Supercharging Reagent for Enhanced Liquid Chromatographic

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Supercharging reagent for enhanced liquid chromatographic separation and charging of sialylated and high-molecularweight glycopeptides for nanoHPLC-ESI-MS/MS analysis Chia-wei Lin, Micha Andres Haeuptle, and Markus Aebi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00938 • Publication Date (Web): 03 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Analytical Chemistry

Title: Supercharging reagent for enhanced liquid chromatographic separation and charging of sialylated and high-molecular-weight glycopeptides for nanoHPLC-ESI-MS/MS analysis Chia-wei Lin1, Micha A. Haeuptle2,§, Markus Aebi1,* 1

Institute of Microbiology, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland

2

LimmatTech Biologics AG, CH-8952 Schlieren, Switzerland

§Current address: Molecular Partners AG, CH-8952 Schlieren, Switzerland *Correspondence: Prof. Markus Aebi, ETH Zürich, Institute of Microbiology, Vladimir-Prelog-Weg 4, 8093 Zürich, Switzerland E-Mail: [email protected] Phone: +41 44 632 64 13, Fax: +41 44 632 11 48,

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Abstract: Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of P. aeruginosa covalently linked to S. flexneri type 2a O-antigen (Sf2E) produced by engineered E.coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reverse-phase LC solvents improves sensitivity, charging and chromatographic resolution for acidic and hMW glycopeptides.


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Introduction Bacterial O-antigens are large cell-surface polysaccharides composed of multiple repeating units1. Glycoconjugate vaccines are produced by covalently linking such pathogen-specific polysaccharides to an antigenic protein carrier, and are one of the safest and most effective methods of protecting against bacterial infection 2,3. However, the analytical tools to characterize these high-molecular-weight (hMW) glycopeptides bearing multiple glycan chains with variable repeating units are lacking. Likewise, sialylation of the terminal glycans is an abundant and important modification of mammalian glycoproteins but is often excluded from analytical throughputs due to technical limitations. Glycoproteomics using mass spectrometry-based analytical techniques is a rapidly developing field and yields structural information regarding the peptides as well as the covalently linked glycan. It is possible to obtain quantitative and glycosylation site-specific information of a given glycoprotein

4,5

. However,

glycopeptides with charged (e.g. sialylated) or large glycans are intrinsically difficult to detect due to the low ionization propensity and the high molecular mass of the glycopeptides. One possibility to overcome these limitations is to increase the ionization efficiency in the electrospray ionization process. This would improve on the one hand the detection of charged (sialylated) glycopeptides and on the other hand the identification of glycopeptides with very large glycans in an increased charge state. According to Lord Raleigh’s studies, the charge state of molecules with electricity can be estimated by following equation: total charge= ZRe= 8π(ε0γR3)1/2 (R: the radius of charging possible for a spherical droplet; γ: surface tension)6. However, the complex chemical environment at the end of the droplet lifetime produces deviations from theory. It has been demonstrated that the charge state for analytes during electrospray ionization is determined by many factors, such as the physical properties of the compound, buffer conditions used for electrospray, or the opening diameter of emitter just to name a few 7-10. Therefore, charge state alterations are experimentally accessible. In 2003, Williams and colleagues invented the term “supercharging” to describe the increased charging observed in spectra obtained from solvents 3 ACS Paragon Plus Environment


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supplemented with a variety of chemicals

11,12

. Among them, m-nitrobenzyl alcohol (m-NBA) and

dimethyl sulfoxide (DMSO) are well known to enhance the charge states of macromolecules11,12, i.e. protein and peptides 11,12. In addition, the peptide identification rate is significantly improved when the mobile phase contains DMSO as co-solvent. This improvement is due to charge state coalescence13. Furthermore, it has been reported that m-NBA added into the mobile phase in a liquid chromatographymass spectrometry (LC-MS) system increased the chromatographic separation of proteins and peptides 14

. Thus, the supplementary chemicals in the LC mobile phase not only influence the charge states but

also the chromatographic separation. To examine the effect of m-NBA additive in the LC buffer system, commercial glycoproteins were first applied in this study. Our data showed that with the help of m-NBA, the charge states of sialylated glycopeptides increased and the chromatographic separation of neutral and sialylated glycopeptides revealed a significant improvement. Next, we applied this system for the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of P. aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered E. coli. The type 2a antigen consists of multimer of one ß-D-GlcNAc and three α-L-Rha with non-stoichiometric glucosylation 15,16

. The presence of m-NBA increased the charge state of the large glycoconjugated glycopeptides

allowing us to characterize peptides with covalently linked glycan chains of up to 20 repeating units (98 monosaccharides). Moreover, our results demonstrated that incorporation of m-NBA into reverse-phase LC solvents improves sensitivity, charging and chromatographic resolution for sialylated and large glycopeptides.


