Capillary Electrophoresis Mass Spectrometry for the Characterization

Article pubs.acs.org/ac

Capillary Electrophoresis Mass Spectrometry for the Characterization of O‑Acetylated N‑Glycans from Fish Serum Roxana G. Jayo,† Jianjun Li,‡ and David D. Y. Chen*,† †

Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada Human Health Therapeutics, National Research Council Canada, Ontario, Canada

‡

S Supporting Information *

ABSTRACT: O-Acetylated N-glycans from fish serum of Atlantic salmon (Salmo salar) are characterized by capillary electrophoresis (CE) in conjunction with both laser-induced fluorescence (LIF) and mass spectrometry (MS) detection methods. Glycans derivatized with negatively charged fluorescent label 8-aminopyrene-1,2,6-trisulfonate (APTS) were separated to obtain a CE-LIF profile of the complex glycan mixture, and the profile concurs with that obtained by using electrospray mass spectrometry. The identity of the APTS-labeled glycans was confirmed by CE−MS. The same glycans can be identified also in their native state by CE−MS without derivatization. The structural variations of O-acetylated sialic acid isomers in fish serum glycans are investigated by CE− MS/MS. Selected ion monitoring provided useful structural information of the underivatized glycans from fragmentation spectra. New complex fish serum glycans that are not reported previously were observed and characterized. These methods may be useful not only for the characterization of acetylation of complex glycans but also to study other types of glycan modifications, as well as to allow determination of overall glycan composition in glycoproteins.

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of neuramidase action,13,14 bacterial antigenicity,15 binding of influenza C viruses to cells,16,17 modulation of the alternative pathway of complement activation,18 and cell−cell interactions.10,13 The most abundant sources of O-acetylated Neu5Ac are bovine submandibular gland mucin and human colon mucosa.19 O-Acetylation of Neu5Ac has also been identified on certain salivary mucins,11 neural gangliosides,20 rat liver membranes,21 cysteine proteinase inhibitors from Atlantic salmon skin,22 and kininogens from spotted wolffish and Atlantic cod.23 Recently, Liu et al.24 investigated the change of O-acetylated sialic acids in complex N-glycans in the sera of Atlantic salmon (Salmo salar) as a response to varying periods of long-term handling stress. However, structural details were difficult to elucidate because of the possible presence of isomers.24 Most commonly used analytical methods require releasing the sialic acids from glycoconjugates by enzymatic or chemical hydrolysis, followed by a combination of reduction and fluorescence labeling with 1,2-diamino-4,5-methylenedioxybenzene (DMB) and subsequent separation by reversed-phase high-performance liquid chromatography (RP-HPLC) with fluorescence or mass spectrometric (MS) detection.25,26 However, serious limitations have been encountered when Oacetylated sialic acids are analyzed. Problems associated include incomplete release of O-acetylated sialic acids from the

rotein N-glycosylation is the most common post-translational modification in eukaryotes and bacterial glycoproteins.1,2 In this type of glycosylation, the oligosaccharides, having a core pentasaccharide structure composed of GlcNAcβ1−4GlcNAcβ1−4Manα1−3Man(α1−6Man), are linked to the protein backbone via the amide nitrogen of Asn side chains in a tripeptide recognition site Asn-X-Ser/Thr, where X is any amino acid residue except for Pro or Asp.3 N-Linked oligosaccharides are classified into high-mannose, complex, and hybrid type, depending on the antennas attached to the external mannose residues of the common pentasaccharide core.4,5 The composition and structure of the carbohydrate moieties at the glycosylation sites have many important roles in defining the biological and biophysical properties of the proteins.4−8 The family of sialic acids includes over 40 naturally occurring, nine-carbon carboxylated sugars frequently found as the terminal units of glycoproteins and glycolipids.4,5 The two most dominant species are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is the most diversely substituted sugar known in nature.9,10 One of the better studied modifications of Neu5Ac is the addition of Oacetyl esters to hydroxyl groups at the 4, 7, 8, and/or 9 positions, giving rise to a great variety of possible compounds and isomers.5,10,11 O-Acetylation of Neu5Ac is highly tissueand species-specific and is perhaps the most common modification found on sialic acids due to its involvement in a growing number of biological and pathophysiological phenomena.10,12 Typical examples include the partial or complete block © 2012 American Chemical Society

