Assignment of Saccharide Identities through Analysis of Oxonium Ion

Article pubs.acs.org/jpr

Assignment of Saccharide Identities through Analysis of Oxonium Ion Fragmentation Profiles in LC−MS/MS of Glycopeptides Adnan Halim,†,⊥ Ulrika Westerlind,§ Christian Pett,§ Manuel Schorlemer,§ Ulla Rüetschi,† Gunnar Brinkmalm,‡ Carina Sihlbom,∥ Johan Lengqvist,∥ Göran Larson,† and Jonas Nilsson*,† †

Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, ‡Department of Neurochemistry, Institute of Neuroscience and Physiology, University of Gothenburg, SE-41345 Gothenburg, Sweden § Gesellschaft zur Förderung der Analytischen Wissenschaften e.V. ISAS−Leibniz Institute for Analytical Sciences, Dortmund 44227, Germany ∥ Proteomics Core Facility, University of Gothenburg, SE-40530 Gothenburg, Sweden S Supporting Information *

ABSTRACT: Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure−function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography−tandem mass spectrometry (LC−MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing Nacetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC−MS/MS-based glycoproteomic studies. KEYWORDS: glycoproteomics, oxonium ion, LC−MS/MS, glycopeptide, O-glycosylation

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INTRODUCTION Glycoproteomics aimed at site-specific characterization of protein glycosylation is an emerging field of research.1,2 Generally, proteomic samples are subjected to protease digestion, and the N- and/or O-glycosylated peptides (Nand O-glycopeptides) are purified from the vast majority of unglycosylated peptides by the use of lectins,3−6 carbohydrate affinity chromatography,7−9 or carbohydrate-specific chemistry10−12 and are then analyzed using liquid chromatography− tandem mass spectrometry (LC−MS/MS). In addition, using a targeted glycoproteomic approach, purified glycoproteins may be analyzed by LC−MS/MS as intact glycoproteins13−15 or by analysis of peptides and glycopeptides after a protease digestion of the purified glycoproteins.1,16−20 Glycopeptides represent a special challenge in structural analysis of covalently modified peptides since (i) a wide variety of glycans may modify the same amino acid sequence and (ii) the glycosidic bonds are prone to fragmentation upon the generally used collision-induced dissociation (CID). Thus, two different types of fragmentation techniques are usually used for complete glycopeptide analysis. Using electron capture/transfer dissociation (ECD/ETD) techniques for fragmentation of ionized glycopeptides leaves the glycan part intact (except for © 2014 American Chemical Society

the loss of the HexNAc N-acetyl group from the precursor ion and loss of Neu5Ac from the precursor and glycopeptide fragment ions), whereas the peptide backbone is fragmented into the c- and z-series of ions, and the site-specific glycan attachment site(s) can be pinpointed.16,21 ECD/ETD works best when glycopeptide precursor ions have a low mass-tocharge ratio and are composed of single N-acetylhexosamine (HexNAc) units, such as in the case of O-GlcNAc glycosylation of intracellular proteins (GlcNAcβ-O- structure)22,23 or in SimpleCell-based analysis of GalNAcα-O- sites.3,24 Although ECD/ETD may also be used for the identification of more complex N- and O-glycopeptides,25−27 no information aside from the site-specific attachment of the glycan mass is made available. Conversely, for CID using ion trap MS/MS, stepwise glycosidic fragmentation dominates, and the peptide part generally remains intact, which may be used to investigate the glycan sequence of, e.g., hexose (Hex) and HexNAc units. CID-MS3 fragmentation of the (partially) deglycosylated peptide ion obtained in the MS2 step might, however, also be used for identification of the peptide.10,11,25,28 In contrast, Received: August 29, 2014 Published: October 30, 2014 6024

