ionization tandem mass spectrometry of N-glycans derivatized with isonicotinic hydrazide and its biotinylated form.

Research Article Received: 8 February 2014

Revised: 29 April 2014

Accepted: 26 May 2014

Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 1745–1756 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6955

Matrix-assisted laser desorption/ionization tandem mass spectrometry of N-glycans derivatized with isonicotinic hydrazide and its biotinylated form Stephanie Bank1, Eberhard Heller1, Elisabeth Memmel2, Jürgen Seibel2, Ulrike Holzgrabe1 and Petra Kapková1* 1 2

Institute of Pharmacy and Food Chemistry, Julius-Maximilians-University Würzburg, Am Hubland, 97074 Würzburg, Germany Institute of Organic Chemistry, Julius-Maximilians-University Würzburg, Am Hubland, 97074 Würzburg, Germany

RATIONALE: Successful structural characterization of glycans often requires derivatization prior to mass spectrometric analysis. Here we report on a new derivatization reagent for glycans, biotinylated isonicotinic hydrazide, allowing glycan analysis by both mass spectrometry (MS) and biochemically. Fragmentation behavior in MS and its use in structural elucidation were investigated and compared with other labels. METHODS: Glycans, released from ribonuclease B and ovalbumin, were derivatized with hydrazine labels (isoniazid (INH), biotinylated isonicotinic hydrazide (BINH) and biotinamidocaproylhydrazide (BACH)). In addition, native counterparts and 2-aminobenzamide (2-AB) derivatives were prepared. Comparative matrix-assisted laser desorption/ ionization tandem time-of-flight (MALDI TOF/TOF) experiments were carried out to investigate the fragmentation pattern of the derivatives. Finally, the capability of BINH derivatives to bind lectins was explored. RESULTS: Generally, derivatization provided beneficial enhancement in the mass spectrometric signal intensity as compared to native counterparts. The mass spectrometric fragmentation varied with the kind of label used. The most significant structure-revealing ions (cross-ring cleavages) were observed in the spectra of BINH derivatives, whereas mainly glycosidic cleavages were found with native form of glycans and 2-AB derivatives. CONCLUSIONS: Hydrazine derivatization provided the means to obtain structurally informative fragment ions. Due to BINH derivatization, specific fragments of the isomers allowed the identification of diverse glycans. The derivatization reaction can be carried out without the need for purification. The biotin residue of BINH enabled for biochemical studies, i.e. protein–glycan interactions. Copyright © 2014 John Wiley & Sons, Ltd.

Analysis of the glycan is a substantial part of the glycoprotein characterization. Usually, a combination of various chromatographic and spectroscopic methods is required in order to explore the posttranslational modification. Its complete characterization still represents a challenging analytical task. Derivatization has a long-standing tradition in the analytical pathway of glycan analysis. In former times, chemical labeling was used to improve the volatility of sugars for gas chromatography by silylation of hydroxyl groups.[1] Later on, the characterization of carbohydrates by other analytical methods e.g. high-performance liquid chromatography (HPLC), UV spectroscopy and fluorescence were shown to profit from derivatization.[2,3] Thus, fluorophoric or chromophoric labels have been introduced in glycans in order to improve the sensitivity of these analytes. Additionally, the hydrophobicity of the analytes has been increased often resulting in a better separation of

Rapid Commun. Mass Spectrom. 2014, 28, 1745–1756

Copyright © 2014 John Wiley & Sons, Ltd.

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* Correspondence to: P. Kapková, Department of Pharmacy and Food Chemistry, Am Hubland, 970 74 Würzburg, Germany. E-mail: [email protected]

the carbohydrates on the widely used reversed-phase material.[4] In the course of the development of new analytical methods, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) emerged as sensitive methods in the structural elucidation of oligosaccharides. Even though the analysis of native glycans can be performed directly, derivatizations were applied to enhance the signal sensitivity via improving the ionization.[5,6] Moreover, in tandem mass spectrometric (MS/MS) analyses, the fragmentation behavior of derivatized carbohydrates was shown to be influenced by the type of the label used.[7] The most frequently applied derivatization approach is the reductive amination via Schiff-base formation, mostly accompanied by the reduction step in order to attain a stable secondary amine linkage. The widely used tags include aryl amine reagents such as 2-aminobenzamide (2-AB), 2-aminopyridine, and 2-aminoacridone.[6,8] Similarly, hydrazine reagents which form Schiff-base products with the aldehyde of the sugar were applied.[9–13] These derivatives are more stable than the Schiff-base product from an amine-aldehyde reaction even without reduction.[14] In this way, the resultant linkage is glycosylhydrazide, having almost the native conformation at the reducing end.[15] This proved to be very important when using hydrazine glycan derivatives in