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Experimental section Preparation of purified glycoproteins Purified proteins, IgG 1 from human myeloma plasma (Millipore, USA), fetuin and asialofetuin (SigmaAldrich), were loaded onto Microcon YM-30 (Millipore, USA) 30,000 MWCO centrifugal filter device. Proteins were processed by Filter-assisted sample preparation (FASP) 17. Briefly, proteins were reduced by 50mM dithiothreitol (AppliChem, Germany) in 50mM ammonium bicarbonate buffer (pH 8.5) at 37⁰C for 1 hour, followed by alkylation by 65mM iodoacetamide (Sigma-Aldrich, USA) at 37⁰C in the dark for 1 hour. After four times washing of the filter device with ammonium bicarbonate buffer, proteins were digested using sequencing-grade modified trypsin (Promega, USA) in the ratio of 80:1 at 37⁰C overnight. All digested peptides and glycopeptides were collected by centrifugation and dried in a SpeedVac. Samples were desalted by Zip-Tip C18 (Millipore, USA) prior to nanoLC-MS/MS analysis. Bacterial strains and expression of bioconjugates The Sf2E glycoconjugate was expressed in an Escherichia coli W3110 strain, wherein the wild-type Oantigen cluster was replaced by the O-antigen cluster originating from the S. flexneri 2a strain CCUG29416 (Culture Collection University of Göteborg, Sweden) under control of its native constitutive promotor. Additionally, the W3110 gtrs gene was replaced by the S. flexneri 2a gtrII gene coding for the glucosyltransferase GtrII, catalyzing the addition of a branching Glc to the third Rha

18

. The resulting

strain genotype is E. coli W3110 ΔwaaL ΔaraBAD gtrS::gtrII ΔrfbW3110::rfbCCUG29416. For expression of

the

glycoconjugate,

this

strain

was

transformed

with

the

plasmids

encoding

the

oligosaccharyltransferase PglB and the EPA protein carrier as described earlier 19, with the exception that ampicillin was exchanged by kanamycin for genetic selection. Sf2E glycoconjugate was produced in a 150 L fermenter (B. Braun Biotech International, Germany) by inoculation of 60 L batch medium (yeast extract (Becton Dickinson, USA) 24 g/L, soy peptone 5 ACS Paragon Plus Environment


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(Organotechnie, France) 12 g/L, glycerol 25 g/L, phosphate buffer 14.9 g/L, Spectinomycin 80 mg/L and trace elements) at an OD600 of 0.005 with the expression strain described above. Fermentation was performed at pH 7.0 and 35°C under aerobic conditions. At an OD600 of 45, glycoconjugate formation was induced by the addition of L-Arabinose (0.1%) and IPTG (1 mM). For the subsequent 24 hours feed medium (yeast extract 66 g/L, soy peptone 33 g/L, glycerol 350 g/L, phosphate buffer 14.9 g/L, Spectinomycin 80 mg/L, trace elements, L-Arabinose 0.2% and IPTG 1mM) was constantly added leading to a total fermentation volume of 100 L. The cells were harvested by tangential flow filtration (TFF) on a Hollow Fiber 500 kDa cut-off unit (Spectrum Labs, USA). Primary recovery of the glycoconjugate from the periplasm was performed by osmotic shock. The shocked cells were clarified by TFF using a 0.2 µm Hollow Fiber module (Spectrum Labs, USA). Cell harvest and osmotic shock were performed at an appropriate scale. Purification of bioconjugates The Sf2E periplasmic extract was loaded onto a Q ceramic hyperD F (Pall Corporation, USA) column and eluted by single step elution from 50 to 204 mM NaCl in the presence of 10 mM Tris pH 8.5. The eluate was further purified by binding to a Butyl Sepharose HP (GE Healthcare, USA) column in K2HPO4 127.5 g/L, KH2PO4 63.5 g/L buffer followed by a step elution in a K2HPO4 47.5 g/L, KH2PO4 23.7 g/L buffer. A Source 15Q (GE Healthcare, USA) column was equilibrated with 50 mM NaCl in 10 mM BisTris pH 6.5. Sf2E was loaded and eluted by applying a gradient to 200 mM NaCl in 10 mM BisTris pH 6.5. The resulting eluate was concentrated using a Hollow Fiber mini 10 kDa (Spectrum Labs, USA) TFF module prior to the final purification step performed on a Superdex 200 (GE Healthcare, USA) column in 1 x Tris buffered saline (25 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl), which corresponds to the final glycoconjugate formulation. The polysaccharide content of the Sf2E conjugate was determined by the Anthrone assay20 , while the protein content was determined using a commercial BCA assay kit (Thermo Scientific, USA). 6 ACS Paragon Plus Environment

