shape. The diamond on the left side represents α2,3-linked SA, and the one on the right side is α2,6-linked SA. Tri3SA(3,6,6) is the abbreviation of triantennary N-glycans with one α2,3-linked SA residue and two α2,6-linked SA residues.

samples were dissolved in 20 μL of 0.1 M GP solution prepared in a water/methanol/acetic acid mixture (6:3:1, vol/vol/vol). After incubation at room temperature for 30 min, the sample was dried for further use. MALDI-TOF MS Analysis of Glycans. 0.5 μL of glycan solution prepared in 50% methanol was mixed with 0.5 μL of DHB matrix on a 384-well μFocus MALDI plate (Shimadzu, Columbia, MD). The DHB matrix solution was prepared by dissolving 20 mg of DHB in 1 mL of 50% ACN solution (vol/vol) containing 0.05% TFA. Glycans were analyzed by a Shimadzu AXIMA Confidence Mass Spectrometer (Shimadzu, Columbia, MD) in the positive reflectron mode. Online RP-HPLC-MS and MS/MS Analysis. Detailed analysis of sialylglycans was performed on an electrospray ionization linear ion trap quadrupole mass spectrometer (ESILTQ-MS) coupled with an HPLC system (LTQ XL, Thermo Scientific, USA). To perform LC-MS analysis, the glycan derivatives from 5 mg of glycoprotein sample were dissolved in 20 μL of water, and a 10-μL aliquot was injected using a Surveyor automatic sampler in the partial loop injection mode. The RP-HPLC separation was achieved using a Sinochrom ODS-BP column (4.6 mm × 250 mm, 5 μm) (Elite Corporation, Dalian, China) at ambient temperature (25 °C) with the following gradient: solvent A, ACN; solvent B, 10 mM ammonium acetate (pH 5.5); time = 0 min (t = 0), 1% A, 99% B; t = 30, 1% A, 99% B; t= 40, 9% A, 91% B; t = 100, 13.5% A, 86.5% B; t = 160, 18% A, 82% B; t = 175, 23% A, 77% B; the total flow rate of mobile phase was 800 μL/min. Glycans were analyzed by ESI-MS in the positive mode with a mass

range from m/z 850 to m/z 2000. The spray voltage was set at 4 kV, with a sheath gas (nitrogen gas) flow rate of 20 arb., an auxiliary gas (nitrogen gas) flow rate of 10 arb., a capillary voltage of 37 V, tube lens voltage of 250 V, and capillary temperature of 300 °C. For MS/MS analysis, the glycans were fragmented by collision-induced decomposition (CID), with helium (He) as the collision gas. A normalized collision energy degree ranging from 30 to 45 and an isotope width of m/z 3.00 were used. Activation Q was set at 0.25 and activation time at 30 ms. The MS and MS/MS data were recorded using the Xcalibur software (Thermo) and interpreted in detail using the GlycoWorkbench software. The glycan structure corresponding to each peak during RP-HPLC-MS was preliminarily assigned according to the online MS and MS/MS data. The SA linkage types of separated isomers as d0-aniline nonselectively-labeled GP derivatives were identified according to multiple data, such as their retention times that are different from other structurally defined isomers, biosynthetic rules of glycans in certain species, MS/MS data of different types of ion adducts of each glycan, MS/MS data of larger or smaller structurally related glycans or some other co-eluted glycans when considering the microheterogeneity of natural glycans, EIC peak area increased when adding some structurally defined glycans, a comparison with their selectively, isotopically derivatized forms coanalyzed by RP-HPLC-MS in molecular weights, MS/MS data and shapes, elution order and area ratios of EIC peaks, etc. The results can be confirmed using different types of experimental data, which can support each other by conclusion.



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Figure 2. MALDI-TOF mass spectra of FBS N-glycan derivatives. (A) Native N-glycans released from FBS. (B) FBS N-glycan derivatives obtained by nonselective amidation of SA residues with aniline. (C) FBS N-glycan derivatives obtained by nonselective amidation of SA residues with aniline and reducing-end labeling with GP. (D) FBS N-glycan derivatives generated by selective amidation of SA residues with d5-aniline and reducing-end labeling with GP. (E) Equimolar mixture of d0- and d5-aniline nonselectively labeled sialylglycans from FBS as GP derivatives.