S. Bank et al. biochemical studies.[15,16] A practical advantage of hydrazine derivatization in comparison to the aryl-amine reduction was found to be the possibility of omitting the cleanup procedure.[10,11,13,17] Consequently, the labeling can be carried out quickly, very easily and with minimal sample loss. Different hydrazine labels and their variants were investigated and developed in order to find an optimum tag for glycan derivatization. Derivatization with carboxymethyl trimethylammonium hydrazide (Girard’s reagent T) was performed in order to set a permanent charge to the reducing end of some neutral oligosaccharides which were then analyzed by means of MALDI-TOFMS.[18] Other cationic hydrazides based on Girard’s reagent T were synthesized following this objective and applied to N-linked glycans.[9] However, later studies have shown that neutral labels perform better than cationic reagents when coupled to Nlinked glycans.[19,20] Guanidine hydrazide derivatives demonstrated detection of N-linked glycans even in the presence of other bioanalytes, e.g. peptides.[13] Biotinylated tags have emerged as an additional class of labels that allow exploitation of the affinity of the derivatized sugar for carbohydrate-binding proteins.[21–24] These labels incorporate often UV activity and bioaffinity.[16,25] In this regard, glycans derivatized with a biotinylated hydrazide tag could be used in both mass spectrometric and functional studies, e.g. interaction studies with carbohydrate-binding proteins.[10,26] In this study, we have performed the derivatization of Nglycans with isonicotinic hydrazide (INH) and its biotinylated version (BINH). The glycan fragmentation and intensity was investigated by MALDI-TOF MS/MS and compared to glycans of BACH (biotinamidocaproylhydrazide) and 2-AB derivatives. We further investigated a lectin-binding property of BINH derivatives.

EXPERIMENTAL Materials and methods Hen ovalbumin (grade V), ribonuclease B, 2,5-dihydroxybenzoic acid (DHB), 2-aminobenzamide (2-AB), biotinamidocaproylhydrazide, streptavidin-coated glass slides (PolyAn®, Berlin, Germany), and ConA-Alexa Fluor®647 were purchased from Life Technologies (Darmstadt, Germany). Isoniazid (INH), super-DHB and mannose were obtained from Fluka (Buchs, Switzerland). BINH was synthesized according to the procedure described in the Supporting Information. PNGaseF and GlycoClean™ cartridges were obtained from Europa Bioproducts Ltd. (Cambridge, UK). Deionized water prepared by means of a Milli-Q purification apparatus (Millipore®, Eschborn, Germany) was used throughout. Deglycosylation of glycans from the glycoprotein

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PNGase F-protocol: 500 μg of glycoprotein was denatured in water (40 μL) and reaction buffer 5× (10 μL) supplied with the deglycosylation kit by heating at 100 °C for 10 min. After cooling, oligosaccharides were released from glycoprotein (ovalbumin, ribonuclease B) by 2 μL PNGase F enzyme (incubation time: 18 h, temperature 37 °C). Deglycosylated

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protein was precipitated by the addition of 200 μL of ethanol, kept on ice for ~1.5 h and centrifuged down to a pellet. The glycans, dissolved in the supernatant, were dried in vacuo and further processed and analyzed or derivatized as described below. The analyzed portion was ~5 pmol. Derivatization with INH, BINH and BACH 4 mg of the respective label: INH, BINH or BACH was diluted in 500 μL 50% methanol (derivatization solution). Glycans released from glycoproteins were treated with 5-fold excess of the derivatization solution and dried in vacuo at 30 °C. Then, 40 μL 95% methanol were added and incubated at 90 °C for 1.5 h. Subsequently, the mixture was evaporated to dryness and dissolved in 100 μL 50% methanol (sample solution). Derivatization with 2-AB 50 μL of 2-AB-derivatization solution composed of 2-aminobenzamide (5 mg), NaBH3CN (7.5 mg), DMSO (500 μL) and acetic acid (200 μL) was added to the glycans released from glycoprotein. After the incubation at 65 °C for 2.5 h, 2-AB derivatives were purified with GlycoClean™ cartridges according to the protocol provided by the manufacturer. The glycan derivatives were then dissolved in 10 μL water and subjected to MALDI analysis. MALDI-TOF mass spectrometry Mass spectrometric analyses were performed using a Ultraflex TOF/TOF (Bruker Daltonics, Bremen, Germany) mass spectrometer equipped with a LIFT™ MS/MS facility. DHB (80 mg/mL in 30% acetonitrile) and super-DHB (10 mg/mL in 50% acetonitrile) were used as matrices. 0.5 μL of the matrix was applied to the spot. Subsequently, 0.5 μL of derivatized N-glycans was spotted onto the MALDI target plate. After air-drying at room temperature, the MALDI target was introduced into the ion source. Spectra were acquired in positive ion reflectron mode using the Flex Control software. Following settings were used for MS (shots, 50; frequency, 50; laser power, 50–80%, scan range, m/z 200–3200) and MS/MS (shots, 200; frequency, 50; laser power, 100–150%, scan range, m/z 200–3200, window range, 16 Da). MS/MS experiments were performed under laser-induced dissociation. The mass spectra were obtained from the recorded raw data using Flex Analysis software. External calibration was carried out using singly charged monoisotopic peaks of angiotensin II, substance P and ACTH 18–39 fragment. Graphics for the mass spectra were created with GlycoWorkbench Software version 1.3.[27] Lectin-binding assay with BINH derivatives BINH-derivatized glycans of ribonuclease B (released from 1 mg of glycoprotein) were diluted in 50 μL of sample buffer (20 mM TRIS, pH 7.4, containing: 150 mM NaCl, 0.05% Tween 20, 1% BSA) and 0.5 μL were applied to the streptavidin-coupled glass slide which was washed before with Millipore® water. After 1 h incubation, the slide was washed again with water and blocked for 2 h with blocking-buffer (100 mM TRIS, pH 9, containing: 150 mM NaCl, 0.05% Tween 20, 0.5% BSA, 1% biotin). Subsequently,