dx.doi.org/10.1021/pr500898r | J. Proteome Res. 2014, 13, 6024−6032

Journal of Proteome Research

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beam-type CID on quadrupole29−31 and C-trap equipped Orbitrap instruments, which use higher-energy collisional dissociation (HCD),3,26,32 results in simultaneous fragmentation of both the glycan and peptide parts, which can be utilized for the identification of glycopeptides in a single MS/MS step. The collision cell of the HCD unit33 also allows acquisition of fragment spectra covering the full m/z 100−2000 region, thus enabling the simultaneous analysis of low-molecular-weight saccharide oxonium ions, the whole range of peptide backbone b- and y-ions, low-molecular internal fragments, and amino acid immonium ions. In current glycoproteomics, the level of structural detail obtained by positive mode CID, HCD, and ECD/ETD fragmentation, and recently negative mode CID as well,34 of glycopeptides is generally limited to the identification of, for instance, the Hex/HexNAc- and peptide sequences. It is, however, of great importance to be able to distinguish between GalNAc- and GlcNAc-substituted peptides because these modifications represent quite different cellular machineries of glycosylation. The O-GlcNAc glycosylation of proteins is an important regulator occurring within cells and thus it is important to identify the O-GlcNAc attachment sites. Analogously, the overexpression of GalNAcα1-O- (Tn antigen) on extracellular proteins is a tumor marker.35 However, LC−MS/MS studies aimed at identifying either modification is limited to assigning it as GlcNAc or GalNAc based only on the predicted cellular localization of the protein. However, this designation is challenged by the fact that O-GlcNAc has recently also been described for extracellular proteins.36,37 In addition, analysis of extracellular GalNAc-type O-glycosylations using SimpleCells has also identified potential O-GlcNAc modifications originating from intracellular compartments.24,38 To this end, the simultaneous presence of both extracellular and intracellular Oglycosylations has been described in a large-scale glycoproteomic study aimed at identifying O-GlcNAc.27 Thus, having a simple, reliable method to distinguish between isomeric GalNAc and GlcNAc modifications would be very important. At present, precise saccharide identification and linkage analysis of glycoproteins is typically performed using various wet-lab procedures and by NMR spectroscopy39,40 or including chemical or enzymatic glycan release from proteins with subsequent MS/MS disassembly of permethylated saccharides.41−43 However, such methodologies require several wetlab procedures and the use of dedicated MS setups and are also decoupled from attachment site-specific analysis. Also, ion mobility spectrometry (IMS)−MS has emerged with great potential to address glycan and glycopeptide linkage analysis,44,45 including the characterization of glycosaminoglycans.46 In IMS−MS, the ions are separated on the basis of their crosssection/charge ratio rather than only on their m/z values, which enables separation of structural epimers based on their drift time. Although IMS−MS is able to resolve epimeric glycopeptides and saccharide oxonium ions following CID, the methodology has not yet been applied to a complex mixture of glycopeptides. A simple way to possibly capture structural information on glycans is to analyze the beam-type and ion trap CID fragmentation spectra of saccharide oxonium ions present at low mass (m/z 100−400) generated from glycopeptides during the collisional activation.47 HCD spectra of synthetic GalNAcαO- and GlcNAcβ-O-substituted glycopeptides have been analyzed, but differences in the saccharide oxonium ion spectra have not been reported.22 However, after careful analysis of the

published HCD spectra, we observed certain spectral differences. Thus, to determine whether these differences are consistently reproducible, we analyzed a collection of 21 different O-GalNAc- and O-GlcNAc-substituted peptides and investigated the dependence on the collision energy used. In addition, elongated structures were used to assay if additional glycosidic linkages influenced the oxonium ion spectral profiles. We found that there are indeed unique profiles in the resulting saccharide oxonium ion spectra that depend critically on glycan structure. We show that the observed fragmentation signatures could be used to identify saccharide identities and glycosidic linkage positions on native glycopeptides found in human cerebrospinal fluid (CSF)10,32 and urine25 samples.

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MATERIALS AND METHODS The synthetic glycopeptides 1, 5, 9−11, 16−21 have been previously described,48−50 and glycopeptides 2, 3, 5−8, 12−14, 18, and 19 are described in the Supporting Information. Synthetic glycopeptides were dissolved (2 μM) in water containing 0.02% formic acid. Desialylated glycopeptides from human transferrin (Sigma), CSF, and urine were prepared as described.10,11,25,32 Human CSF samples were in-solution trypsin digested (Promega) according to the Protease Max protocol (Promega). Sialylated glycopeptides were then purified using strong anion exchange (SAX) chromatography.51 HCD of Synthetic O-Glycopepeptides