RESULTS AND DISCUSSION Principle of the Glycoqueuing Analytical Strategy. This novel analytical method is proposed for the differentiation and quantification of sialylglycans at the isomeric level, which are required for the studies on their biofunctions and the search for sialylglycan biomarkers of many diseases. To realize the proposed method, two major experimental steps should be performed, including RP-HPLC-MS analysis of the mixture of d0- and d5-aniline nonselectively labeled sialylglycans from different glycoprotein samples as GP derivatives for differentiation and quantification of glycan isomers (Figure 1A), and RP-HPLC-MS/MS analysis of the mixture of d0-aniline nonselectively-labeled GP derivatives and SA-linkage-specific d5-aniline-labeled GP derivatives of each sialylglycan sample for identification of SA residue linkages and branching structure of each nonselectively derivatized glycan isomer (Figure 1B). For the nonselective derivatization, reducingglycans are subjected to isotopic labeling of SA residues by amidation with either d0- or d5-aniline, removal of non-sialylated glycans through fractionation using a C18 SPE column and reducing-end derivatization with GP. The obtained sialylglycan derivatives allow comprehensive analysis by RPHPLC-MS, including high-resolution differentiation of glycan isomers based on the subtle difference in their hydrophobic nonreducing end structures (glycoqueuing), high-sensitivity MS detection enabled by the stable positive charge of GP, and accurate quantification based on MS signal intensity ratios

between different isotopic forms of each glycan isomer. As for SA-linkage specific derivatization of sialylglycans, α2,3-linked SA residues generate lactone structures, while α2,6-linked SA residues are modified by amidation with d5-aniline, followed by reducing-end derivatization with GP. When the mixture of d0-aniline nonselectively-labeled GP derivatives and SAlinkage-specific d5-aniline-labeled GP derivatives of each sialylglycan sample is analyzed by RP-HPLC-MS/MS, the extracted ion chromatogram (EIC) and online MS/MS data of each glycan isomer can be obtained, and their structure details in SA residue linkage and branching can be identified by comparison between the nonselectively and selectively derivatized glycans in molecular weight, retention time, peak area ratio, peak area increased and peak shape. Therefore, this strategy enables isomer-specific structural identification and quantification of sialylglycans. Nonselective Derivatization of Sialylglycans and Glycoqueuing Separation. To evaluate the feasibility and reliability the proposed method for nonselective derivatization and high-resolution separation of sialylglycans, FBS N-glycans were analyzed in detail as a model glycan sample. Initially, we successfully realized the SA residue amidation and GP labeling of sialyllactose (SL), showing the feasibility of the nonselective derivatization method (Figure S1 in Supporting Information). Then the obtained derivatization method was applied to FBS Nglycans (Figure 2). When native


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Figure 3. The UV chromatogram (A) and TIC (B) obtained during RP-HPLC-MS analysis of nonselectively labeled sialylated Nglycans released from FBS. Bi- or Tri- represents the number of antennas of N-glycans, and nSA represents the number of SA residues. Iso-n represents the isomer number of each glycan composition.

FBS N-glycans were analyzed by MALDI-MS, target glycans exhibited multiple ion forms, and the loss of SA was found (Figure 2A). After amidation with aniline, the FBS N-glycans were detected as intact molecular ions, without any SA residues lost (Figure 2B). When the aniline-amidated FBS N-glycans were labeled with GP, the obtained mass spectrum generated a series of [M]+-form molecular ion signals, greatly improving the MS detection sensitivity and resulting in the discovery of four N-glycan compositions with Neu5Gc residues in very low abundance (Figure 2C). Therefore, the non-selective derivatization method can not only stabilize sialylglycans, but also improve the MS detection sensitivity. To distinguish sialylglycan isomers, a method for RPHPLC-MS analysis of the non-selectively derivatized sialylglycans was developed. The non-selectively derivatized FBS N-glycans exhibited forty-one chromatographic peaks in the ultraviolet (UV) chromatograms and total ion chromatograms (TICs) obtained from RP-HPLC-MS analysis (Figure 3). All of the target MS signals corresponding to these chromatographic peaks in online mass spectra were [M+Na]2+ form molecular ions. Based on the online MS and MS/MS data, these chromatographic peaks were assigned to sixteen different N-glycan compositions15,16, which could be separated into four groups (1SA, 2SA, 3SA and 4SA) according to the number of SA residues of each glycan. Interestingly, the four groups of FBS N-glycans were eluted in a regular order (glycoqueuing) during RP-HPLC-MS analysis, which can be utilized for rapid assignment of chromatographic peaks. Moreover, many chromatographic peaks corresponding to the rare N-glycans with Neu5Gc residues were also clearly observed, indicating the ideal separation resolution and good detection sensitivity of the proposed analytical method. Identification of Sialylglycan Isomers by Selective Derivatization. To identify the SA linkage types of separated isomers, we performed selective derivatization of sialylglycans as GP derivatives by α2,6-linkage specific amidation of SA residues with d5-aniline and subsequent RP-HPLC-MS/MS analysis of the mixture of d0-aniline nonselectively-labeled GP derivatives and d5-aniline selectively-labeled GP derivatives of