Received: July 20, 2012 Accepted: September 12, 2012 Published: September 12, 2012 8756

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Sample Preparation. N-Glycans present in serum of juvenile Atlantic salmon (S. salar) were obtained from two groups of fish: control and stress. Fish in the stress group were subjected to daily long-term handling stress (15 s out of water) for a period of 1−4 weeks, as described previously.24 Dried glycan samples were stored at −20 °C. The dried samples were resuspended in 100 μL of purified water before labeling with APTS. APTS Labeling. N-Glycans of Atlantic salmon from the control group of fish sera were derivatized with APTS by reductive amination via Schiff base formation according to the manufacturer’s instructions for the glucose ladder. Hereto, 50 μL of fish serum glycan solution was dried down in a SpeedVac vacuum centrifuge (Eppendorf, Germany). To the dried glycan, 2 μL of 0.2 M APTS diluted in 15% acetic acid and 2 μL of 1 M sodium cyanoborohydride/THF were added. The resulting solution was vortexed and incubated overnight at room temperature in the dark. Following incubation, the excess APTS reagent was removed by precipitating the labeled glycans with ice-cold acetone and centrifuging at 12 000 rpm for 15 min. After removing the supernatant, the APTS-labeled glycan pellet was washed with additional ice-cold acetone and reconstituted in 100 μL of distilled water. This sample solution was used directly for all CE-LIF and CE−MS analysis. Capillary Electrophoresis. Capillary electrophoretic analyses of the APTS-labeled N-glycans were carried out with a Beckman Coulter P/ACE MDQ system (Beckman Coulter Inc., Fullerton, CA) equipped with a laser-induced fluorescence detection module. The argon ion laser was operating at 488 nm for excitation, and emission was monitored at 520 nm, which provides optimal sensitivity for APTS-labeled N-glycans. Separations were performed using neutral, hydrophilic N− CHO-type capillaries (65 or 50.2 cm total length × 40 cm length to detector × 50 μm i.d. × 360 μm o.d.) obtained from Beckman Coulter Inc. (Brea, CA). Separations were carried out in reverse-polarity mode. Background electrolytes (BGEs) were composed of 0.2−2.0% formic acid or acetic acid, 10−50 mM ammonium acetate, ammonium formate, or 25−50 mM εaminocaproic acid, as specified in the Results and Discussion section. The APTS-labeled N-glycans were injected for 10 s at a pressure of 1 psi, which corresponds to a volume of 24 nL. Mass Spectrometry. CE−MS analysis were performed using a Finnigan LCQ* Duo ion trap mass spectrometer (Thermo Scientific, Waltham, MA) operating in the negative ion mode. The CE−ESI-MS interface with a flow-through microvial was developed in our laboratory and has been described previously.37−40 Electrospray voltage was set at −3.5 kV, and the temperature of the heated capillary at the MS inlet was set at 200 °C. The detector scan range was 900−1300 m/z. The trap injection time was set at 50 ms. The MS scanning parameters were optimized using the “Autotune” function of the LCQ Xcaliber software (Thermo Fisher Scientific, Waltham, MA) by continuous infusion of APTS-labeled glucose ladder standard. The modifier solution was delivered by a syringe pump (Harvard Apparatus, Holliston, MA) at a flow rate of 0.3 μL/min and was composed of 10 mM ammonium acetate (pH 3.17) containing 75% 2-propanol and methanol (2:1 ratio). The samples were injected at 1.0 psi for 10 s, corresponding to a total volume of 19 nL. The same interface setup and solutions were used for CE− ESI-MS/MS with a Prince CE system (Prince Technologies, The Netherlands) coupled to an API 3000 triple-quadrupole mass spectrometer (Applied Biosystems/Sciex, Concord,