Synthetic glycopeptides (4 pmol) were injected onto an analytical 230 mm × 75 μm i.d. PicoFrit column (New Objective Inc., Woburn, MA, USA) packed in-house with 3 μm reversed-phase particles (ReproSil-Pur C18-AQ, Dr. Maisch GmbH) operated in a trap-column configuration (45 mm × 100 μm, in-house packed as above). The chromatography was developed at a flow rate of 200 nL/min using an Easy nLC1000 chromatograph interfaced to a Q Exactive hybrid instrument (Thermo Fisher Scientific), using a gradient with 7−35% B over 5 min, 35−80% B over 5 min, and a final hold step at 80% for 5 min. Solvents A and B were 0.2% formic acid in deionized water and 100% acetonitrile, respectively. Full MS data and fragment spectra were collected from the Q Exactive instrument operated in positive ion mode. For each run, five different so-called top 3 experiments (each with a specific normalized collision energy (NCE) setting) were run in parallel. Thus, two consecutive injections per peptide were needed to cover the NCE range from 10 to 50% in 5% increments. Each experiment was set to use an inclusion list for the 2+ and 3+ ions of each specific peptide. Specifically, full scan (MS1) spectra were acquired over the m/z range 400− 1800 at a resolution setting of 35 000 (full width at halfmaximum at m/z 200), with the automatic gain control (AGC) target value set to 3 × 106 and a maximum injection time of 100 ms. MS/MS spectra were acquired from m/z 50 for the three most abundant precursors at a resolution setting of 17 500 for maximal AGC and injection time settings of 1 × 106 and 50 ms, respectively, using a precursor isolation window of 2 m/z units. Full MS data and fragment spectra were additionally collected using a hybrid linear ion trap Orbitrap mass spectrometer (Velos, Thermo Fisher Scientific) operated in positive ion mode at 1.6 kV. For each run, eight parallel HCD scan experiments (each with a specific NCE setting) were run. Thus, the LC−MS/MS analysis per peptide covered the NCE range from 10 to 50% in 5% increments. Each experiment was set to use an inclusion list for the 2+ and 3+ ions of each 6025



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Figure 1. Saccharide oxonium ion spectra of synthetic O-glycopeptides. Synthetic O-glycopeptides with the sequence PAHGVTSAPDTRPAPGSTAP substituted with (A) GalNAcα1-O-, (B) Galβ3GalNACβ1-O-, (C) Neu5Acα6GalNAcα1-O-, (D) GlcNAcβ1-O-, (E) Galβ3GlcNAcβ3GalNAcα1-O-, and (F) Galβ4GlcNAcβ3GalNAcα1-O-, at the underlined Thr residue, were subjected to HCD at normalized collision energy (NCE) levels of 10−50% at 5% intervals on the Q Exactive. The oxonium ion HCD spectra at m/z 100−220 (m/z 100−300 for the Neu5Acα6GalNAcα1-O-substituted peptide in panel C) at the 30% energy level are shown to the left, and the oxonium ion intensity profiles are shown to the right. The precursor structures are shown in boxes.

value of the standard deviation was ±3% for all measurements shown.

specific peptide. Specifically, full scan (MS1) spectra were acquired over the m/z range 400−2000 at a resolution setting of 30 000. MS/MS spectra were acquired from m/z 100 for the included precursors at a resolution setting of 7500 using a precursor isolation window of 4 m/z units.

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RESULTS AND DISCUSSION

Intensity Profiles of Saccharide Oxonium Ions of Synthetic O-Glycopeptides

CID-MS3/MS4 Method

Synthetic O-glycopeptides,48−50,52,53 originating from the sequence PAHGVTSAPDTRPAPGSTAP from human mucin1 (MUC1), with Thr-6, Ser-7, Thr-11, Ser-17, or Thr-18 (underlined) substituted with either Tn-antigen, T-antigen (Core 1), Sialyl Tn-antigen, GlcNAcβ1-O-, Type 1 terminated Core 3, or Type 2 terminated Core 3 structures, were analyzed. Glycan structures (boxed) are depicted in Figure 1 according to the Consortium for Functional Glycomics recommendations. The glycopeptide structures (peptides 1−21) and their syntheses are presented in Supporting Information Section S1. Most of the synthetic glycopeptides were derivatized Nterminally with a poly(ethylene glycol) (PEG) spacer for surface immobilization and additional use in microarray applications.53 The HCD normalized collision energy (NCE) settings on the hybrid quadrupole Orbitrap mass spectrometer (Q Exactive) were varied from 10 to 50% at 5% intervals, and the relative intensities of the saccharide oxonium ions originating from HexNAc and Neu5Ac (Table 1) at m/z 100−366 were plotted against the NCE (Figure 1 and Figure