each sialylglycan sample. For the SA-linkage specific derivatization of sialylglycans, two different reactions should be highly selectively achieved, including the lactonization of α2,3-linked SA residues without aniline and amidation of α2,6linked SA residues with aniline14,18 (Figure 1B). The derivatization specificity and lactone stability were verified based on the complete conversion of 3'-SL to its lactone form (Figure S2A and S2C) and 6′-SL to the aniline-amidated derivatives (Figure S2B and S2D). In contrast with literaturereported methods14,18, our analytical strategy features mild reaction conditions and a good stability of lactone derivatives of sialylglycans. When FBS N-glycans were selectively modified with d5-aniline and labeled with GP, the obtained results (Figure 2D) were consistent with previous reports14,18, except the newly observed glycans with Neu5Gc residues. This further demonstrated the good derivatization specificity of the method for α2,3- and α2,6-linked SAs. The RP-HPLC-MS/MS analysis of the mixture of d0-aniline nonselectively-labeled GP derivatives and d5-aniline selectively-labeled GP derivatives of FBS sialylglycans generated essential data for detailed structural elucidation of sialylglycan isomers. The structure of each sialylglycan isomer separated during RP-HPLC-MS analysis of the nonselectively

Figure 4. Identification of isomeric structures of the FBS N-glycan H6N5Ac3 (Tri-3Ac) by RP-HPLC-MS/MS analysis of the mixture of d0-aniline nonselectively-labeled GP derivatives and SAlinkage-specific d5-aniline-labeled GP derivatives of FBS sialylglycans. EICs of d0-aniline nonselectively-labeled GP derivative of Tri-3Ac (A) and SA-linkage-specific d5-anilinelabeled GP derivatives of Tri-3Ac(6,6,6) (B), Tri-3Ac(3,6,6) (C), Tri-3Ac(3,3,6) (D), Tri-3Ac(3,3,3) (E) are provided. All of the glycan fractions have been analyzed by MS/MS.



Figure 5. FBS sialylated N-glycan structures detected using the glycoqueuing analytical strategy. The lines in different colour represent the glycan groups containing different number of SA residues. The blue number represents the elution order of different glycan structures during RP-HPLC-MS analysis. H, Hexose; N, Nacetylhexosamine; Ac, Neu5Ac; Gc, Neu5Gc.

labeled FBS N-glycans (Figure 3) was identified by online MS/MS data (Figure S3) as well as a comparison of EICs between the nonselectively and selectively labeled glycan samples (Figure 4 and Figure S4 to Figure S18 in Supporting Information). The obtained parameters that are required for certification of glycan structure are provided in Table S2, and the identified N-glycan structures of FBS are shown in Figure 5. For instance, the FBS N-glycan H6N5Ac3 (abbreviated as Tri3Ac) nonselectively labeled with d0-aniline and GP exhibits four major peaks (Iso-1, 5, 7, and 8) and four minor peaks (Iso2, 3, 4, and 6) in EIC (Figure 4A). In contrast, the EICs of d5aniline selectively-labeled GP derivatives of the Tri-3Ac generate a series glycan peaks with different molecular weights and retention time (Figure 4B to 4E). The varied molecular weights provide information regarding the exact number of α2,3-linked and α2,6-linked SA residues of each selectively derivatized glycan isomer, the online MS/MS data allow the identification of branching structures of selectively derivatized glycan isomers, and the relationship between EIC peaks of the d0-aniline nonselectively labeled glycan isomers and d5-aniline selectively labeled glycan isomers in retention time and area can be used to confirm the detailed structure of the d0-aniline nonselectively labeled glycan isomers. As a result, we found the glycan structure Tri-3Ac(6,6,6) corresponding to the EIC in Figure 4B exhibits only one peak (Iso-1). Its retention time in Figure 4B is very similar to that in Figure 4A, due to the tiny difference in hydrophobicity between d0- and d5-aniline, indicating the EIC peak Iso-1 in Figure 4A corresponds to the glycan structure Tri-3Ac(6,6,6). The glycan structure Tri3Ac(3,3,3) corresponding to the EIC in Figure 4E also exhibits only one peak with the smallest retention time, due to the lack of d5-anilinie structure. In contrast, other glycan structures,