MS of N-glycans derivatized with biotinylated isonicotinic hydrazide the microslide was washed with saline-sodium-citrate buffer (15 mM NaCl, 1.5 mM sodium citrate, pH 7) and three times with water. 40 μL of lectin (ConA labelled with Alexa 647) was added to the slide and again incubated for 1 h in the dark. Then, the glass slide was washed in three steps with PBST (pH 7.4) with Tween 20 (containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.1% Tween 20), PBS pH 7.4 (containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) and water. Afterwards, the slide was spin dried and the emission of the slide was recorded in a fluorescent scanner at 670 nm. The same lectin-binding assay with mannose-BINH and lactose-BINH was followed. Three different concentrations (10 μg/mL, 25 μg/mL, and 75 μg/mL) were analyzed.

Figure 1. Structures of labels used in this study: isoniazid (INH), biotinylated isoniazid (BINH), biotinamidocaproylhydrazide (BACH), and 2-aminobenzamide (2-AB).

RESULTS AND DISCUSSION N-Glycans released from ribonuclease B and hen ovalbumin were labeled with INH and BINH. A derivatization reaction occurs between the reducing terminal sugar and the hydrazide of the label (Scheme 1). Derivatized carbohydrates were analyzed by means of MALDI-TOF and MALDI-TOF/ TOF MS. Profile spectra and fragmentation behaviors of biotinamidocaproylhydrazide (BACH), 2-AB, INH and BINH derivatives were compared with those of the underivatized counterpart (Fig. 1). BACH as label for derivatization of carbohydrates in mass spectrometry was presented and investigated in our previous work.[10] Here, more in-depth MS/MS analysis on glycans was performed. 2-AB was chosen for comparison studies as this is up to now one of the most used labels for derivatization of oligosaccharides. MALDI-MS of N-glycans Glycans enzymatically released from ovalbumin and ribonuclease B were derivatized with INH, BINH, BACH and 2-AB and compared with the underivatized glycan pool. The glycans formed sodiated molecular ions. Resulting profiles for ovalbumin are shown in Figs. 2 and 3. In the case of underivatized glycans (Fig. 2), 22 out of 26 known ovalbumin glycans could have been registered. The highest glycan belonging to the ovalbumin was the one with m/z 2028.8. The other higher glycans originate from the usual co-isolates of the ovalbumin, as investigated by Harvey et al.[28] As can be seen, derivatized ovalbumin glycans showed a higher signal intensity than the native ones, especially the high-mass glycans. For example, the complex

glycan with m/z 2313.9 (Fig. 2) was observed after derivatization with significantly higher intensity. For comparison, see the spectra after derivatization in Fig. 3 (INH: m/z 2432.9; BINH: m/z 2775.4; BACH: m/z 2667.4; 2AB: m/z 2434.0). Moreover, three additional glycan structures could have been detected after derivatization: two complex and one hybrid glycan. These are highlighted in the Fig. 3 with an asterisk. Also signal intensities of the MS spectra of ribonuclease B high-mannose derivatives were enhanced when compared with the native ones (rel. intensity of underivatized glycans: 5 × 106, INH and 2-AB: 7 × 106, BINH and BACH: 8 × 106). Especially the glycans with higher number of mannose units (7, 8 and 9) profited from the derivatization and gave up to three times higher signals in comparison with their native counterparts (data not shown). The strongest effect in the enhancement of the signal intensity was observed with INH and BINH derivatization of the 9-mannose glycan. MS/MS of high-mannose glycans The MS/MS spectrum of the native high-mannose glycan Man6GlcNAc2 ([M+Na]+; m/z 1419.5) exhibited mainly Band Y-ions and some cross-ring fragments (Fig. 4). The cleavages were assigned according to the nomenclature of Domon and Costello.[29] The high-mannose character of this glycan was reflected in the presence of the Y-ions formed by loss of mannose residues from the molecular ion. Losses of one (Y4, m/z 1257.4), two (Y3ß, m/z 1095.4), three (Y3α, m/z 933.3) or six mannose residues (Y2, m/z 447.1), but not four




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Scheme 1. Reaction of the reducing end of the Man5GlcNAc2 glycan with the isoniazid tag.