glycosidic linkages, poor derivatization of some species, de-Oacetylation and migration of O-acetyl groups, and complete destruction of chemically unstable substituted sialic acids during isolation and purification of the glycoconjugates. Furthermore, even if O-acetylated sialic acids could survive the isolation and purification steps, the linkage information to define the position of each substituent in the glycan chain is lost.17,19,27 Therefore, new methods that allow the detection of O-acetylated sialic acids without the liberation of glycans are of great interest. In recent years, profiling of intact glycans containing O-acetyl sialic acids has also become increasingly important for complete characterization of acidic glycoproteins. However, due to the structural variability and lability of O-acetylated N-glycans at the glycosylation sites of glycoconjugates, their analysis remains a challenging task. Capillary electrophoresis (CE) has shown superior capability in separating positional and linkage isomers.28,29 Typically, Nglycans enzymatically released from glycoproteins using peptide-N-glycosidase F (PNGase F) are labeled and characterized by CE with laser-induced fluorescence (LIF) detection.30,31 A frequently used labeling reagent is 8aminopyrene-1,2,6-trisulfonate (APTS) due to its high fluorescent yield and its ability to remain ionized over a wide pH range.32−35 Although CE-LIF allows glycoprofiling, identification of new glycans is not straightforward. Combining the resolving power of CE with the high sensitivity and information-rich MS detection could be a powerful tool for glycan analysis. Isomer identification and structure determination for glycans can be achieved simultaneously by CE−ESIMS/MS. Because O-acetylated N-glycans from fish serum are highly sialylated and remain negatively charged over a broad pH range, they can be CE-separated and MS-detected in negative electrospray ionization (ESI) mode without the need for derivatization. There are only a few reports in the literature dealing with the CE−MS of intact glycans containing Oacetylated sialic acids.24,36 However, comprehensive glycan analyses down to the level of isomeric differentiation for the characterization of native oligosaccharides have not been achieved. In this study, a combined approach for the analysis of APTS-derivatized and underivatized O-acetylated N-glycans enzymatically released from fish serum of Atlantic salmon (S. salar) is developed. CE-LIF was used for the separation of APTS-labeled O-acetylated N-glycans under acidic conditions. The results were compared with those obtained by CE−ESIMS of the derivatized and underivatized glycans for online mass and composition determination. Furthermore, CE−ESI-MS/ MS was used to confirm the O-acetylation of sialic acids of isomeric glycans that were baseline-separated with our methodology.

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MATERIALS AND METHODS Materials. A carbohydrate analysis kit, including APTS labeling dye and glucose ladder standard, was obtained from Beckman Coulter Inc. (Brea, CA). Sodium cyanoborohydride (1 M in tetrahydrofuran) and ε-aminocaproic acid were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). All chemicals and solvents were of analytical grade or better. Formic acid, acetic acid, ammonium acetate, methanol, ethanol, acetone, and 2-propanol were purchased from Fisher Scientific (Nepean, ON, Canada). All aqueous solutions were prepared using purified water (18.2 MΩ·cm−1 ) with a Milli-Q purification system (Millipore, Bedford, MA, U.S.A.). 8757

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Figure 1. (A) CE-LIF separation of APTS-labeled N-glycans of control fish serum. Peaks 1a, 2a, 3a, 4a, and 5a are APTS-labeled glycan peaks. (B) Base peak electropherogram of underivatized O-acetylated N-glycans of control fish serum acquired using the LCQ* Duo ion trap MS. BGE was 40 mM ε-aminocaproic acid with 20% methanol. Peaks 1b, 2b, 3b, 4b, and 5b correspond to the peaks 1a, 2a, 3a, 4a, and 5a, respectively, in panel A. The migration time (above) and the observed m/z (below) are indicated for each peak.

