Fragmentation Characteristics of Neutral N-Linked Glycans Using a

Anal. Chem. 2004, 76, 2343-2354

Fragmentation Characteristics of Neutral N-Linked Glycans Using a MALDI-TOF/TOF Tandem Mass Spectrometer Elaine Stephens,* Sarah L. Maslen, Luke G. Green,† and Dudley H. Williams

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K. CB2 1EW

Glycosylated proteins are widespread components of cellular surfaces and extracellular matrixes, in which their sugar chains are implicated in a variety of important biological functions. These functions include modulation of the conformation and stability of proteins and control of the half-life of proteins and cells. Additionally, the saccharide chains can act as ligands for specific binding events that mediate protein targeting, cell-cell and cell-matrix interactions.1-4 Glycoproteins often have more than one glycosylation site, each with its own diverse array of glycan variants (glycoforms) linked via an asparagine residue (N-linked glycosylation) or a serine or threonine residue (O-linked glycosylation).

Therefore, complete structural characterization of a glycoprotein is an extremely challenging task and requires the identification of the site of glycosylation, the determination of the constituent monosaccharides of each glycoform, their sequence, the branching pattern, and the hydroxyl groups involved in the linkage of one residue to another. No single mass spectrometric technique is yet able to determine all of these parameters, but several techniques used in combination, supported by knowledge of the biosynthetic pathways of oligosaccharides,5 can be employed to deduce the complete structure.6 Sequence and branching information can be gained from fragment ions produced by fast atom bombardment mass spectrometry (FAB)7 or liquid secondary ion mass spectrometry (LSIMS)8 on permethylated derivatives. However, more recently, highly sensitive mass spectrometers, which include ESI-MS on Q-TOF instruments, are capable of fragmenting oligosaccharides and glycopeptides by low-energy CID and can easily provide detailed sequence and branching information at the low-femtomole level.9,10 Composition and linkage information are currently best determined by gas chromatography-mass spectrometry of derivatized hydrolysates, which is limited in sensitivity. In view of this latter limitation, many investigators have explored the use of MS/MS for the determination of linkage positions. Such information can be gained from cross-ring fragment ions that are particularly abundant in collision-induced dissociation (CID) spectra induced by high-energy collisions. These high-energy CID experiments have traditionally been conducted by FAB-MS or LSIMS on tandem four-sector magnetic instruments or by linkedscanning on double-focusing instruments.11 However, a hybridsector mass spectrometer, fitted with a matrix-assisted laser desorption/ionization (MALDI) source and an orthogonal acceleration TOF analyzer has been shown to provide some linkage

* Corresponding author. Phone: +44 1223 336688. Fax: +44 1223 336913. E-mail: [email protected]. † Current address: F. Hoffman-La Roche, Pharmaceuticals Division, PRBDCI, Discovery Chemistry, Bldg. 065/412, CH-4070, Basel, Switzerland. (1) Varki, A. Glycobiology 1993, 3, 97-130. (2) Rudd, P. M.; Wormald, M. R.; Stanfield, R. L.; Huang, M.; Mattsson, N.; Speir, J. A.; DiGennaro, J. A.; Fetrow, J. S.; Dwek, R. A.; Wilson, I. A. J. Mol. Biol. 1999, 293, 351-366. (3) Rudd, P. M.; Woods, R. J.; Wormald, M. R.; Opdenakker, G.; Downing, A. K.; Campbell, I. D.; Dwek, R. A. Biochim. Biophys. Acta 1995, 1248, 1-10. (4) Helenius, A.; Aebi, M. Science 2001, 291, 2364-2369.

(5) Kornfeld, R.; Kornfeld, S. Annu. Rev. Biochem. 1985, 54, 631-664. (6) Dell, A.; Morris, H. R. Science 2001, 291, 2351-2356. (7) Dell, A.; Thomas-Oates, J. Analysis of Carbohydrates by GLC and MS; CRC Press: Boca Raton, FL, 1989. (8) Poulter, L.; Burlingame, A. L. Methods Enzymol. 1990, 193, 661-688. (9) Stimson, E.; Hope, J.; Chong, A.; Burlingame, A. L. Biochemistry 1999, 38, 4885-4895. (10) Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; Bordoli, R. S.; Hoyes, J.; Bateman, R. H. Rapid Commun. Mass Spectrom. 1996, 10, 889896. (11) Boyd, R. K.; Beynon, J. H. Org. Mass Spectrom. 1977, 12, 163.