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The purified glycoproteins were processed according to the FASP method and the resulting (glyco)peptides were desalted by Zip-Tip C18 prior to MS analysis. Glycopeptide analysis by nanoHPLC-MS/MS with m-NBA additive in the mobile phase All m-NBA experiments were performed on a calibrated LTQ-Orbitrap Velos mass spectrometer (Thermo Fischer Scientific, Germany) coupled to an Eksigent-Nano-HPLC system (Eksigent Technologies, USA). Peptides were resuspended in 2.5% acetonitrile (ACN) and 1% formic acid (FA) and loaded on a homemade fritted column (75 µm × 150 mm) packed with reverse phase C18 material (ReproSil-Pur 120 C18AQ, 1.9 µm, Dr. Maisch GmbH, Germany). Two buffer solutions were used in this study: 1) aqueous buffer (Buffer A) containing 0.1% FA in water and 2) organic buffer (Buffer B) containing 0.1% FA in ACN. The digested samples were eluted with a flow rate of 300 nL per min. A 100min gradient was employed that included 20 min at 2% B to load sample, then went from 2 to 31% solvent B over 52min, up to 44% B over a further 10 min, and up to 98% B over 8 min before back to 2% B. For EPA glycopeptides analysis, samples were loaded by 2% B for 20 min and eluted in a gradient from 2 to 10% of B for 30 min, 10 to 35% for 25 min, and then 98% B for 10 min before back to 2% B. During m-NBA experiments, the mobile phases were modified as follows: 1) 0.5% m-NBA: 5% ACN/94.4% water (Buffer A) and 5% water/ 94.4% ACN (Buffer B) with 0.5% m-NBA and 0.1% FA ; 2) 1% m-NBA: 7.5% ACN/ 91.4% water (Buffer A) and 7.5% water/ 91.4% ACN (Buffer B) with 1% m-NBA and 0.1% FA (Buffer B). For MS analysis, one scan cycle comprised of a full scan MS survey spectrum, followed by up to 10 sequential HCD MS/MS on the most intense signals above a threshold of 2000. Full-scan MS spectra (700–2000 m/z) were acquired in the FT-Orbitrap at a resolution of 60,000 at 400 m/z, while HCD MS/MS spectra were recorded in the FTOrbitrap at a resolution of 15,000 at 400 m/z. HCD was performed with a target value of 1e5 and stepped collision energy rolling from 35, 40 and 45 V was applied. Automatic gain controlTM (AGC) target values were 5e5 for full FTMS. For all experiments, dynamic exclusion was used with 1 repeat count, 15 s repeat duration, and 60 s exclusion duration. 7 ACS Paragon Plus Environment


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For CID-MS/MS analysis of large glycoconjugated glycopeptides deriving from Sf2E, samples were analyzed on a calibrated LTQ-Orbitrap Fusion mass spectrometer (Thermo Fischer Scientific, Germany) coupled to an Easy-nLC 1000 UHPLC system (Thermo Fischer Scientific, USA). The same batch of samples were loaded on a self-made fritted column (75 µm × 150 mm) packed with the same material as previously described and eluted by a gradient from 0 to 10% of B in 30 min, 35% in 25 min, 98% B in 3 min. For MS analysis, one scan cycle comprised of a full scan MS survey spectrum, followed by up to 20 sequential HCD and CID MS/MS in parallel on the most intense signals above a threshold of 2000. Fullscan MS spectra (700–2000 m/z) were acquired in the FT-Orbitrap at a resolution of 120,000 at 400 m/z, while HCD and CID MS/MS spectra were recorded in the FT-Orbitrap at a resolution of 30,000 at 400 m/z. HCD was performed with a target value of 2e5 and stepped collision energy rolling from 35, 40 and 45 V was applied and CID was performed with a target value of 2e5 and 30% NCE was applied for fragmentation. AGC target values were 5e5 for full FTMS. For all experiments, dynamic exclusion was used with 1 repeat count, 15 s repeat duration, and 60 s exclusion duration. Data analysis XCalibur 3.0 was used for data processing. To identify each glycoform, all MS/MS spectra were examined manually for the presence of glycan oxonium ions, [HexNAc]+ 204.09, [HexNAc+Hex]+ 366.12, [NeuAc]+ 292.10, [NeuAc-H20]+ 274.09, their corresponding Y1 ions, [Peptide+HexNAc]+ and the known structures from previous studies. To show the elution profile under different buffer systems, extracted ion chromatography (XIC) of each glycoform was plotted by its corresponding m/z with a mass tolerance of 5 ppm. All MS spectra were the sum of the individual spectrum across the XIC peak. For Sf2E glycoconjugate, the deconvolution of MS and MS/MS spectra was performed by XtractRaw file using the following parameters: The resolution was 15,000 at m/z 400 and S/N ratio should be larger than 2. All peaks were annotated manually.


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Results and discussions The effect of m-NBA on glycopeptide analysis To understand the effect of m-NBA additive in the mobile phase for glycopeptide separation and analysis, we used human IgG1 as a model protein. It is characterized by a single glycosylation site in the Fc domain and contains both neutral and sialylated glycans

21

. IgG1 samples were trypsinized and applied to

nanoLC-MS/MS analysis. Due to the insolubility of m-NBA in 0.1% FA aqueous solution, both aqueous and organic buffer solutions were modified as detailed in the experimental section. For each condition, three technical replicates were performed intradaily and interdaily. The effect of 0.5% and 1% m-NBA on the extracted ion chromatography (XIC) and the corresponding MS spectra of neutral and sialylated glycopeptides from human IgG 1 are depicted in Figure 1. First, we noted a significant effect on the separation efficiency of glycopeptides differing in the presence of a sialic acid residue. Increasing the mNBA concentration resolved the glycopeptides N-glycosylated with either CoreFuc(GlcNAcGal)GlcNAc or CoreFuc(GlcNAcGal)GlcNAcNeuAc significantly (Figure 1A). Neutral glycopeptides were showen to coelute regardless of the presence of m-NBA. We concluded that the presence of m-NBA had no influence on their separation efficiency (Supplementary Figure S1). In addition, the charge state of the peptide bearing CoreFuc(GlcNAc)2 was found to be +2 when the normal buffer system was applied and shifted to a triply charged state in the presence of m-NBA (Figure 1B). Moreover, for sialylated glycopeptides, the quadruply charged precursor ions were only observed (Figure 1C) and the absolute intensity of triplycharged neutral and sialylated glycopeptides increased when using m-NBA as additive. All observed m/z are summarized in the Supplementary Table 1. We concluded that m-NBA did not only increase the charge states but also the sensitivity towards neutral and sialylated glycopeptides. In addition, it also improved the chromatographic separation of sialylated glycopeptides. Previously, it has been shown that carboxylate anions feature a higher basicity compared to uncharged amine and guanidine groups in the gas phase 22-24. We proposed that this, at least partially, accounts for 9 ACS Paragon Plus Environment