1153.07 (1153.04), and 1111.10 ([M − 2H]2−) which are in agreement with samples of control fish sera according to Liu et al.24 Clearly, the presence of a set of doubly charged peaks differing by 42 Da, due to the presence of different numbers of acetyl groups in the sialic acids, can be observed. Therefore, the method allows the detection of mobility differences due to only 42 Da in high molecular mass analytes (2000−3200 Da). As expected, heavier glycans bearing the same negative charge showed a lower mobility and are detected later than smaller glycans. This is illustrated by the migration order of the fish serum glycans containing zero, one, and two acetyl groups (at m/z 1111.10, 1132.02, and 1153.04, respectively) as can be observed for peaks 3b, 4b, and 5b in Figure 1B. Earlier migration of N-glycans at m/z 1131.8 and 1153.07 is discussed in the following paragraph. Although some degree of overlapping is observed in the first dimension corresponding to CE separation, glycans can be resolved in the second dimension corresponding to MS detection, showing the excellent separation power of CE−MS. Interestingly, two pairs of peaks corresponding to biantennary glycans containing one and two O-acetyl groups (at m/z 1132.0 and 1153.0, respectively) were baseline-separated and provided evidence of structurally different isomeric species present in N-glycans of fish serum. The observed isomers, peaks 1b/4b and 2b/5b in Figure 1B, could be due to positional and/ or linkage isomers that have different electrophoretic mobilities and therefore migrate with different velocities. The baseline separation achieved for the isomers, as can be observed in the base peak electropherograms in Figure 2, parts A and B, demonstrates in more detail the separation power of the CE− MS method. The major reason for an improved selectivity of the native isomeric species could be the use of ε-aminocaproic acid as buffer. Isomeric species may have distinctively selective interactions with the BGE, resulting in different hydrodynamic sizes and consequently dissimilar electrophoretic mobilities, yielding a baseline separation. Liu et al.24 has reported that the major ion at m/z 1153.0 corresponds to a biantennary complex glycan with two O-acetyl groups at its terminal sialic acid residues. However, they were not able to determine if two mono-O-acetylated sialic acids or a di-O-acetylated sialic acid was present as the terminal units in the complex glycan. Moreover, only one glycan species was reported at m/z 1132.0.24 In order to identify the isomeric species already separated, CE−MS/MS analyses were performed using the API

Canada). Electrospray voltage was set at −3.5 kV. The MS/MS spectra were acquired with a dwell time of 5.0 ms per step of 1 Th; the precursor ion was selected at low resolution (about 5 Th window), and the Q3 peak width was set to approximately 2.0 Th.

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RESULTS AND DISCUSSION CE-LIF. CE-LIF separation with MS-amenable buffers gave good separation of APTS-labeled glucose ladder standard sample, as shown in the Supporting Information (Figure S1); the ladder can also be analyzed by MS.37 However, the optimum buffer condition for CE-LIF analysis of fish serum glycans was found to be different from that of the standard glucose sample. Figure 1A shows the CE-LIF electropherogram for the separation of APTS-labeled N-glycans of fish serum using 25 mM ammonium acetate buffer (pH 3.1) which gave an acceptable current of 12 μA at −20 kV. According to Liu et al.,24 the glycoprofile of control fish serum contains only three N-glycans corresponding to O-acetylated biantennary oligosaccharides. Interestingly, the glycoprofile in Figure 1A shows five species with distinctive electrophoretic mobilities that can be partially separated. In order to verify their identity, CE−ESIMS was performed on the APTS-labeled O-acetylated Nglycans used, as described below. CE−MS of Native N-Glycans of Fish Serum. Attempts of using CE−MS for APTS-labeled N-glycans of control fish serum did not produce optimum results because the CE current under CE-LIF conditions was too high, as discussed in the Supporting Information. In this section we evaluated the CE− MS response of underivatized, native O-acetylated N-glycans present in four samples of control fish serum, relying on the dissociation of sialic acids that would provide the charges needed for electrophoretic migration and ESI ionization. Moreover, this approach was useful for preserving the structural characteristics of the natural low-abundance O-acetylated Nglycans. At pH 3.03 in the negative ion mode, higher ionization efficiencies were observed in comparison to the positive ion mode (data not shown). The separation was carried out under reverse polarity and worked for all the N-glycans released from fish serum which migrated under electrophoretic conditions according to the extension of chain length. For example, in Figure 1B, a set of ions were observed at m/z 1131.8 (1132.02), 8758