The fragmentation characteristics of native and permethylated oligosaccharides using a matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight tandem mass spectrometer are described. The use of two MALDI matrixes, r-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), is shown to control the nature and extent of fragmentation observed in collision-induced dissociation experiments on synthetic oligosaccharides. CHCA promotes the occurrence of glycosidic cleavages, whereas DHB promotes a wide range of fragmentations. These latter fragmentations include glycosidic cleavages, cross-ring cleavages, and the formation of “internal” cleavage ions, which are derived from elimination of substituents from around the pyranose ring. This extensive fragmentation is shown to facilitate the detailed structural characterization of high-mannose and hybrid-type N-glycans purified from avidin. Importantly, the cross-ring fragments reveal linkage information, unambiguously define antennae substitutions, and differentiate isomeric glycoforms.

10.1021/ac030333p CCC: $27.50 Published on Web 03/06/2004

© 2004 American Chemical Society

Analytical Chemistry, Vol. 76, No. 8, April 15, 2004 2343

Figure 1. MALDI-TOF/TOF CID spectra of the [M + Na]+ ion of synthetic oligosaccharide Man3GlcNAc2Gal2. MS/MS spectra of the native sugar using (A) CHCA and (B) DHB as the matrix. (C) MS/MS spectrum of the permethylated sugar using DHB as the matrix.

information due to high-energy cross-ring cleavages of native oligosaccharides and glycopeptides.12,13 A conventional MALDI reflectron-type instrument can provide sequence information of oligosaccharides by post-source-decay (PSD), where metastable decomposition taking place after acceleration in the source region can be observed with a stepping reflectron mirror.14 Although useful for sequence determination, the cross-ring fragments giving linkage information are not observed. Furthermore, it has been shown that when argon is introduced into the collision cell of a MALDI-TOF reflectron instrument, cross-ring fragment ions are still not formed efficiently.15 Although this MALDI technique has been useful in terms of gaining oligosaccharide sequence and branching information, it has limitations due to the requirement to “stitch” together a series of separate scans for different regions of the fragment ion spectrum, due to defocusing by the reflectron. One recent mass spectrometer design that overcomes many of the limitations of PSD and CID in a conventional MALDI is the TOF/ (12) Harvey, D. J.; Bateman, R. H.; Green, M. R. J. Mass Spectrom. 1997, 32, 167-187. (13) Kuster, B.; Naven, T. J. P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 1645-1651. (14) Tang, X.; Ens, W.; Standing, K. G.; Westmore, J. B. Anal. Chem. 1988, 60, 1791-1799. (15) Mechref, Y.; Baker, A. G.; Novotny, M. V. Carbohydrate Res. 1998, 313, 145-155.

2344 Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

TOF,16,17 in which high velocity ions produced in a high-vacuum pulsed MALDI-TOF are selected with a timed ion gate, collided at kiloelectronvolt energies with gas atoms or molecules, and then further accelerated for analysis in a two-stage reflectron TOF. This instrument can provide high-energy CID spectra of peptides17 and oligosaccharides18 and is superior to the earlier generation of sector-based tandem mass spectrometers using FAB or LSIMS ionization. In this paper, we investigate the fragmentation of native and permethylated synthetic oligosaccharides and permethylated Nlinked glycans from avidin using a MALDI-TOF/TOF instrument. We report observations in oligosaccharide fragmentation patterns and show this mass spectrometric technique to be a sensitive, convenient and rapid method for determining sequence, branching, and linkage information of native and permethylated oligosaccharides. EXPERIMENTAL SECTION Materials and Reagents. Native synthetic oligosaccharides were dissolved in water to a final concentration of 10 pmol/µL, (16) Vestal, M. L.; Juhasz, P.; Hines, W.; Martin, S. A. Mass Spectrometry in Biology and Medicine; Humana Press: Totowa, NJ, 2000. (17) Medzihradszky, K. F.; Campbell, J. M.; Baldwin, M. A.; Falick, A. M.; Juhasz, P.; Vestal, M. L.; Burlingame, A. L. Anal. Chem. 2000, 72, 552-558. (18) Mechref, Y.; Novotny, M. V.; Krishnan, C. Anal. Chem. 2003, 75, 48954903.