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the effect of the m-NBA on the sialylated glycopeptides. Therefore, we postulate that the number of sialic acid on its carrier peptide in an m-NBA containing electrosprayed droplet determines the most favorable charge state of the analyzed glycopeptides. Moreover, the application of m-NBA results in a superior chromatographic separation of glycopeptides differing only by the presence of a single sialic acid (Figure 1A), which enhances the quality of LC based analyses of complex eukaryotic glycopeptide samples. Since m-NBA introduces different functional groups into the reverse-phase separation system, it may alter the binding affinity between C18 material and carboxyl/acetyl group on the glycan part via hydrogen bonding, aromatic pi-stacking/ pi-cation interaction and ionic interactions. This could partially explain why the sialylated glycopeptides were retained longer on the reverse phase when using m-NBA as an additive than neutral ones. Although, the separation of neutral and sialylated glycopeptides was satisfying in a buffer system containing 1% m-NBA, the initial ACN concentration in the aqueous phase (7.5% ACN) was too high to obtain a sufficient chromatographic efficiency when the same buffer was applied to proteins featuring more than one potential N-glycosylation site (data not shown). In addition, technical limitations caused by 1% m-NBA in the mobile phase prompted us to focus in the subsequent studies on buffer systems containing 0.5% m-NBA. The influence of m-NBA for multiply sialylated glycopeptides To further study the effect of m-NBA on neutral and monosialylated glycopeptides as well as its effect on multiply sialylated glycopeptides, bovine fetuin and asialofetuin, containing three N-linked and five Olinked glycosylation sites 25-27, were subjected for nanoLC-MS/MS analysis. Like IgG1, the charge state of glycopeptides with neutral and monosialylated glycans improved (Supplementary Figure S2). For glycopeptides containing multiple sialic acid residues, MS profiles of glycopeptides bearing N-linked bi-, tri- and tetra-sialylated glycans were analyzed (Figure 2). The major charge states of peptides Nglycosylated with Core(GlcNAcGal)3NeuAc3 were +4 in absence and +5 in the presence of m-NBA. In 10 ACS Paragon Plus Environment

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addition, the same precursors with six to eight protons were also observed to a lesser extent. However, in the presence of m-NBA, the higher charge states of multiply sialylated glycopeptides were primarily formed (Figure 2B). Next, we examined the influence of m-NBA on O-linked glycopeptides derived from bovine fetuin (Figure 3). The most intense charge state of the O-glycopeptides bearing (GalNAcGal)3NeuAc2 was +5 under normal conditions but became +6 when m-NBA was applied, higher charge states (z>6) were also observed for the same precursors. To the best of our knowledge, this is the first time that the peptide with tetrasialic acids can be detected by direct on-line LC-MS analysis without prior enrichment or fractionation 26,27. Our results (summarized in the Supplementary Table 1) indicated that the presence of m-NBA improved the ionization of charged (sialylated) glycopeptides during the ionization process. m-NBA enabling the analysis of large glycoconjugated glycopeptides Since the presented analysis of N-glycopeptides demonstrated that m-NBA improves the ionization process, we reasoned that the same procedure might facilitate the analysis of glycopeptides featuring large, covalently linked glycans. We evaluated this hypothesis using an new class of glycoconjugates for which no appropriate analytical approaches are currently available, namely recombinantly produced glycoconjugates decorated with long chains of bacterial O-antigens

28,29

. To this end, a genetically

modified version of exotoxin A of P. aeruginosa, featuring two engineered consensus sites for Nglycosylation was expressed in an E. coli strain (the sequence is shown in the Supplementary Figure S3) capable of transferring the non-acetylated O-antigen of S. flexneri type 2a to a carrier protein. This glycoengineering approach is described elsewhere

30,31

and constitutes a promising approach for the

development of vaccine candidates against bacterial infections 32,33. The resulting glycoconjugates were purified by applying a series of chromatographic steps. Immunoblot as well as size-exclusion HPLC analyses confirmed proper preparation of pure glycoprotein consisting of either one or two large S. flexneri type 2a O-antigen polysaccharide chains linked to exotoxin A of P. aeruginosa (data not shown) 3. 11 ACS Paragon Plus Environment


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The purified Sf2E was trypsinized using the FASP method and applied to LC-MS/MS analysis. First, the sample was analyzed in the absence of m-NBA. Since the N-glycosidic linkage connects the polypeptide to the reducing end of GlcNAc, the presence of oxonium ions of HexNAc, [M+H]+=204.0868 after HCD fragmentation would indicate the presence of N-glycopeptides in MS/MS