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components in three consecutive days with five injections on each day were in the range of 0.15−0.31%. Of particular importance is that fact that under the ionization and detection conditions of the N-glycans, the in-source decay by desialylation is nonexistent, which allows detection of native O-acetylated complex type oligosaccharides. These studies clearly demonstrate that the CE−ESI-MS approach is an excellent tool for the analysis of low-abundance acidic glycan species without the need of extra labeling steps. Identification of Isomeric O-Acetylated Species of Native N-Glycans. To further explore the identity of the isomeric species separated, native O-acetylated N-glycans were subjected to CE−ESI-MS/MS using the API 3000 triplequadrupole MS. In-source fragmentation was promoted by using a high sample cone voltage to produce collision-induced dissociation (CID) of selected ions, using nitrogen as the neutral collision gas.41 The high voltage was experimentally determined by comparing the intensities of the fragment ions, and a cone voltage of 75 V was selected as the optimum. The in-source fragmentation was carried out using full MS in the range of m/z 200−400 to determine the extent of acetylation of sialic acids. Inset a in Figure 2A shows the CE−ESI-MS/MS spectrum of precursor ion at m/z 1153.07. The fragment ions observed at m/z 290.0 and 373.5 correspond to terminal unmodified Neu5Ac and di-O-acetylated Neu5Ac, respectively. Inset b in Figure 2A shows the CE−ESI-MS/MS spectrum of precursor ion at m/z 1153.04 with fragment ions at m/z 290.0 and 331.9, corresponding to terminal unmodified Neu5Ac and mono-Oacetylated Neu5Ac, respectively. In both cases, the fragments generated were produced by glycosidic cleavages of terminal Neu5Ac from the glycan antenna region. These fragmentation results confirmed that the biantennary complex type oligosaccharides, with two additional O-acetyl groups and nominal m/z 1153.0, were originated from two structural isomers containing two mono-O-acetylated Neu5Ac and another isomer with a Neu5Ac residue and a di-O-acetylated Neu5Ac. This observation is in agreement with those previously proposed by Liu et al.24 and, in addition, provides conclusive evidence on the degree and distribution of O-acetyl groups on the sialic acids present as terminal units in mixtures of N-glycans from fish serum. CE−MS/MS analyses were also performed on precursor ions at m/z 1131.8 and 1132.0 corresponding to the mono-Oacetylated biantennary N-glycans of fish serum. In both cases the same fragments were obtained as observed in the insets c and d of Figure 2B, which correspond to Neu5Ac and mono-Oacetylated Neu5Ac at m/z 290.0 and 331.8, respectively. A weak peak at m/z 271.8 was also observed and could be the result of a water loss adduct from the fragment ion at m/z 290.0. According to previous studies in the literature, the major mono-O-acetylated sialic acid species identified was Neu5,9Ac2, while Neu5,7Ac2 and Neu5,8Ac2 were also present but in lower abundance leading to the occurrence of isomers for di-Oacetylated Neu5Ac.17,21,22,27 The observed mono-O-acetylated Neu5Ac at nominal m/z 1132.0 could be composed of a mixture of two possible sialic acid species, carrying O-acetyl groups in 9- and 7- or 8-OH of Neu5Ac leading to the occurrence of two isomeric species. However, at this stage we are not able to identify the mono-O-acetylated isomers in CE− ESI-MS/MS electropherograms, shown in the insets c and d of Figure 2B. We were not able to find studies in the literature

Figure 2. Extracted ion electropherogram showing baseline separation for isomeric species at (A) m/z 1153.04 and (B) m/z 1132.02. CE− MS spectra were acquired using the LCQ* Duo ion trap MS. In all cases, [M − 2H]2− was observed. The insets show the CE−MS/MS spectra for the isomeric species acquired using the API 3000 triplequadrupole MS: fragment ions from precursor ions at (a) m/z 1153.07 (peak 2b), (b) m/z 1153.04 (peak 5b), (c) m/z 1131.8 (peak 1b), and (d) m/z 1132. 02 (peak 4b) were observed.