Scheme 1. (A) Glycosidic Cleavages and (B) Cross-Ring Cleavages Found in the MS/MS Spectra of the Synthetic Oligosaccharide Man3GlcNAc2Gal2a

a The fragmentation scheme is based on that proposed by Domon and Costello.23 R ) H and R ) Me for native and permethylated structures, respectively.

whereas their permethylated counterparts were dissolved in methanol to a concentration of 5 pmol/µL before being mixed 1:1 (v:v) with matrix solution. R-Cyano-4-hydroxycinnamic acid solution (CHCA) (Applied Biosystems) was mixed 1:1 with oligosaccharide solution and spotted directly onto the target plate and allowed to air-dry. 2,5-Dihydroxybenzoic acid (DHB) (Fluka) was prepared as a saturated solution in 50% methanol in water and mixed 1:1 with the analyte. A 0.5-µL portion of this solution was spotted onto the MALDI target and dried rapidly in a desiccator in order to produce small DHB crystals for easy automated spectral acquisition. A 250-µg portion of avidin (egg white) (Sigma) was reduced and alkylated in 6 M guanidine

hydrochloride, precipitated with ice-cold methanol, redissolved in 4 M urea, and digested with porcine trypsin (Promega) as described previously.9 The resulting glycopeptides were purified by analytical reversed-phase HPLC using standard protocols. HPLC fractions were screened for signals corresponding to glycopeptides by MALDI-TOF-MS. Glycopeptide-containing fractions were dried down and digested with peptide-N-glycosidase F (EC 3.5.1.52) (Roche) for 16 h in 50 mM ammonium bicarbonate buffer (pH 8.4). The released oligosaccharides were separated from their peptides by purification using a C18 Zip-Tip (Millipore). The aqueous fraction containing released avidin glycans was Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

2345

Figure 2. Plausible structures for clusters of fragment ions accompanying Y3, Z3, and oligosaccharide Man3GlcNAc2Gal2.

permethylated using the NaOH permethylation procedure.19 The derivatized glycans were redissolved in 15% acetonitrile in water and further purified using a C-18 Zip-Tip, where they were eluted into 60 µL of 75% acetonitrile. Mass Spectrometry. All mass spectra were recorded on a 4700 Proteomics analyzer with TOF/TOF optics (Applied Biosystems). This MALDI tandem mass spectrometer uses a 200-Hz frequency-tripled Nd:YAG laser operating at a wavelength of 355 nm. Averages of 2000 and 5000 laser shots were used to obtain the MS/MS spectra in CHCA and DHB, respectively. For MS/ MS experiments, the collision energy, which is defined by the potential difference between the source acceleration voltage (8 kV) and the floating collision cell (7 kV), was set at 1 kV. Inside the collision cell, the selected oligosaccharide ions were collided with argon at a pressure of 2 × 10-6 Torr.

1,5X

3

of (A) native and (B) permethylated synthetic

MS/MS of Synthetic Oligosaccharides. The fragmentation characteristics of native and permethylated synthetic oligosaccharides were evaluated using two common MALDI matrixes, R-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). CHCA is a relatively “hot” matrix that induces

unimolecular decomposition20,21 and is used for the analysis of peptides, but it can also be employed for the analysis of small neutral oligosaccharides. DHB is a “cooler” matrix,22 suitable for the analysis of neutral sugars and most peptides. All oligosaccharides gave abundant [M + Na]+ signals, and these ions were chosen for MS/MS in each case. The CID spectrum of the [M + Na]+ ion of the native synthetic sugar Man3GlcNAc2Gal2 using CHCA as the matrix and argon as the collision gas is shown in Figure 1 (panel A), and the fragmentations producing the major ions are indicated in Scheme 1A. The spectrum is dominated by glycosidic cleavages (B and Y ions, Domon and Costello nomenclature23) that are observed for all glycosidic bonds in the molecule and, consequently, provide useful sequence information. The Y-type cleavage at m/z 906.1 (Y2) gives rise to the major fragment ion, probably due to preferred cleavage adjacent to GlcNAc, an observation also common to FAB spectra.19 Important cross-ring fragments, normally derived from high-energy collisions, which provide linkage information, are minor compared with glycosidic cleavages. For example, only small signals were detected for 3,5A2 and 3,5A4. These data show that use of the “hot” matrix CHCA promotes the formation of fragment ions derived from cleavage of the glycosidic bond.

(19) Dell, A.; Khoo, K.-H.; Panico, M.; McDowell, R. A.; Etienne, A.; Reason, A. J.; Morris, H. R. In Glycobiology. A practical approach; Fukuda, M., Kobata, A., Eds.; 1992; Vol. 125, pp 187-222. (20) Stimson, E.; Troung, O.; Richter, W.; Waterfield, M. D.; Burlingame, A. L. Int. J. Mass Spectrom. Ion Processes 1997, 169/170, 231-240.

(21) Cordero, M. M.; Cornish, T. J.; Lys, I. A.; Cotter, R. J. Rapid Commun. Mass Spectrom. 1995, 9, 1356-1361. (22) Karas, M.; Bahr, U.; Strupat, K.; Hillenkamp, F.; Tsarbopoulos, A.; Pramanik, B. N. Anal. Chem. 1995, 67, 675-679. (23) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

RESULTS AND DISCUSSION


Figure 3. MALDI-TOF/TOF CID spectra of the [M + Na]+ ion of synthetic oligosaccharide Man3GlcNAc2Gal2Fuc4. MS/MS spectra of the native sugar using (A) CHCA and (B) DHB as the matrix. (C) MS/MS spectrum of the permethylated sugar using DHB as the matrix.