34,35

. Consequently, this

diagnostic ion was used to detect the presence of glycopeptides during reverse phase chromatography (Figure 4A). Thereby, two groups of glycopeptides were observed; the first group eluted between 50 and 54 min and the second between 54 and 59 min. The two glycopeptide groups differed in their peptide backbone, since for group one the peptide sequence DNNNSTPTVISHR was confirmed, while group two corresponded to an incompletely cleaved version of the same glycopeptide, HDLDIKDNNNSTPTVISHR. The broad elution range resulted from different oligosaccharide chain length: the longer the chain length, the earlier the elution time. A representative MS spectrum of the second group recorded at 55.83 min is shown in Figure 4B. To determine the overall glycosylation profile of this N-glycopeptide, every other MS spectra between 54 to 59 mins were deconvoluted separately and the resulting spectra were merged (Figure 4C). The molecular weight of the glycopeptides ranged from 3 to 17kDa, suggesting to correspond to 1 to 17 oligosaccharide repeating units with non-stoichiometric glucosylation of the Nlinked glycan. The deduced glycan structures are summarized in Table 2. A comparable analysis was done for the second glycosylation site of the protein eluting in lower amounts in the range of 30 to 34.5 minutes (Supplementary Figure 4). To confirm the identity of each precursor, all HCD MS/MS spectra were first extracted based on the presence of HexNAc oxonium ion and Y1 ions and then manually assigned. Two HCD MS/MS spectra at m/z 755.86 (z=4) and 1335.17 (z=5) are shown in Figure 5 A and B, respectively. The m/z of the first ion corresponds to the peptide sequence HDLDIKDNNNSTPTVISHR glycosylated with GlcNAc-Rha-Rha-RhaGlcNAc. The corresponding HCD MS/MS spectrum, comprising the consecutive series of y ions deriving from the peptide backbone and Y1, the peptide linked to HexNAc, confirmed the identity as N-linked 12 ACS Paragon Plus Environment

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glycopeptide. The putative glycan composition of the second detected glycopeptide (m/z 1335.17, z=5) was assigned as (GlcNAc-Rha-Rha-Rha)2 combined with (GlcNAc-Rha(Glc)-Rha-Rha)4 attached to the HDLDIKDNNNSTPTVISHR peptide backbone. In the resulting HCD spectrum, the most intense fragments derived from cleavage of the glycosidic linkages. In addition, the Y1 ion at m/z 1190.08 (z=2) corresponding to [peptide+HexNAc+2H]2+ confirmed the identity as an N-linked glycopeptide. The proposed glycan composition suggests that two of the repeating units are not glucosylated. However, as HCD is a relatively harsh fragmentation method the exact location of the repeating units lacking the glucose within the oligosaccharide chain could not be determined. Therefore, a collision induced dissociation (CID) fragmentation method generating mostly fragment ions arising from glycosidic bond cleavage was applied

36,37

. CID MS/MS spectrum measured on an Orbitrap system (Figure 5C) showed

that the non-reducing end units were preferentially glucosylated. Finally, the effect of m-NBA on the analysis of these glycopeptides was examined. The same amount of glycopeptides was applied to nanoLC-MS/MS in the absence and the presence of 0.5% m-NBA. The intensity of two major charge states for each precursor were used to evaluate the effect of m-NBA and the results are summarized in Table 2. Obviously, high molecular weight glycopeptides are ionized more efficiently in the presence of m-NBA, resulting in an increased sensitivity of the established analytical system. Due to the presence of m-NBA in the mobile phase, glycopeptides with glycan chains comprising up to 20 repeating units (98 monosaccharides) were observed (Table 1). Until now, no analytical tool is able to determine the maximal length of glycan chains on this glycoconjugate unambiguously. The addition of m-NBA presents a supplementary information in comparison with using normal buffer systems. Thus, it may lead us to a closer approach to the real structures of this glycoconjugate.



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Conclusions The presented study provides a novel analytical approach for the detailed structural analysis of charged and hMW glycopeptides by combining the application of the supercharging reagents m-NBA with a state of the art nanoHPLC-ESI-MS/MS method. Consistent with previous reports on other applications 12-13, the m-NBA containing mobile phase lead to extensive supercharging of intact N- and O-linked glycopeptides (Figure 1-3), especially when sialylated. In addition, the use of m-NBA regarding the characterization of recombinant glycoproteins that carry long, O-antigen carbohydrate chains was studied. Using a classical buffer system, glycopeptides bearing 1 to 17 repeating units of the S. flexneri type 2a O-antigen were detected and the detailed analysis of data obtained from CID and HCD fragmentation allowed us to define in detail the exact localization of non-stoichiometrically occurring glucosylation of the polysaccharide (Figure 5). In this study, we were able to obtain good quality of HCD or CID MS/MS spectra up to 14 repeating units. Our results suggest that no glucose was present as a branching hexose on the first repeating unit from reducing end. The addition of m-NBA to the developed analytical system promoted supercharging of these large glycoconjugated glycopeptides and increased therewith the intensity of the precursor ions featuring higher charge states. This enabled the identification of glycopeptides carrying polysaccharide chains of unprecedented length, up to 98 monosaccharides. Thus, the addition of m-NBA to the mobile phase represents a new dimension in MS-based analysis of glycopeptides.


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Acknowledgments The authors would like to thank the Functional Genomics Centre Zurich for providing all the support on mass spectrometers and Dr. Michael Kowarik and Dr. Timothy G. Keys for critical reading of the manuscript. This work was supported by ETH Research Grant No. 07 10-1 to M.A..