S. Bank et al.

Figure 2. MALDI mass spectrum of underivatized glycans released from chicken ovalbumin.

Figure 3. MALDI mass spectra of INH-, BINH-, BACH- and 2-AB-derivatized glycan pool of ovalbumin.

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or five, were observed. Prominent ions were also B-ions involving loss of one (B4, m/z 1198.5) or two Nacetylglucosamines (B3, m/z 995.2) from the reducing end of the glycan. Other ions (internal fragments) arising from combination of B- and Y-type cleavages were observed (e.g. m/z 1036.3, 874.3, 712.2, 550.2, 833.3). Fragmentation of this glycan shows relatively high abundance of the internal B3/Y3 fragment ion (D-ion, m/z 671.2), which results from loss of two mannose residues from the 3-antenna of the B3-ion. Thus, this reflects the distribution pattern of the mannose residues between the antennae referring to the presence of


the three mannose residues in the 6-antenna. This pattern of fragments is consistent with observations of Harvey[30] and Rouse et al.[31] for this type of glycans. Some cross-ring cleavages in the core GlcNAcs were observed (3,5A4, 3,5A5); however, they were not diagnostically useful. The MS/MS spectrum (Fig. 5) of the INH-derivatized Man6GlcNAc2 glycan ([M+Na]+; m/z 1538.6) showed a similar pattern of glycosidic cleavages as the native glycan. Loss of one (Y4, m/z 1376.4), two (Y3ß, m/z 1214.5), three (Y3α, m/z 1052.4) and six (Y2, 566.3) mannose residues was observed, whereas losses of four and five mannose residues were



MS of N-glycans derivatized with biotinylated isonicotinic hydrazide

Figure 4. MALDI-MS/MS spectrum of underivatized Man6GlcNAc2 glycan released from ribonuclease B. Symbols used for the structural formulae: blackfilled squares (■) GlcNAc; gray-filled circles ( ) mannose. Arrows with continuous and dashed lines indicate mass differences representing mannose (162 u) or N-acetylglucosamine (203 u) residue, respectively, and do not necessarily imply the fragmentation pathway. Short arrows indicate loss of water.


with the B3-ion at m/z 671.2 is the D-ion and defines the composition of the 6-antenna. As can be seen, with BINH derivatization the number of cross-ring cleavages distinctly increased. The most dominant signal of the spectrum besides the peak involved with the BINH label (m/z 486.3) was the cross-ring cleavage of the chitobiose GlcNAc (0,2A5) at m/z 1318.4. Further cleavages of the terminal GlcNAcs were relatively weak but were present at m/z 1329.5 (1,4A5), 1272.4 (3,5A5), 1258.5 (2,4A5), 1170.4 (1,5A4), 1115.4 (0,2A4), 630.2 (3,5X0) and 530.2 (1,5X0). Diagnostically helpful cross-ring fragments of the core mannose appeared at m/z 953.3 (0,2A3) and 583.2 (3,5A3), whereas the latter one indicates the presence of three mannoses on the 6’-antennae and had higher intensity than the fragment of the non-biotinylated INH derivative. Two cross-ring fragments of the antennae were registered at m/z 1644.6 (3,5X3) and 1436.6 (0,2X3). The spectrum of the BACH derivative of Man6GlcNAc2 is also shown in Fig. 5. Most of the fragment ions were common to the spectrum of the underivatized glycan and of the INH derivative, whereas the intensity of the signals was comparable with INH derivatization, i.e. the abundance of the spectrum was higher than that of the native glycan. Glycosidic cleavages from the molecular ion ([M+Na]+, m/z 1773.0) were observed after the loss of one, two, three and six mannose residues. Further fragment ions were produced by the loss of the mannose from the B3- (m/z 995.3) or B4-ion (m/z 1198.3). The B3-ion (m/z 995.3) and the D-ion (m/z 671.2) with its water loss were easy detected. Beside glycosidic fragments, some cross-ring cleavages of the A- and X-type were present. Cleavage of the chitobiose core gave ions such as 3,5A4, m/z 1069.4; 0,2A4, m/z 1115.3; 1,5A4, m/z 1170.4; 2,4A5, m/z 1258.5; 3,5A5, m/z 1272.5; 0,2A5, m/z 1318.4; 0,2X0, m/z