3000 triple-quadrupole MS based on the adequate separation selectivity achieved. It is important to note that CE−ESI-MS detection of native N-glycans and CE-LIF detection of APTS-labeled N-glycans yielded the same glycoprofile. The five migrating species could be clearly seen in both the LIF and MS base peak electropherograms. In order to establish a correspondence between peaks detected with both methods, a quantitative assessment of the total peak area was made for the LIF trace (Figure 1A) and the MS base peak electropherogram (Figure 1B). In both cases, the percentage of a specific oligosaccharide present in the mixture was calculated from the area of the peak of that glycan relative to the total area of the five glycans, which is defined as 100%. The approximate percent peak area of the total glycans for peaks 1a−5a in Figure 1A is as follows: peak 1a = 2.87%, peak 2a = 2.86%, peak 3a = 13.79%, peak 4a = 37.12%, and peak 5a = 43.33%, while for peaks 1b−5b in Figure 1B peak 1b = 1.93%, peak 2b = 2.95%, peak 3b = 14.26%, peak 4b = 36.03%, and peak 5b = 43.02%. The correlation shown between the relative peak areas gives us confidence to compare the peaks from the LIF trace and MS base peak electropherograms. Reproducibility of the CE−ESI-MS method was also evaluated in terms of the migration times of the N-glycans present in four samples of control fish serum. The migration time percent relative standard deviations (% RSDs) for the five 8759

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Table 1A. Identification and Annotation of the Biantennary (Column on the Left) and Triantennary (Column on the Right) NGlycans Present in Fish Serum Observed as [M − 2H]2− and [M − 3H]3−, Respectivelya

a Composition and structural schemes are given in terms of N-acetylglucosamine (blue squares), mannose (green circles), galactose (yellow circles), and sialic acid (purple diamonds). For ease of identification, the numbers of O-acetyl groups present on sialic acids are included as subscripts in the N-glycan composition.

Figure 3. Base peak electropherograms showing separation of isomeric species for bi- and triantennary oligosaccharides present in samples taken from (A) weeks 1 and 4, (B) week 2, and (C) week 3 of the handling-stress experiment. Data was acquired using the API 3000 triple-quadrupole MS.

where the locations of O-acetyl groups in native, unreleased sialic acids were definitively reported. Analysis of Potential N-Glycans Isomers. A further application of the CE−ESI-MS/MS method for the separation and identification of glycan isomers present in control fish serum was to study the potential occurrence of isomeric species in 10 samples of fish serum subjected to periods of stress from 1 to 4 weeks. Each sample was analyzed in three consecutive days with three injections per sample. In addition to the previously observed biantennary oligosaccharides with zero, one, and two O-acetylated Neu5Ac, three and four O-acetyl groups in the terminal sialic acids were also detected. Moreover, triantennary structures ([M − 3H]3−), containing from zero up to six O-acetylated Neu5Ac, were also observed. Table 1A shows the glycan composition and proposed structure for each of the N-glycans detected. A list of the bi- and triantennary glycans observed in fish serum samples, according to the week of the stress experiment, is shown in the Supporting Information (Table 1B).

Figure 3 shows the base peak electropherograms for the separation of bi- and triantennary oligosaccharides present in samples taken from weeks 1−4 of the handling-stress experiment. Several peaks at the same m/z were identified and baseline-separated, both for bi- and triantennary glycans. These peaks, which correspond to isomeric species, are possibly due to the distribution of O-acetyl groups in Neu5Ac, as discussed before. In general, three types of electropherograms were observed showing a distribution pattern, depending on the week of the stress experiment. The electropherograms of week 1 and week 4 samples show baseline separation for isomers of bi- and triantennary oligosaccharides. The electropherogram of week 2 sample displays isomer separation for bi- and triantennary glycans, with an increased number of isomers for biantennary glycans. Week 3 sample only shows isomers corresponding to biantennary glycans, while isomers of triantennary species were not detected. Interestingly, the pattern for isomer distribution not only shows the occurrence of O-acetylated Neu5Ac in bi- and triantennary glycans but also 8760