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Reference: (1) Lerouge, I.; Vanderleyden, J. FEMS Microbiol. Rev. 2002, 26, 17-47. (2) Vella, M.; Pace, D. Expert Opin. Biol. Ther. 2015, 15, 529-546. (3) Ravenscroft, N.; Haeuptle, M. A.; Kowarik, M.; Fernandez, F. S.; Carranza, P.; Brunner, A.; Steffen, M.; Wetter, M.; Keller, S.; Ruch, C.; Wacker, M. Glycobiology 2016, 26, 51-62. (4) Hong, Q.; Lebrilla, C. B.; Miyamoto, S.; Ruhaak, L. R. Anal. Chem. 2013, 85, 8585-8593. (5) Hang, I.; Lin, C. W.; Grant, O. C.; Fleurkens, S.; Villiger, T. K.; Soos, M.; Morbidelli, M.; Woods, R. J.; Gauss, R.; Aebi, M. Glycobiology 2015, 25, 1335-1349. (6) F.R.S., L. R. Philosophical Magazine Series 5 1882, 14, 184- 186. (7) Kamel, A. M.; Brown, P. R.; Munson, B. Anal. Chem. 1999, 71, 5481-5492. (8) Li, Y.; Cole, R. B. Anal. Chem. 2003, 75, 5739-5746. (9) Hogan, C. J., Jr.; Biswas, P. J. Am. Soc. Mass Spectrom. 2008, 19, 1098-1107. (10) Wilm, M. Mol. Cell. Proteomics 2011. (11) Iavarone, A. T.; Jurchen, J. C.; Williams, E. R. Anal. Chem. 2001, 73, 1455-1460. (12) Iavarone, A. T.; Williams, E. R. J. Am. Chem. Soc. 2003, 125, 2319-2327. (13) Meyer, J. G.; E, A. K. J. Am. Soc. Mass Spectrom. 2012, 23, 1390-1399. (14) Chingin, K.; Xu, N.; Chen, H. J. Am. Soc. Mass Spectrom. 2014, 25, 928-934. (15) Simmons, D. A. Biochem. Soc. Trans. 1993, 21, 58S. (16) Perepelov, A. V.; L'Vov V, L.; Liu, B.; Senchenkova, S. N.; Shekht, M. E.; Shashkov, A. S.; Feng, L.; Aparin, P. G.; Wang, L.; Knirel, Y. A. Carbohydr. Res. 2009, 344, 687-692. (17) Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Nat. Methods 2009, 6, 359-362. (18) Lehane, A. M.; Korres, H.; Verma, N. K. Biochem. J. 2005, 389, 137-143. (19) Kampf, M. M.; Braun, M.; Sirena, D.; Ihssen, J.; Thony-Meyer, L.; Ren, Q. Microb. Cell Fact. 2015, 14, 12. (20) Leyva, A.; Quintana, A.; Sanchez, M.; Rodriguez, E. N.; Cremata, J.; Sanchez, J. C. Biologicals 2008, 36, 134-141. (21) Wuhrer, M.; Stam, J. C.; van de Geijn, F. E.; Koeleman, C. A.; Verrips, C. T.; Dolhain, R. J.; Hokke, C. H.; Deelder, A. M. Proteomics 2007, 7, 4070-4081. (22) Grandori, R. J. Mass Spectrom. 2003, 38, 11-15. (23) Prakash, H.; Mazumdar, S. J. Am. Soc. Mass Spectrom. 2005, 16, 1409-1421. (24) Marchese, R.; Grandori, R.; Carloni, P.; Raugei, S. PLoS Comput. Biol. 2010, 6, e1000775. (25) Green, E. D.; Adelt, G.; Baenziger, J. U.; Wilson, S.; Van Halbeek, H. J. Biol. Chem. 1988, 263, 1825318268. (26) Zauner, G.; Koeleman, C. A.; Deelder, A. M.; Wuhrer, M. J. Sep. Sci. 2010, 33, 903-910. (27) Windwarder, M.; Altmann, F. J. Proteomics 2014, 108, 258-268. (28) Feldman, M. F.; Wacker, M.; Hernandez, M.; Hitchen, P. G.; Marolda, C. L.; Kowarik, M.; Morris, H. R.; Dell, A.; Valvano, M. A.; Aebi, M. Proc. Natl. Acad. Sci. U S A 2005, 102, 3016-3021. (29) Kowarik, M.; Numao, S.; Feldman, M. F.; Schulz, B. L.; Callewaert, N.; Kiermaier, E.; Catrein, I.; Aebi, M. Science 2006, 314, 1148-1150. (30) Wacker, M.; Linton, D.; Hitchen, P. G.; Nita-Lazar, M.; Haslam, S. M.; North, S. J.; Panico, M.; Morris, H. R.; Dell, A.; Wren, B. W.; Aebi, M. Science 2002, 298, 1790-1793. (31) Wetter, M.; Kowarik, M.; Steffen, M.; Carranza, P.; Corradin, G.; Wacker, M. Glycoconj. J. 2013, 30, 511-522. (32) Wacker, M.; Wang, L.; Kowarik, M.; Dowd, M.; Lipowsky, G.; Faridmoayer, A.; Shields, K.; Park, S.; Alaimo, C.; Kelley, K. A.; Braun, M.; Quebatte, J.; Gambillara, V.; Carranza, P.; Steffen, M.; Lee, J. C. J. Infect. Dis. 2014, 209, 1551-1561. (33) Hatz, C. F.; Bally, B.; Rohrer, S.; Steffen, R.; Kramme, S.; Siegrist, C. A.; Wacker, M.; Alaimo, C.; Fonck, V. G. Vaccine 2015, 33, 4594-4601. 16 ACS Paragon Plus Environment