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missing in the spectrum. The presence of B-ions (B3, B4) and B/Y internal fragments was identical to the spectrum of the native counterpart. However, the signals of the INH derivative were much more abundant. The loss of 3positioned mannoses from the B3-ion appeared as the D-ion at m/z 671.2, which was accompanied by a loss of water (B3/Z3ß). This ion defines composition of the 6-antenna, which consists of three mannoses. Cross-ring cleavages 0,2A5 at m/z 1318.5 (with highest abundance in the spectrum) and 2,4 A5 at m/z 1258.4 were consistent with β-1-4 linkage of the chitobiose core. Cross-ring cleavage ions of rather weak abundance were those from the core mannose residue (3,5A3, m/z 583.3; 2,4X2, m/z 1154.4). The ion 3,5A3 conveys information about the branching pattern of the antennae. Here, it confirms the aforementioned interpretation that the antenna consisting of three mannose residues is the one at the 6’-position. The MALDI-TOF/TOF spectrum of the BINH derivative of Man6GlcNAc2 shows more fragmentation than the native and the INH-derivatized glycan (Fig. 5). Both, glycosidic and cross-ring cleavages were observed. Here, the complete loss of mannose residues from the antennae could be registered: m/z 1719.1, 1556.9, 1394.9, 1232.8, 1070.8, 908.7, i.e. inclusive the losses of four and five mannoses, which on the contrary were not observed with the native glycan and its INH derivative. Similar behavior was recorded with the analysis of the ABDEAE (4-aminobenzoic acid 2-(diethylamino)ethyl ester) derivatized high-mannose glycans,[32] where the fragmentation spectra contained full series of Y-ions. A prominent B4-ion was present at m/z 1198.5 and fragmented further by losses of mannose residue(s) to give ions at m/z 1036.3, 874.3, 712.2, 550.2 and 388.1. B/Y-cleavage involved

S. Bank et al.

Figure 5. MALDI-MS/MS spectra of INH-, BINH-, BACH- and 2-AB-derivatized Man6GlcNAc2 glycan released from ribonuclease B. For key to monosaccharide symbols and arrows, see legend to Fig. 4.

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477.2 with the latter two being the most dominant of the spectrum. Cross-ring fragments of the core mannose were detected at m/z 407.2, 2,4A3; m/z 583.2, 3,5A3; and m/z 950.3, 0,2 X2. On the basis of the two A-fragments of the core mannose, the branching pattern at the two branch points could be determined (3-antenna, two mannose residues; 6antenna, 3 mannose residues). The difference between the m/z of the 0,2X2-ion and the sodiated molecular ion (less two GlcNAc) could confirm the number of mannose residues present. The mass spectrum of the [M+Na]+ ion from the 2-AB derivative of Man6GlcNAc2 (Fig. 5) shows some differences in comparison to the previous spectra. Here, a smaller amount of cross-ring cleavages was detected with in part also significantly less abundant intensity. In contrast with the BACH spectrum, where the prominent ions originated from cross-ring cleavages, the major ions in the 2-AB spectrum were products of glycosidic cleavages. Thus, the ion 0,2A5 could not have been detected with 2-AB at all. This might be due to the opened terminal sugar ring because of the reducing step in the derivatization procedure. Also other fragments of the terminal GlcNAc could not have been registered. Some weak signals were observed for the second


GlcNAc of the chitobiose core (counting from the reducing terminus) at m/z 1069.1 (3,5A4), 1099.1 (2,5A4), and 1115.6 (0,2A4). Cleavages of the core mannose were detected at m/z 583.3 (3,5A3) and 599.3 (0,3A3), which led to the loss of the 6antenna. Some X-type fragments with rather lower information content were present at m/z 447 (0,2X1) and 1317 (2,4X3). As mentioned previously, mainly glycosidic cleavages were present in the spectrum of the 2-AB derivative and occurred with full coverage of the expected Y-fragments (m/z 1377.5, 1215.4, 1053.4, 891.3, 729.3, 567.2) and B- and of B/Y-fragments (B4, m/z 1198.4; 1036.3; 874.3; 712.2; 550.2; 388.1; B3, m/z 995.3; 833.3; 671.2). The highest intensity was shown by the B4-ion (m/z 1198.4) and the Y2-ion (m/z 567.2). The B3-ion (m/z 995.3) and the D-ion (m/z 671.2) were also relatively intense. However, the overall intensity of the 2-AB derivative fragment spectrum was lower than that of the hydrazide derivatives (INH, BINH and BACH). Looking at the all MALDI-TOF/TOF spectra of the investigated high-mannose glycan Man6GlcNAc2, it could be said that the derivatization brought significant improvement in the fragmentation in terms of the generation of informative fragments. With underivatized glycan, the intensity of the spectrum was rather low and mostly