In the CID-MS3/MS4 experiments full MS data and fragment ion spectra were collected using an LTQ FT Ultra instrument (Thermo Fisher Scientific) operated in positive ion mode and coupled to a nano-LC system. Precursor ion scans were acquired over the m/z range 300−2000 at a resolution setting of 50 000. CID-MS2 spectra were recorded for the most intense precursor ion in each full scan, at a NCE of 30% and an isolation width of 5 m/z units. The experiment was set to use an inclusion list for the 3+ ions of each glycopeptide as well as the 4+ ion for HexNAc-substituted peptides. An ion product list containing the m/z 204.09 and m/z 366.14 ions were used for MS3 and MS4 selections. Preparation of Oxonium Ion Profile Diagrams

The mean value of the intensities from four measurements of the m/z 126, 138, 144, 168, 186, 204, 274, 292, and 366 oxonium ions were normalized against the mean value of the most intense one and plotted as percentage values. The mean 6026



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weaker; sometimes, m/z 144 may be nonexistent. At the same time, GalNAc produces a significant m/z 126 ion, whereas m/z 138 and m/z 144 are present at about the same level and are 50−70% weaker than that of m/z 126. The different profiles were consistently observed at NCE values routinely used for glycopeptide analysis.

Table 1. Identities and Masses of Saccharide Oxonium Ions 126 138 144 168 186 204 274 292 366

126.055 138.055 144.065 168.066 186.076 204.087 274.092 292.103 366.140

[C6H7NO2]+ [C7H8NO2]+ [C6H10NO3]+ [C8H10NO3]+ [C8H12NO4]+ [C8H14NO5]+ [C11H6NO7]+ [C11H18NO8]+ [C14H24NO10]+

[HexNAc − C2H6O3]+ [HexNAc − CH6O3]+ [HexNAc − C2H4O2]+ [HexNAc − 2H2O]+ [HexNAc − H2O]+ [HexNAc]+ [Neu5Ac − H2O]+ [Neu5Ac]+ [HexHexNAc]+

Galβ3GlcNAc- versus Galβ4GlcNAc-Modified Glycopeptides

The oxonium ion intensity profiles of the Type 1 glycopeptides having a terminal Galβ3GlcNAc structure (16) (Figure 1E) were similar to those of the GlcNAcβ1-O-substituted glycopeptides, showing distinct intensities of the m/z 204 and m/z 138 ions (cf. Figure 1D) and demonstrating that Gal substitution at the GlcNAc C-3 position did not affect the oxonium ion profile. However, for the corresponding Type 2 glycopeptide having a terminal Galβ4GlcNAc structure (19) (Figure 1F), the m/z 204 ion intensity levels were dramatically lower at all NCE levels, whereas the m/z 138 and m/z 168 oxonium ions were very prominent already at the 15% NCE level. Therefore, this oxonium ion intensity profile was clearly distinguishable from the GlcNAcβ1-O- and Type 1 glycopeptides. Because the internal GalNAcα1-O- structure of the Type 1 and Type 2 glycopeptides might affect the oxonium ion intensity profile, we also used CID-MS2/MS3 to selectively characterize the oxonium ions originating from the terminal Galβ3GlcNAc and Galβ4GlcNAc moieties. Thus, the [Galβ3GlcNAc]+ and [Galβ4GlcNAc]+ ions at m/z 366 in the CID-MS2 spectra, collected at the 30% NCE level, were selectively subjected to CID-MS3 (m/z 854.74 → m/z 366 → transitions, Figure S3A,B), and both resulted in oxonium ion spectra very similar to those obtained using only HCD. The measurements demonstrated that oxonium ions originating from the GalNAcα1-O- part did not significantly affect the oxonium ion spectra of the Type 1 and Type 2 glycopeptides. Analogously, CID-MS3 of the [Galβ3GalNAc]+ ion at m/z 366 from the Core 1 glycopeptide (m/z 819.40 → m/z 366 → transition, Figure S3C) resulted in an intensity profile similar to that of the m/z 100−220 region of the HCD spectra of the Core 1 glycopeptide, except that m/z 204 was more prominent using CID (cf. Figure 1B). Therefore, it can be concluded that the fragmentation profiles, to a major extent, are related to the glycan structure rather than whether HCD or CID is used to perform the dissociations. The observed differences are currently not fully understood, and we are now conducting multistage MSn experiments, including the use of isotopelabeled glycopeptides, in order to structurally investigate the decomposition of the various oxonium ions.