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including Tri-3Ac(3,6,6) and Tri-3Ac(3,3,6), exhibit more EIC peaks corresponding to isomers with different branching structures (Figure 4C and 4D), which have been identified by online MS/MS. The more α2,6-linked SA residues the glycans contain, the larger retention time they have. According to the percentage of each glycan isomer in their total amount, the structural correspondence between the EIC peaks of glycans shown in Figure 4A and those in Figure 4C to 4E was confirmed. These results together show that the Tri-3Ac component has four groups of isomers with different SA-linkages, including Tri-3Ac(6,6,6), Tri-3Ac(3,6,6), Tri-3Ac(3,3,6), and Tri3Ac(3,3,3). As nonselectively labeled derivatives, these glycan isomers are eluted according to a regular order, in which the more α2,6-linked SA residues an isomer has, the earlier it will be eluted, showing a typical glycoqueuing elution pattern that are very useful for structural identification of glycan isomers. Identification of more detailed branching structures of each separated glycan isomer relies on online MS/MS fragmentation. For example, the glycan structure Bi-2Ac (3,6), a group of SAlinkage isomers of the N-glycan composition H5N4Ac2 (abbreviated as Bi-2Ac), was differentiated as two branching isomers (Figure S19). Similarly, the isomers of other FBS Nglycan compositions were identified in detail using this method and found to share the same elution regularity with Tri-3Ac, demonstrating the ideal reliability of the glycoqueuting strategy. Noteworthy, we have also successfully analyzed a series of unusual FBS N-glycans with Neu5Gc residues at the isomeric level during this process, indicating the high detection sensitivity of our method. Totally, forty-one FBS sialylated N-glycan structures have been successfully separated andidentified, including twentyeight N-glycans with Neu5Gc residues (Figure 5). Additionally, we also concluded that the nonselectively derivatized sialylglycans is separated by RP-HPLC in a glycoqueuing pattern, which is closely related with the total number of SA residues and the ratios between α2,3- and α2,6-linked SA residues of each glycan. Verification of Isomer-Specific Quantification Features of the Strategy. The feasibility and reliability of the isomerspecific quantification strategy based on isotopic labeling and glycoqueuing were investigated. Two equal aliquots of FBS

Figure 6. Linear correlations between experimentally detected EIC peak area ratios and theoretical molar ratios of d0-aniline-labeled FBS sialylated N-glycans to the corresponding d5-aniline derivatives (n = 3 replicates).


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Figure 7. Quantitative comparison between HS and HCCS in sialylated N-glycans at the isomeric level using the glycoqueuing strategy. (A) EIC peak area ratios of each sialylated N-glycan structure between HCCS and HS. Error bars, s.d. (n = 3 replicates). (B) Comparison between HS and HCCS in the relative abundance of each sialylated N-glycan structure. C) Quantitative distribution of different types of sialylated Nglycans of HS and HCCS. P values were calculated using Student’s t-test. * represents significant difference (P < 0.05); ** represents extremely significant difference (P < 0.01).

N-glycans were separately labeled with d0- and d5-aniline by nonselective derivatization, followed by a mix of them in different molar ratios (10:1, 5:1, 3:1, 1:1, 1:3, 1:5 and 1:10 of d0- to d5-aniline derivatives) and quantitative analysis by MALDI-TOF and RP-HPLC-MS. As a result, the detected mean value of the signal intensity ratio between d0- and d5aniline derivatives of each glycan composition in the MALDITOF mass spectra of the equimolar mixture is close to the theoretical value 1.00 (Figure 2E), indicating the good accuracy of the isotopic labeling based quantification method. Isomerspecific quantification of FBS N-glycans were further performed by RP-HPLC-MS, and the obtained EIC peak area ratios between the d0- and d5-aniline derivatives of each glycan