MS of N-glycans derivatized with biotinylated isonicotinic hydrazide glycosidic fragments were observed. However, the D-, D–H2O- and B3-ions were still present. After derivatization with INH and BACH the intensity of the spectrum was enhanced and cross-ring cleavages of the core mannose (e.g. 3,5 A3 with INH and BACH, 2,4A3 with BACH) were registered beside glycosidic fragmentation, which provided helpful information about the assignment of the antennae. In the spectrum of BINH, 3,5A3 and 0,2A3 appeared besides other various cross-ring fragments. Moreover, glycosidic fragments involved with loss of four or five mannose residues, which occur very rarely with native glycans,[30] were detected in the spectra of the BINH and 2-AB derivatives. After all, spectra of biotinylated derivatives (BINH and BACH) showed the highest number of cross-ring cleavages both peripheral and of the core mannose. Indeed, on the basis of the spectrum of the native glycan it was possible to resolve the structure of

the glycan. After hydrazine derivatization, however, the abundance of the spectrum increased favorably and a higher number of diagnostically significant cross-ring cleavages appeared in the spectra of the derivatizatized counterparts. MS/MS of complex glycans Native and derivatized N-glycans of ovalbumin (labeled with INH, BINH, BACH and 2-AB) were subjected to MALDITOF/TOF fragmentation experiments. Special attention has been given to the task, how the derivatization influences the occurrence and the intensity of the isomer specific fragments in the spectra of the single derivatives. In the spectra of the complex biantennary bisecting glycan Man3GlcNAc6 (MW 1722.5), the presence of three isomers is expected (Table 1). Cleavages characteristic only for one of the isomers were

Table 1. Core mannose cross-ring fragments of type A for single isomeric glycans of Man3GlcNAc6



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Specific ions belonging to only one of the isomers are highlighted in gray.


S. Bank et al. depicted in the spectra with pictogram and the number (I, II or III) of the isomer. Unspecific fragments (common to more than one isomer) are annotated with pictogram of the isomer II. Figure 6 shows the underivatized glycan Man3GlcNAc6. Dominant cleavages were glycosidic fragments. The most intensive ions were generated through the loss of one terminal N-acetylglucosamine (m/z 1542.6) and through B4and B3-cleavages (m/z 1524.6 and 1321.5). Fragmentation of the glycan could be observed both from the molecular ion and the B3-fragment. For all three isomers the corresponding D-ion (m/z 753.3 (I), m/z 956.3 (II), m/z 1159.5 (III)) could be detected. The presence of bisecting GlcNAc could be confirmed in two isomers, where the loss of 221 u from the B3/Y3-ion appeared at m/z 532.2 (I) and m/z 735.3 (II). A specific fragment for isomer III appeared at m/z 1583.6. This one originates from elimination of one mannose which can be consistent only with the isomer III. Some other cross-ring cleavages were observed (A4-, X3- and X4-ions); however, these were not of high diagnostic significance. Derivatization with INH considerably improved the signal intensity of the spectrum and the number of glycosidic and cross-ring cleavages rose (Fig. 7). Dominant signals belonged to loss of one GlcNAc (m/z 1661.9), B4-cleavage (m/z 1524.5) and B3-ion (m/z 1321.5). In contrast to underivatized glycan, also a prominent cross-ring cleavage in the chitobiose core (0,2A5, m/z 1644.4) was observed. With the INH derivative, similarly to what was observed with glycosidic fragmentation of the high-mannose glycan Man6GlcNAc2, fragments with one and two mannoses on the chitobiose core were missing. Specific cleavages of isomer III were observed at m/z 1702.9 (Y4, loss of one mannose) and B2-cleavage of the 6-antenna (m/z 794.3). D-ions of all three isomers were detected at m/z 753.2 (I), 956.3 (II), and 1159.3 (III) and thus composition of the 6-antenna for each of the isomers was recognizable. The

losses of 221 mass units (loss of bisecting GlcNAc) from D-ions gave rise to corresponding Z-ions with m/z 532 (isomer I), m/z 735 (isomer II), and m/z 938 (isomer III). Further cross-ring cleavages were involved with GlcNAcs of the chitobiose core: m/z 1381.5 (2,4A4), m/z 1395.5 (3,5A4), m/z 1441.6 (0,2A4), m/z 1584.7 (2,4A5), m/z 1598.7 (3,5A5) or with the core mannose. Here, an X-cleavage at m/z 1236.5 was registered, which can be attributed to more than one fragmentation option: 1,3X2 (isomer I), 2,4X2 or 0,4X2 (isomer II). Further, A-fragments of the core mannose appeared at m/z 868.2 (3,5A3) and 1279.5 (0,2A3), whereas the first is very interesting, as this one can derive only from isomer II. Similar fragmentation regarding glycosidic cleavages was observed (Fig. 7) with the biotinylated derivative of INH (BINH). The spectrum was dominated by losses of GlcNAc residues from the B4-, B3- and the molecular ion. Besides diagnostic D-ions of the three isomers (m/z 753.3 (I), m/z 956.3 (II), m/z 1159.4 (III)), the loss of 221 mass units for all of the isomers was observed, which provided the confirmation of the existence of the bisecting GlcNAc residue in the glycan. Further, Y-cleavage of one terminal mannose residue yielded for isomer III specific fragment at m/z 2045.0 and B2α-cleavage of the 6-antenna the ion at m/z 794.4. Specific A-fragments of single isomers were those from the core mannose (3,5A3): m/z 665.2 (I), m/z 868.4 (II), and m/z 1071.1 (III). In addition to A-ions, X-fragments (3,5X2) of the core mannose for isomers I and II were detected at m/z 1375.6 and 1361.6, respectively. Generally, more cross-ring cleavages were observed in the case of BINH than with the INH derivative. The fragmentation spectrum of the BACH-derivatized Man3GlcNAc6 glycan (Fig. 7) showed some differences in terms of the glycosidic cleavages when compared with its INH and BINH counterparts. Glycosidic fragments with one and two mannoses on the chitobiose core (m/z 963.5 and 1124.8) appeared in the spectrum, whereas a gap arose