S1). The peptides that were glycosylated at Thr-6 were also subjected to HCD on a hybrid linear ion trap Orbitrap mass spectrometer (Velos) (Figure S2), from which it was evident that the oxonium ion profiles of the Q Exactive (Figure 1) and Velos (Figure S2) instruments differed in the normalized energies, i.e., the HCD spectra run at 30% energy level were similar to the ones run at 25% on the Q Exactive. Thus, instrument-specific settings of the energy level should be adjusted in order to obtain similar spectra using different instruments. GalNAc- versus GlcNAc-Modified Glycopeptides

At the 30% NCE level, the saccharide oxonium ions for the Tn glycopeptide (1) (Figure 1A, left) were [HexNAc]+ at m/z 204, its loss of H2O at m/z 186, and loss of one more H2O into m/z 168; [HexNAc-C2H4O2]+ at m/z 144 and its loss of H2O into m/z 126; and finally [HexNAc − CH6O3]+ at m/z 138. See Table 1 for a list of exact masses and molecular compositions of the typical saccharide oxonium ions observed. At the 10−20% NCE levels, the intact m/z 204 ion and the m/z 186 ion predominated (Figure 1A, right), but the intensity of the m/z 126 ion took over above the 25% NCE level and became the major ion at and above the 30% level. Conversely, the m/z 138 and m/z 144 ions increased in relative intensity at and above the 25% NCE level but were present at lower intensities compared to that of the m/z 126 ion. A similar oxonium ion intensity profile was evident for the Core 1 disaccharide (5) (Figure 1B) and included an intact disaccharide [HexHexNAc]+ ion (m/z 366) at the lower NCE levels. Also, the sialyl Tn-antigen glycopeptide (9) resulted in a similar intensity profile (Figure 1C) but additionally included the [Neu5Ac]+ (m/z 292) and [Neu5Ac − H2O]+ (m/z 274) oxonium ions, present at gradually lower intensities as the NCE level was raised (Figure 1C, right). The similarities of oxonium ion intensity profiles of these three glycopeptides (Figure 1A−C) indicate that Gal or Neu5Ac substitutions did not alter the GalNAc fragmentation profile. In the case of GlcNAcβ1-O-substituted peptides (12, Figure 1D), the m/z 138 ion was persistently more intense than the m/z 186 and m/z 126 ions at the 25−50% NCE levels. Additionally, the intensity of the m/z 144 ion was considerably less pronounced (≤20%) compared to the intensity of this ion for the GalNAc-modified peptides (60−80% relative intensity) at the ≥30% NCE levels. Two commercially available glycopeptides with GTTPSPVPTTSTTSAP (4, from Mucin5) and TAPTSTIAPG (15, from transcription factor CREB) sequences substituted with Tn antigen and GlcNAcβ1-Ostructure, respectively, showed intensity profiles comparable with the corresponding Mucin-1 glycopeptides (Figure S1A,D), proving that the peptide’s N-terminal PEG spacer did not influence the fragmentation profiles. In summary, for GlcNAc, m/z 138 is the major product, and the others are significantly

The GlcNAc/GalNAc Ratio

The difference in oxonium ion spectral profiles of GlcNAcβ1O- and GalNAcα1-O-substituted peptides must be due to the axial [GalNAc]+ or equatorial [GlcNAc]+ C-4 hydrogen configurations, where the decomposition pathway leading to m/z 138 and m/z 168 is preferred for GlcNAcβ1-O-, and m/z 126, together with m/z 144, is preferred for GalNAcα1-Osubstituted peptides. On the basis of this qualitative observation for the decomposition of HexNAc-generated oxonium ions, we introduced the (m/z 138 + m/z 168) to (m/z 126 + m/z 144) relative abundance ratios, referred to as the GlcNAc/GalNAc ratio (Figure 2), in order to distinguish whether the decomposition is GlcNAc- or GalNAc-like and thus to assign the saccharide identities. The GlcNAc/GalNAc ratio was 0.4− 0.6 for the Tn, T, and Sialyl Tn glycopeptides throughout the 6027