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follows the trend of the distribution of O-acetylated species reported by Liu et al.,24 i.e., week 2 shows a significant increase of O-acetylation in comparison to week 3, while weeks 1 and 4 have the same acetylation pattern. The distribution of Oacetylated Neu5Ac in bi- and triantennary glycans may lead to an increased number of isomeric species that show a characteristic CE profile. Of particular interest is the occurrence of three isomers for biantennary oligosaccharides in the week 2 samples, as seen in Figure 3B. To evaluate the possibility of obtaining structural information, CE−ESI-MS/MS was also performed under the optimum conditions. Comparison of the relative intensities of the fragment ions indicates that the most intense peak of biantennary glycans at week 2 could correspond to Neu5,7,9Ac3 followed by Neu5,8,9Ac3 (see Figure 3B). Also the 8-O-acetyl derivative, Neu5,7,8Ac3, regularly occurs, with lower abundance from the 7-O-acetyl migration.11,21,42 This potentially could correspond to the first peak of biantennary glycans at week 2, which explains the isomeric distribution of the acetylated species (see Figure 3B). Certainly, more effort needs to be put into the interpretation of MS/MS spectra; however, this is beyond the scope of this paper and requires the extensive characterization of individual standards of O-acetylated Neu5Ac (not commercially available) in order to compare fragmentation patterns and to assign a specific signature for each isomer. The potential of this approach to separate and detect even less abundant components with high sensitivitypreviously inaccessible due to overlapping of isobaric structurescan be considered as a significant contribution in the progress of detailed glycan analysis.

it was shown that these structural isomers can be distinguished and assigned by CE−ESI-MS/MS. Further identification of linkage isomers will potentially require strategic glycosidase digestion but was not possible at this stage. However, we believe that the approach discussed in this study could be extended to other types of complex oligosaccharides and probably to all kinds of N-glycans.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 604-822-0878. Fax: 604-822-2874. E-mail: chen@ chem.ubc.ca. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from Beckman Coulter Inc., Brea, CA, USA, and the Natural Sciences and Engineering Research Council of Canada. The fish serum sample was obtained from Dr. Laura Brown and Dr. Stewart Johnson of Fisheries and Oceans Canada.

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REFERENCES

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CONCLUDING REMARKS A simple and effective separation method using CE−MS was developed for the analysis of enzymatically released Oacetylated N-glycans in Atlantic salmon (S. salar) fish serum. Characteristic CE glycoprofiles for both control and stress serum glycans were obtained. The CE method allowed unraveling the complexity of the glycans showing detailed information about the degree of O-acetylation present in the sialic acids. The use of hydrophilic N−CHO capillaries along with an acidic zwitterionic buffer allows successful CE separations with ESI-MS and ESI-MS/MS detection of mixtures of complex N-glycans containing minor modifications. Although comparable CE-LIF and CE−ESI-MS glycoprofiles for APTS-derivatized and native glycans were obtained, it has been demonstrated that there is no need for derivatization or extra sample preparation when CE−ESI-MS is used for the analysis of glycans containing sialic acids. However, since derivatization with APTS is a relatively simple procedure, it can be used as a starting point for CE profiling of glycan mixtures containing neutral and charged species. Clearly, the advantages of using mass spectrometry include valuable direct molecular weight and structural information for glycan identification with the potential of quantitative evaluation for glycan pools. The CE−ESI-MS method developed was used to obtain baseline separation of isomeric species and can be a new tool for glycomic analysis. Moreover, CE−MS/MS provides an isomer-specific profile of O-acetylated Neu5Ac in fish serum glycans to unequivocally determine glycan structure for different degrees of O-acetylation. Isomer-specific analysis revealed up to three different isomers for disialylated bi- and triantennary oligosaccharides with highly reproducible migration times that have not been previously observed. In particular, 8761

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