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Figure 6. MALDI-MS/MS spectrum of underivatized Man3GlcNAc6 glycan released from hen ovalbumin. Symbols used for the structural formulae: black-filled squares (■) GlcNAc; gray-filled circles ( ) mannose. Arrows with continuous lines and dashed lines indicate differences in the mass of the mannose or N-acetylglucosamine residue, respectively.




MS of N-glycans derivatized with biotinylated isonicotinic hydrazide

Figure 7. MALDI-MS/MS spectra of INH-, BINH-, BACH- and 2-AB-derivatized Man3GlcNAc6 glycan released from hen ovalbumin. For key to monosaccharide symbols and arrows, see legend to Fig. 4.


The spectrum of the Man3GlcNAc6-2-AB derivative (Fig. 7) had similar fragmentation features with the spectrum of the underivatized counterpart. All three isomer-specific D-ions were present in the spectrum, but the D-221 shift appeared only with isomers I and II. From the isomer III specific fragments, only the Y-ion at m/z 1703.6 with relatively low intensity was observed. The second possible fragment indicating the composition of the 6-antenna of isomer III was not detected. In the range of cross-ring cleavages of the chitobiose core, only a small number of fragments was found (m/z 1381.5, 3,5A4; m/z 1441.5, 0,2A4; m/z 1615.6, 3,5A5). A specific fragment of isomer II involved with the core mannose (3,5A3) was recorded at m/z 868.3. X-cleavage of the core mannose (m/z 1237.4) could originate either from isomer I (1,3X2) or isomer II (2,4X2, 0,4X2). Other isomer-specific fragments did not appear. Glycosidic fragmentation of this derivative resembled that of the INH derivative. Similarly, fragments B3 (m/z 1321.5), B4 (1524.5) and Y4 (m/z 1662.6) belonged to the intensive signals of the available spectrum. However, the dominant peak of the spectrum was the one at m/z 1820.8, which resulted from the cleavage in the 2-AB reagent. After investigation of the MS/MS spectra of the Man3GlcNAc6 glycan derivatives, it can be concluded that derivatization brought a significant improvement in terms



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in the spectrum by reason of missing fragments expected at m/z 1286 and 1327. The fragmentation from the B3-ion (m/z 1321.5) proceeded completely as observed with the previous derivatives. The presence of the bisecting GlcNAc was reflected by the presence of D-ions for each of the three isomers and their further fragmentation by loss of 221 mass units corresponding to ion D-GlcNAc. However, the specific ions of isomer III (Y-cleavage of one mannose and detachment of the 6-antenna) were not detectable in this spectrum as it was with INH and BINH derivatives. The cross-ring fragment 0,2A5 (m/z 1644.6) had here as in spectra before the highest signal intensity. Apart from that, various isomeric unspecific cross-ring fragments involved with the chitobiose core were observed in the spectrum (2,4A4, m/z 1381.5; 3,5A4, m/z 1395.6; 0,2A4, m/z 1441.6; 2,4 A5, m/z 1584.7; 3,5A5, m/z 1598.7) or with the core mannose (m/z 1237.5, 1,4X2 (I) or 0,3X2 (II); m/z 1263.4, 2,5 A3; m/z 1279.5, 0,2A3). Isomer-specific cleavages of the 3,5 A3 fragment of the core mannose occurred only with isomer II (m/z 868.4) and isomer III (m/z 1071.5). They were of rather low intensity but still in evidence. X-ions complementary to 3,5A3 fragments which in part were available with the BINH derivative were after BACH derivatization not detectable.

S. Bank et al.

Figure 8. (A) Schematic representation of the lectin-binding assay. (B) Visualization of the fluorescence signal after reaction of (I) BINH-labeled glycans from ribonuclease B and (II) mannose-BINH (25 μL/mL) with concanavalin A, (III) background.