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ratio was 1.5−3.5 for the GlcNAcβ-O-, Type 1, and Type 2 terminated glycopeptides, and it was 0.2 for the Tn and Core 1 substituted glycopeptides (Figure 2B). Thus, by using this approach, it was possible to determine the HexNAc identity as being either GlcNAc or GalNAc. A similar approach has previously been used for the identification of ammonium adducts of native monosaccharides, where the (m/z 168)/(m/z 126) intensity ratio was used to distinguish GlcNAc from GalNAc.54 A drawback of the CID-MS3/MS4 method for distinguishing Tn antigen (GalNAcα-O-) from GlcNAcβ-O- on glycopeptides is that the mass difference between m/z 204 and the precursor (m/z 765 for the 3+ ions of the HexNAc-substituted MUC1 peptides) often may be close to 25% (204/765 = 0.27), which is the lowest observable limit that is practically possible to detect for low mass product ions generated from CID. This LTQ-specific limitation is due to the compromise of using a high voltage for retaining the precursor ions in the trap, as the same high voltage simultaneously leads to ejection of low mass ions.55 Consequently, because m/z 204 was not observed for CID-MS2 at m/z 765, the [M + 4H]4+ ions at m/z 574, which had intensities of about 5% compared to those of the [M + 3H]3+ precursors, had to be used (Figure 2B). The CID-MS4 analysis of m/z 204 for the HexHexNAc-substituted peptides did not have the same limitation since 366/819 = 0.45, and the m/z 366 ion was prominent in the CID-MS2 spectra. Oxonium Ion Spectrum Profiles of Desialylated N- and O-Glycopeptides from Native Proteomic Samples

We next analyzed the m/z 100−220 regions of HCD spectra, collected at a NCE level of 35% using the Velos Orbitrap, with samples originating from human urine and CSF. The NCE level of 35% was chosen in order to provide abundant peptide fragmentation in the presence of the glycans. These N- and Oglycopeptides had been enriched by the use of a sialic acidspecific capture-and-release protocol, where sialic acid is a prerequisite for the enrichment but is removed during the release of the glycopeptides from the hydrazide beads.10,11 The [M + 3H]3+ precursor for HexHexNAc-O-substituted 342AVAVTLQSH-350 glycopeptide from protein YIPF3 was observed in both CSF and urine samples at m/z 645.83 (Figure 3A). The appearance of the HCD induced oxonium ion spectrum at m/z 100−220 was very similar to that of previously observed synthetic Core 1 glycopeptides (cf. Figure 1A) and showed a GlcNAc/GalNAc ratio < 1, indicating that the native glycopeptide indeed has a Galβ3GalNAcα1-O- structure. A comparable intensity profile was obtained using CID-MS3 at m/z 366 (Figure 3B), which is consistent with the synthetic Core 1 glycopeptides (cf. Figure S3C). Furthermore, the HCD spectra of a HexNAc-substituted AVAVTLQSH peptide (Figure 3C), present from urine samples, showed a similar intensity profile as that of the synthetic Tn glycopeptides (cf. Figure 1A), with a significant m/z 126 ion demonstrating that the glycopeptide was substituted with a GalNAcα1-O-. A similar oxonium ion spectrum was observed by CID-MS3 fragmentation of the [HexNAc]+ ion at m/z 204 (Figure 3D). A Hex2HexNAc2 glycoform of this peptide was also present in the urine samples,25 and the HCD spectra (Figure 3E) showed an oxonium ion profile with a GlcNAc/GalNAc ratio > 50, which was also valid for CID-MS2/MS3 at m/z 366 (Figure 3F). Thus, the glycopeptide has either Core 2 [Galβ3(Galβ4GlcNAcβ6)GalNAcα1-O-] or a Type 2 extended Core 1 [Galβ4GlcNAcGalβ3GalNAcα1-O-] structure because they,

Figure 2. GlcNAc/GalNAc ratios for HCD and CID-MS3/MS4 spectra of synthetic glycopeptides. (A) HCD-generated GlcNAc/ GalNAc ratios for the synthetic O-glycopeptides in Figure 1 and Figure S1. An expansion is shown at the top, and the full range is shown at the bottom. (B) CID-MS3/MS4-generated GlcNAc/GalNAc ratios for synthetic O-glycopeptides.