isomer mixed in different molar ratios were calculated. Except the glycan structures in very low abundance, fourteen major glycan isomers in higher abundance (shown in Figure 3B) exhibit an excellent linear relationship between the experimental peak area ratios and the corresponding theoretical molar ratios (mixing ratios) ranging from 0.1 to 10 (main correlation coefficient R2 ≥ 0.9900) (Figure 6). Moreover, the CV values of most peak area ratios remain below 20%, showing an acceptable reproducibility of the quantification method. However, the standard error values increase significantly with changes of the theoretical molar ratios far from 1.0. Especially, an increase of the theoretical molar ratio to 1:15 or 15:1 will result in a standard error larger than 25.0%, with a markedly



reduced correlation coefficient (data not shown). Therefore, to ensure the quantification accuracy of the method, a 100-fold linear dynamic range of the molar ratios of d0- to d5-aniline derivatives of sialylglycans (0.1–10) is recommended. Isomer-Specific Quantitative Comparison of Sialylated N-Glycans between Bovine and Rabbit IgG. To validate the ability of the newly developed method for the quantitative comparison between two homologous glycoproteins, isomerspecific relative quantification of sialylated N-glycans of bovine and rabbit IgG was performed. The N-glycans released from bovine and rabbit IgG were separately labeled with d0and d5-aniline by nonselective derivatization, followed by the mixing of the isotopically labeled glycan samples in equal ratios in three different ways described as follows: (i) d0- and d5aniline derivatives of bovine IgG sialylated N-glycans; (ii) d0and d5-aniline derivatives of rabbit IgG sialylated N- glycans; (iii) d0-aniline derivatives of bovine IgG sialylated N-glycans and d5-aniline derivatives of rabbit IgG sialylated N-glycans. As a result, the equimolar mixture of d0- and d5-aniline derivatives of bovine IgG sialylated N-glycans presents 9 pairs of peaks in the MS profile (Figure S20A), while the equimolar mixture of d0- and d5-aniline derivatives of rabbit IgG sialylated N-glycans generates 15 pairs of peaks in the MS profile (Figure S20B). All of these peaks give the signal intensity ratios close to the expected value 1.00 between the d0and d5-aniline derivatives, demonstrating the good accuracy of the method. In contrast, the equimolar mixture of d0-aniline derivatives of bovine IgG sialylated N-glycans and d5-aniline derivatives of rabbit IgG sialylated N-glycans produces 5 pairs of peaks with multiple MS intensity ratios between the light and heavy isotope forms, 4 single peaks of d0-anline derivatives and 10 single peaks of d5-aniline derivatives (Figure S20C), showing the large difference between the two samples in sialylated N-glycan compositions. The glycan compositions of these MS peaks were assigned according to literature reports6,12,23. To quantitatively compare glycan isomers, the equimolar mixture of d0-aniline derivatives of bovine IgG sialylated N-glycans and d5-aniline derivatives of rabbit IgG sialylated N-glycans was further subjected to RP-HPLC-MS analysis (Figure S21 to S23), and the structure of each separated glycan isomer was elucidated by SA-linkage specific derivatization and online RP-HPLC-MS/MS analysis (Figure S24 to S56). As shown in Table S4 and Figure S57, 12 sialylated N-glycan structures of bovine IgG and 30 sialylated N-glycan structures of rabbit IgG were successfully differentiated, and all of the SA residues of them were identified to be α2,6-linked. The isomer-specific relative quantification was achieved based on EIC peak areas, and the obtained data also demonstrated the extremely difference between the two homologous IgGs in isomeric structures of sialylated N-glycans (Figure S57 and Table S4). Obviously, the good applicability of the new analytical method is revealed. Isomer-Specific Quantitative Comparison of Sialylated N-Glycans between HS and HCCS. In order to elucidate the applicability of this analytical strategy to more complicated biological samples, an isomer-specific quantitative comparison of sialylated N-glycans between HS and HCCS was performed for the search of serum N-glycan biomarker candidates. The Nglycans of HS and HCCS were released, labeled and mixed in the way as those of bovine and rabbit IgG. As shown in Figure S58, each of the three nonselectively labeled glycan mixtures with an equal serum volume ratio generates 21 pairs of peaks of sialylated N-glycan compositions in MALDI-TOF MS profiles,