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of the detection and generation of certain specific fragments. Whereas the fragmentation of the native glycan brought only D-ions of the isomers and the isomer III specific loss of one mannose, in the spectrum of the INH derivative additional cross-ring cleavage of isomer II (3,5A3) and the B2α fragment of isomer III were recognizable. The number of unspecific cleavages rose as well. BACH derivatization brought all three D-ions, 3,5A3-cross-ring fragments which pointed to isomers II and III and some other ring fragments of the core mannose. The spectrum of the 2-AB derivative showed D-ions, core mannose cleavage specific for isomer II (3,5A3) and the glycosidic cleavage Y4β referring to isomer III. The best result was due to derivatization with BINH. Although the intensity of the spectrum was relatively low, all necessary fragments (D-ions, 3,5A3-ions; Y4β and B2α of isomer III), which are important for the identification of the three isomers of the glycan Man3GlcNAc6, appeared in the spectrum. This glycan was analyzed in its native form also by Harvey[30] However, on the basis of the D-ions, only one of the expected isomers could have been identified. Another fragmentation analysis of the phenylhydrazine (PHN), 2-AB and 1-phenyl3-methyl-5-pyrazolone derivatives of this complex glycan[11,33] showed that the 2-AB derivative was the one with the highest spectral intensity, but disclosed the least number of cross-ring cleavages. In that study, the PHN derivative was the one which provided the most meaningful fragments for elucidation of the structure of the isomers present. If compared with PHN derivatives, derivatization with BINH delivered higher numbers of specific fragments characterizing the single isomers of the analyzed complex bisecting glycan. Beside all three D-ions with 221 u loss and B2α of isomer III, also Y4 of isomer III and all 3,5A3-ions of the core mannose were observed, although with relatively low intensity. According to Devakumar et al.,[34] permethylation analysis in combination with photodissociation provides also valuable structural information. However, derivatization of the carbonyl group at the reducing terminal of a sugar is advantageous compared to the permethylation of hydroxyl groups because it is an easy and fast option of a derivatization, with minimal amounts of starting material.[19] Also, with the presence of the intact pyranose ring at the reducing end of the carbohydrate derivative, it would be


possible, if desired, to regenerate the free oligosaccharides under mild acidic conditions.[21,35] A favorable aspect of using BINH is that this label is biotinylated and provides the possibility to use its derivatives both in glycan–protein interaction studies and structural analyses. Lectin interactions with BINH-labeled glycans It is known that the capability of carbohydrate derivatives to interact with lectins or other carbohydrate-binding proteins depends on the label.[15,21] Experiments with the trimannosyl core showed that Man3 elicited a binding signal with ConA when it was presented as the BNAH (biotinyl-L-3-(2-naphthyl)alanine hydrazide) but not the BAP (2-amino-6-amido-biotinylpyridine) derivative.[21] In this regard, the capability of BINH derivatives to interact with lectins was investigated. BINH derivatized high-mannose glycans released from ribonuclease B and saccharides mannose and lactose were reacted with streptavidin-coated glass slides (PolyAn©) and probed with Alexa Fluor 647-conjugated ConA (Fig. 8). ConA recognizes internal and non-reducing terminal α-D-mannosyl groups and to a certain extent also glucose. In the binding experiments, high-mannose glycans of ribonuclease B and the BINH-labelled mannose showed binding to ConA, whereas lactose-BINH elicited no signal. Differently from observations with biotinylated derivatives BNAH and BAP,[21] where the minimum structure bound to ConA was Man3, binding of one mannose residue (e.g. BINH-mannose) to ConA was observed in our study. Thus, the initial lectin-binding experiments performed herein showed that BINH-labelled carbohydrates are recognized by plant lectins of corresponding specificity and have the potential to be used for lectin-carbohydrate studies.

CONCLUSIONS Here we present a comparative MALDI tandem mass spectrometric study on carbohydrates derivatized by hydrazine labels (INH, BINH, BACH), the amine-containing label 2-AB and non-derivatized counterparts. INH and BINH are new tags used for the first time.



MS of N-glycans derivatized with biotinylated isonicotinic hydrazide Generally, spectra of hydrazine derivatives displayed beside Y- and B-type glycosidic cleavages specific fragment ions, e.g. cross-ring ions, revealing more structural information on glycans than the mainly glycosidic cleavage products that dominated the spectra of native glycans and 2-AB derivatives. Thus, hydrazine derivatization provided structurally informative fragment ions without the need for a considerably complex derivatization procedure such as permethylation. BINH derivatives provided the most detailed information on the structure of the investigated glycans. MS/MS spectra of these derivatives showed cross-ring cleavages providing useful structural information also in the case of isomeric relatives. As the BINH label incorporates biotin, it bears also bioaffinity and, thus, the potential to be used in interaction studies. In this context, successful binding of BINH-derivatized ribonuclease B glycans and mannose (BINH-mannose) to ConA was observed. These initial lectin-binding experiments with concanavalin A indicated that BINH derivatives would be probes for exploring the N-glycan binding specificities of novel carbohydrate-binding proteins.

Acknowledgements This work was supported by the Fond der Chemischen Industrie and the Universitätsbund Würzburg. Prof. Dr. Schlosser and Stephanie Lamer (Rudolf Virchow Center, University of Würzburg) are acknowledged for permitting the use of the Ultraflex MALDI mass spectrometer and technical support during the measurements.

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