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(34) Carr, S. A.; Huddleston, M. J.; Bean, M. F. Protein Sci. 1993, 2, 183-196. (35) Huddleston, M. J.; Bean, M. F.; Carr, S. A. Anal. Chem. 1993, 65, 877-884. (36) Morelle, W.; Canis, K.; Chirat, F.; Faid, V.; Michalski, J. C. Proteomics 2006, 6, 3993-4015. (37) Wuhrer, M.; Catalina, M. I.; Deelder, A. M.; Hokke, C. H. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007, 849, 115-128. (38) Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P.; Marth, J. D.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Proteomics 2009, 9, 5398-5399. (39) Allison, G. E.; Verma, N. K. Trends Microbiol. 2000, 8, 17-23.



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Figure legend Figure 1 Chromatographic separation and the charge states of neutral and sialylated glycopeptides from human IgG1 observed under different m-NBA concentrations. (A) XIC of the same human IgG1 peptide, 176

EEQYNSTYR184 (N: N-linked glycosylation site), bearing neutral (upper panel) and acidic glycans (lower

panel) were plotting m/z 1398.56 (z=2) in the upper panel and 1029.75 (z=3) in the lower panel. From left to right, the composition of buffer A is 0.1%FA, 5% ACN with 0.5% m-NBA and 7.5% ACN with 1%mNBA. Y-axis represented the absolute intensity from MS. The same order was also applied for the following MS spectra. MS profiles of the neutral (B) and acidic (C) glycopeptides were summed for each XIC. Y-axis represented the relative intensity from MS and the absolute intensity (AI) of each base peak was shown on the left of its corresponding Y-axis. The glycan structure of the base peak in each spectrum was illustrated in cartoon formula. For residual species, the details were summarized in the Supplementary Table 1. In this study, all glycan symbols conform to the recommendations of Consortium for Functional Glycomics 38. Figure 2 LC-MS profiles for the charge states of glycopeptides with multiple sialic acids from bovine fetuin with and without m-NBA additive. MS profiles (left to right) demonstrated the charge distribution of di-, tri- and tetrasialylated N-linked glycopeptides without m-NBA (A) and with 0.5% m-NBA (B) in the mobile

phase.

The

peptide

backbone

of

these

glycopeptides

was

72

RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR103 with two carbamidomethylated cysteines. In the presence of

m-NBA, the most intense charge state of peptide N-glycosylated with Core(GlcNAcGal)3NeuAc2 is +5 at m/z 1249.14 compared to +4 in the normal buffer system. In addition, the higher charge states of this glycopeptides were also observed, m/z 1041.22 for z=6, 892.53 for z=7 and 781.11 for z=8, respectively. The m/z for each corresponding structure is summarized in the supplementary table 1. The glycans are annotated according to the previous study25. Figure 3 LC-MS profiles for the charge states of O-glycopeptides from bovine fetuin with and without mNBA additive. MS spectra (left to right) demonstrated the charge distribution of di-, tri- and tetrasialylated O-linked glycopeptides without m-NBA (A) and with 0.5% m-NBA (B) in the mobile phase. In the absence of m-NBA, the major charge state of this O-glycopeptides is +5. In the presence of m-NBA, the most intense charge state of peptide, 246

VTCTLFQTQPVIPQPQPDGAEAEAPSAVPDAAGPTPSAAGPPVASVVVGPSVVAVPLPLHR306 (S/T: putative O-

glycosylation site), O-glycosylated with (GalNAc2Gal)3NeuAc2 is +6 at m/z 1282.96 compared to +5 in the normal buffer system. In addition, the tetrasialylated O-glycopeptides were detected (lower panel). The 18 ACS Paragon Plus Environment

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m/z for each corresponding structure was summarized in the supplementary table. The annotation of glycans is based on a previous study 27. Figure 4 LC-MS/MS analysis of Sf2E glycosylation (A) The upper panel represents the base peak intensity chromatography (BPI) of tryptic Sf2E (glyco)peptides recorded by nanoRPLC-ESI-MS/MS. The lower panel depicts the XIC generated for oxonium ions of GlcNAc, 204.0868, at the MS/MS level. The peptide backbone of glycopeptide group 1 was identified to be DNNNSTPTVISHR and HDLDIKDNNNSTPTVISHR was confirmed for group 2 (N is the attachment site of N-linked glycans). (B) A representative MS spectrum recorded at 55.8 min corresponding to the group 2 glycopeptides is shown (C) This deconvoluted MS spectrum is the sum of individual MS spectrum recorded in the range of 54 to 59 mins. The N-glycan structures corresponding to [M+H]+=3823.71 and 13901.71, respectively, are shown and the residually identified structures are summarized in Table 2. The Rha was represented by green triangles and each double arrow corresponds exactly to the molecular weight of 803.31 Da corresponding to one S. flexneri type 2

O-antigen unit, GlcNAc-Rha(Glc)-Rha-Rha

15,39

. The detail

structure of each peak is summarized in Table 1. Figure 5 The glycan structures from Sf2E glycopeptides were characterized by HCD and CID fragmentation methods. (A) The HCD MS/MS spectrum for the [M+4H]4+=755.86 ion acquired on an Orbitrap Velos ProTM mass spectrometer confirmed the assumed glycan and peptide composition of this precursor. The yn’ ions correspond to the yn ions in combination with one GlcNAc. The HCD (B) and CID (C) MS/MS spectra of the [M+5H]5+=1335.17 ion measured on an Orbitrap FusionTM mass spectrometer provided complementary information regarding the composition of the peptide bearing six repeating units with incomplete glucosylation. The inserted panel illustrates the deconvoluted spectrum for a higher mass range.