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the obtained intensity ratios of which between the light and heavy isotope forms demonstrates the good accuracy of the method and the significant differences between the two serum samples in the expression level of some sialylated N-glycan compositions. The glycan compositions of these MS peaks were assigned according to literature reports.11,24,25,26,27 For isomerspecific comparison, the three nonselectively labeled glycan mixtures with an equal serum volume ratio were further analyzed by RP-HPLC-MS (Figure S59), and the structure of each separated glycan isomer was identified by SA-linkage specific derivatization (Figure S60) and online RP-HPLCMS/MS analysis (Figure S61 to S92). Totally, 32 sialylated Nglycan structures were successfully differentiated for the both serum samples, and most of the SA residues of them were identified to be α2,6-linked. All of the SA residues are NeuAc, and the elution order of these glycan structures follows the glycoqueuing regularity. During this process, several large Nglycan compositions detected by MALDI-TOF were not observed, due to the relatively low detection sensitivity of ESIMS for larger molecular weights. However, the obtained quantitative data demonstrate significant quantitative differences of some sialylated N-glycan structures between HS and HCCS (Table S5 and Figure 7). As presented in Figure 7A, the isotopic peak area ratios of equal-ratio mixture of d0- and d5-aniline derivatives from either HS or HCCS are close to the expected value 1.00, with most CV values below 10% (n = 3 replicates), demonstrating the good reliability of the quantification method. As to real quantitative comparison, there are 12 HCCS N-glycan structures that significantly different from those of HS in quantity (Figure 7A and 7B). All of these differently expressed N-glycan structures may be potential biomarkers for HCC. Besides, the percentage of different types of N-glycans can also provide valuable information about HCCS (Figure 7C). Obviously, HCCS has a much higher percentage of sialylated N-glycans with core fucosylation but a much lower percentage of N-glycans with α2,3-linked SA residues when compared with HS. Thess results are consistent with those obtained from studies on transgenic mouse model,28 indicating the reliability of the novel analytical strategy.

CONCLUSION This work has developed a novel analytical strategy named glycoqueuing, for the isomer-specific structural identification and quantification of sialylglycans. In this method, the isomeric structures of nonselectively derivatized sialylglycans can be differentiated with high resolution, detected with high sensitivity and quantified with good accuracy by RP-HPLC-MS, and the detailed structural identification of these isomeric structures can be achieved by a comparative analysis with their SA-linkage-dependent derivatives by RP-HPLC-MS/MS. The nonselectively derivatized sialylglycans can be eluted in a regular order closely related with the total SA residue number and the ratio between α2,3- and α2,6-linked SA residues of each glycan during RP-HPLC-MS analysis, facilitating the rapid assignment of isomeric structures. The reliability of the method has been validated in detail by qualitative and quantitative analysis of FBS N-glycans, and its good applicability has been elucidated by the quantitative comparison of N-glycans between bovine and rabbit IgG as well as between HS and HCCS. Compared with other reported procedures, this new method is unique for the quantification of sialylglycans at the isomeric level and has significant advantages in separation resolution and MS detection sensitivity. Additionally, the


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technique is also suitable for the isomer-specific quantification of sialylated O-glycans (Figure S93 to S105 and Table S6 in supporting information). Therefore, this isomer-specific quantification strategy will contribute to glycomics studies and the search for sialylated glycan biomarkers of many diseases.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, results and discussions of isomerspecific quantification of cubilose sialylated O-glycans; EICs of FBS sialylated N-glycans; MALDI-MS spectra, TICs, EICs, histograms and pie charts of bovine and rabbit IgG sialylated Nglycans; MALDI-MS spectra, TICs and EICs of sialylated Nglycomes of HS and HCCS; MALDI-MS spectra, TICs, EICs, histograms and pie charts of cubilose O-glycans; the table showing clinical information of HCC patients; tables of essential datafor the structural identification of FBS sialylated N-glycan isomers and the validation of the quantification accuracy of the strategy; tales showing the quantitative data of bovine and rabbit IgG sialylated N-glycans, HS and HCCS sialylated N-glycans, and cubilose sialylated O-glycans.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Z.W.) * E-mail: [email protected] (L.H.) ORCID Linjuan Huang: 0000-0003-3174-4945 Zhongfu Wang: 0000-0003-1616-5056 Author Contributions 1 These authors contributed equally to this work. Notes The authors declare that this manuscript has no conflict of interest.

ACKNOWLEDGMENT This work was supported by National Key Research and Development Program of China (No. 2018YFD0901101), National Natural Science Fundation of China (Nos. 31670808, 31600647, 31870798) and Natural Science Basis Research Plan in Shaanxi Province of China (No. 2019JM-407).

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Glycoqueuing: Isomer-Specific Quantification for Sialylation-Focused

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