15−50% NCE levels (Figure 2A, top graph). For the GlcNAcβO- glycopeptides, it was 2.0−2.5 throughout the 15−50% NCE levels, and for the Type 1 glycopeptides, it was 5.0 at the 15% NCE level but gradually approached 2.0 at higher NCE levels. Thus, for GlcNAcβ-O- and Type 1, the combined intensities of the m/z 138 and m/z 168 ions are 2-fold more intense than those for the m/z 126 and m/z 144 ions, and approximately the opposite was observed for the GalNAc-O-substituted peptides. The Type 2 glycopeptides had a GlcNAc/GalNAc ratio > 30 at the 10−15% NCE level (Figure 2A, lower graph), although the standard deviation at these low energy levels was substantial and declined from 20 to 10% at the 20−25% NCE levels and then approached 4−5% at the 35% NCE level and was structurally clearly distinguishable from the other glycopeptides. CID-MS3/CID-MS4 at m/z 204 To Distinguish GalNAc from GlcNAc

A similar GlcNAc/GalNAc ratio strategy was assessed using CID fragmentation at an NCE level of 30% using the synthetic MUC1 glycopeptides. CID-MS3 at m/z 204 for the HexNAcsubstituted glycopeptides and CID-MS4 at m/z 204, via the CID-MS3 spectrum of m/z 366, were assayed. See Figure S3A−C for examples of the transitions used for Type 1, Type 2, and Core 1 glycopeptides, respectively. The GlcNAc/GalNAc 6028



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Figure 3. Saccharide oxonium ion spectra of native O-glycopeptides. HCD spectra (left), including a m/z 100−220 expansion, and CID-MS2/MS3 (right) of 342-AVAVTLQSH-350 originating from the C-terminal tryptic peptide of protein YIPF3 that is substituted at Thr-346 with (A, B) Core 1 O-glycan (boxed structure), (C, D) Tn-antigen, and (E, F) Core 2 O-glycan.

presence of saccharide oxonium ions in the HCD spectra (Figure 4). The disialylated Core 1 substituted 210-AATVGSLAGQPLQER-224 peptide from apolipoprotein E (APOE_HUMAN) showed an oxonium ion spectrum with prominent m/z 126, m/z 186, and m/z 204 ions (Figure 4A) and was very similar to the GalNAc-modified glycopeptide, with the same peptide sequence (cf. Figure S4B), as well as to all other GalNAc-modified glycopeptides. Also, the disialylated Core 2 glycopeptide was found, and the HCD spectrum showed, as expected, prominent m/z 138 and m/z 168 ions (Figure 4B). Additionally, for N-glycopeptides, the disialylated complex biantennary substituted 155-VYKPSAGNNSLYR-167 peptide from beta-2-glycoprotein 1 (Figure 4C) was very similar to the desialylated glycopeptide (Figure S4C) and to all other Galβ4GlcNAc-containing glycopeptides, having prominent m/ z 138 and m/z 168 ions. For sialylated glycopeptides (Figure 4), additional saccharide oxonium ions, lacking nitrogen, at m/z 121, m/z 167, and m/z 197 were observed. The presence of the same oxonium ions from the synthetic sialyl Tn-substituted peptides (Figure 1C and Figure S1C), but not for any other of the synthetic peptides, convincingly demonstrated to us that these ions were specific for Neu5Ac. The presence of these Neu5Ac-specific ions in the HCD spectra, including the very prominent m/z 274 [Neu5Ac − H2O]+ and m/z 292