Table 1 Summary of observed glycoforms from Sf2E Table 2 Effect of m-NBA on the peptide with O-antigens



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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Table 1 Summary of observed glycoforms from Sf2E +

Observed m/z* [M+H] Glycan composition 3+ 4+ 1007.47 , 755.86 3020.40 -[GlcNAc-Rha-Rha-Rha]-GlcNAc 4+ 5+ 916.17 , 733.14 3661.67 -[GlcNAc-Rha-Rha-Rha]2-GlcNAc 4+ 5+ 956.68 , 765.55 3823.71 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]-GlcNAc 4+ 5+ 4423.94 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]2 1106.74 , 885.59 4+ 5+ 1116.99 , 893.80 4464.97 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha] -GlcNAc 4+ 5+ 4627.02 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]2-GlcNAc 1157.51 , 926.21 4+ 5+ 1317.82 , 1054.46 5268.27 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]2-GlcNAc 5+ 1086.87 5430.33 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]3-GlcNAc 5+ 6071.58 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]3-GlcNAc 1215.12 5+ 1247.53 6233.62 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]4-GlcNAc 5+ 6874.88 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]4-GlcNAc 1375.78 5+ 6+ 1408.18 , 1173.66 7036.94 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]5-GlcNAc 5+ 6+ 1568.85 , 1307.55 7840.24 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]6-GlcNAc 6+ 7+ 1441.43 , 1235.65 8643.55 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]7-GlcNAc 6+ 7+ 9446.88 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]8-GlcNAc 1575.31 , 1350.41 6+ 7+ 1709.20 , 1465.17 10250.16 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]9-GlcNAc 6+ 7+ 11053.47 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]10-GlcNAc 1843.08 , 1579.92 7+ 8+ 1688.82 , 1482.23 11815.81 - [GlcNAc-Rha(Glc)-Rha-Rha]12 6+ 7+ 12456.90 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]12 2077.01 , 1780.43 6+ 7+ 2210.89 , 1895.19 13260.28 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]13 7+ 8+ 1986.80 , 1742.95 13901.71 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]13 7+ 8+ 14063.61 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]14 2009.95 , 1763.21 7+ 8+ 2101.55 , 1843.36 14704.74 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]14 7+ 8+ 2124.70 , 1863.21 14866.92 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]15 7+ 8+ 15508.16 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]15 2216.31 , 1943.78 7+ 8+ 2239.46 , 1964.04 15670.22 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]16 8+ 9+§ 2044.19 , 1817.17 16311.46 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]16 8+ 9+§ 2144.60 , 1906.42 17114.76 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]17 8+ 9+§ 17918.06 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]18 2245.01 , 1991.79 *: The peptide backbone of these glycopeptides is HDLDIKDNNNSTPTVISHR. The presented glycan compositions were designed based on the available information of O-antigen synthesis

15,39

. The identity of the observed m/z given in bold were confirmed by at least one HCD MS/MS spectrum, while the composition of the

residual observed m/z were assigned based on the molecular weight. §: only observed when m-NBA was present in the buffer system.



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Table 2 Effect of m-NBA on the peptide with O-antigens

-[GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]16

Observed m/z1 1007.473+ 755.864+ 865.404+ 692.535+ 1106.744+ 885.595+ 1495.835+ 1246.696+ 1782.236+ 1527.767+ 2344.786+ 2009.957+ 1817.179+

-[GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]18

1991.799+

Glycan structures - [GlcNAc-Rha-Rha-Rha]-GlcNAc - [GlcNAc-Rha-Rha-Rha]2 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]2 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]5 - [GlcNAc-Rha-Rha-Rha]2-[GlcNAc-Rha(Glc)-Rha-Rha]9 - [GlcNAc-Rha-Rha-Rha]-[GlcNAc-Rha(Glc)-Rha-Rha]14

Buffer without 0.5% m-NBA intensity Percentage2 1.44E6 377.8% 5.44E6 1.98E6 --n.d.3 1.49E6 14.3% 2.14E5 5.39E6 --n.d.3 7.09E6 77.9% 5.53E6 4.81E6 35.3% 1.7E6 n.d --n.d

---

Buffer with 0.5% m-NBA intensity percentage 2.32E6 251.7% 5.84E6 8.85E5 40% 3.54E5 1.02E6 402.0% 4.1E6 1.8E6 78.3% 1.41E6 3.9E6 109.5% 4.27E6 2.13E6 101.9% 2.17E6 5.31E5 ---4 2.25E4

---4

1

Only two major charge states of each precursor were examined.

2

The given percentage of each molecule was calculated based on the following equation:

Sf2E sample was ran in duplicates under each condition. XIC is an abbreviation for Extracted ion chromatography. 3

not detected

4

only observed in the presence of m-NBA


× 100%. The identical


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For TOC only


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Supercharging Reagent for Enhanced Liquid Chromatographic

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