like the synthetic Type 2 glycopeptides, also have terminal Galβ4GlcNAc structures (cf. Figure 1F). We have previously concluded that the Core 2 alternative is the most likely structure because the [HexNAc2]+ (m/z 407) and [HexHexNAc2]+ (m/z 569) oxonium ions were identified in the CIDMS2 spectra25,32 (see also the presence of m/z 569.2 in Figure 3F). For glycopeptides containing both GalNAc and GlcNAc residues, a possible oxonium ion contribution from both epimers is, of course, a possibility, but this was not observed for relevant glycopeptides in this study. For LC−MS/MS experiments of complex mixtures of N- and O-glycopeptides, it is thus possible to assign whether the HCD spectra of unknown glycopeptides have a terminal Galβ4GlcNAc structure (i.e., from Type 2 terminated O-glycopeptides and complex-type N-glycopeptides), resulting in prominent m/ z 138 and m/z 168 ions, or Core 1 disaccharide Oglycopeptides with dominance of m/z 126, m/z 186, and m/z 204 ions. Additional examples of HCD and CID-MS2/MS3 oxonium ion spectra from native glycopeptides are provided in Figures S4 and S5. Oxonium Ion Spectrum Profiles of Sialylated Glycopeptides from Native Proteomic Samples

Next, we analyzed trypsin-digested CSF samples and found sialylated N- and O-glycopeptides based on the prominent 6029



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Figure 4. HCD spectra of sialylated O-glycopeptides originating from CSF proteins. Full HCD spectra (left) and m/z 100−220 expansion (right) of 210-AATVGSLAGQPLQER-224 peptide from Apo E (UniProt: APOE_HUMAN) substituted at Thr-213 with (A) disialylated Core 1 O-glycan (boxed structure) and (B) disialylated Core 2 O-glycan. (C) Full HCD spectra (left) and an m/z 100−220 expansion (right) of 155VYKPSAGNNSLYR-167 peptide from beta-2-glycoprotein 1 (APOH_HUMAN) substituted at Asn-162 with a disialylated complex biantennary Nglycan.

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[Neu5Ac]+ ions, are a compellingly diagnostic for the presence of sialic acid(s) in the HCD spectra of glycopeptides. In conclusion, analysis of GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβO-terminated synthetic and natively derived N- and Oglycopeptides by LC−MS/MS using both HCD and CIDMS2/MS3 resulted in unique characteristics in the obtained saccharide oxonium ion spectra. In particular, significant differences in the fragmentation signatures of oxonium ion spectral profiles of GlcNAcβ1-O- and GalNAcα1-O-substituted peptides were perceived for the first time. A GlcNAc/GalNAc ratio was introduced to identify the oxonium ion spectral profiles as being either GlcNAc- or GalNAc-like. Therefore, a powerful strategy has been developed to differentiate short mucin-type glycopeptides and structurally similar O-GlcNAcsubstituted glycopeptides. We believe that the knowledge gained in this study will be of importance in future glycoproteomic studies.

ASSOCIATED CONTENT

S Supporting Information *

Section S1: Synthesis and characterization of synthetic glycopeptides. Section S2: Figures S1−S5. Scheme S1: Overview of the synthetic glycopeptides used in the MSfragmentation study. Figure S1: HCD-induced oxonium ion intensity profiles of synthetic O-glycopeptides. Figure S2: Full HCD spectra and oxonium ion intensity profiles on the Velos Orbitrap. Figure S3: CID-MS2 spectra and CID-MS3 at m/z 366 of synthetic glycopeptides. Figure S4: Examples of HCDinduced oxonium ion spectra of native N- and O-glycopeptides. Figure S5: Examples of CID-MS2/MS3 generated oxonium ion spectra of native O-glycopeptides. This material is available free of charge via the Internet at http://pubs.acs.org. 6030



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address ⊥

(A.H.) Copenhagen Center for Glycomics and Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Denmark. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Fredrik Noborn for the LC−MS/MS spectra of CSF samples and Alejandro Gomez-Toledo and Dr. Ammi Grahn for the LC−MS/MS spectra of α-dystroglycan samples. This study was supported by grants from the Swedish Research Council (8266 to G.L.), Alzheimer Foundation, Magn. Bergwall Foundation, and governmental grants to the Sahlgrenska University Hospital. The Inga-Britt and Arne Lundberg Research Foundation; and Knut and Alice Wallenberg Foundation are acknowledged for MS instrumentation funding.

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Assignment of Saccharide Identities through Analysis of Oxonium Ion

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