A second CID spectrum of this compound using DHB as the matrix and argon as the collision gas (shown in panel B), gave a far richer display of fragment ions, as compared to those observed using CHCA. Again, the B and Y fragment ions are detected, although the Y ions no longer dominate the spectrum. The B-type cleavage at m/z 388.1 gives rise to the major fragment ion due to preferred cleavage adjacent to the GlcNAc residue. C ions are also observed in this spectrum and are accompanied by ions containing two fewer hydrogen atoms (C-2), whose formation has been discussed previously.12 In addition, Y ions are also present that are accompanied by ions two Da lower in mass (e.g., Y2 at m/z 906.3 and Y2-2 at m/z 904.3), whose formation probably involves a hydrogen transfer away from the ionic fragment, as indicated for C-2 fragment ions.12 Signals corresponding to Z ions are detected, although these ions may be due to loss of water from Y ions, and permethylation is needed to confirm these assignments. Importantly, cross-ring fragment ions are now particularly abundant using DHB as a matrix, and those present are indicated in Scheme 1B. The 1,5X series are the strongest cross-ring fragments and are found in all constituent monosaccharides. These ions, however, do not yield specific linkage information, because they only contain C-1 from the sugar ring. A second series of X ions (0,2X) provide information about whether the C-2 oxygen is

involved in a glycosidic bond and indicate that GlcNAc in the antennae is 2-linked to R-mannose. Cross-ring fragments comprising the nonreducing part of the molecule (3,5A, 1,3A, and 0,4A) are observed that provide very important linkage information. In particular, the 3,5A2 ion, together with the 2,4X2 cross-ring fragment, shows that GlcNAc is 4-linked to Gal in the antennae. The 3,6linked mannose at the reducing end of the molecule is more difficult to identify because the glycan has two equivalent antennae. Thus, the cross-ring fragments 1,3A4 and 0,4A4 are isobaric, and permethylation is required in order to distinguish them. In addition, two significant signals are apparent below every Z ion fragment, which were attributed to elimination of substituents from the hexose ring (see Figure 2). These attributions provide a plausible and convenient rationalization of the observed masses, but in the absence of isotope labeling experiments, other formalizations are possible. The assignments described above for the native oligosaccharide were corroborated by MS/MS of its permethylated derivative under the same conditions (Figure 1C). In this spectrum, most Y ions are greatly diminished in abundance. Instead, the 1,5X ions give sequence and branching information, because they are formed in each constituent monosaccharide. Minor signals for Z ions are detected, and their associated signals at lower mass are particularly abundant. These latter signals confirm the assignAnalytical Chemistry, Vol. 76, No. 8, April 15, 2004

2347

Scheme 2. (A) Glycosidic Cleavages and (B) Cross-Ring Cleavages Found in the MS/MS Spectra of the Synthetic Oligosaccharide Man3GlcNAc2Gal2Fuc4a

a The fragmentation scheme is based on that proposed by Domon and Costello.22 R ) H and R ) Me for native and permethylated structures, respectively.

ments given for the native sugar, which indicate the loss of substituents from around the pyranose ring (Figure 2). Similar “satellite” signals have been observed associated with B ions for acetylated high-mannose and permethylated monosulfated oligosaccharides analyzed by FAB-MS.19 Cross-ring fragments for the permethylated oligosaccharide are the same as observed for the native sugar, although differentiation of 1,3A4 and 0,4A4 is achieved due to the presence of signals at m/z 764.2 and 750.2, respectively. 2348

Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

The CID spectrum of the [M + Na]+ ion of the tetrafucosylated synthetic structure Fuc4Man3GlcNAc2Gal2 using CHCA as the matrix and argon as the collision gas is shown in Figure 3A, and the fragmentations producing the major ions are indicated in Scheme 2A. Once again, the most abundant fragment ions are B and Y ions that are derived from glycosidic cleavages. However, the sequence of this tetrafucosylated structure is difficult to determine from this fragment ion spectrum due to the occurrence of multiple cleavages that often involve one or more losses of

Figure 4. Plausible structures for clusters of fragment ions accompanying Y3 and Z3 of (A) native and (B) permethylated synthetic fucosylated oligosaccharide Fuc4Man3GlcNAc2Gal2.

fucose. In addition, cross-ring fragments are not detected in this CID spectrum, and consequently, linkage information cannot be obtained. The fragment ion spectrum of this oligosaccharide acquired using DHB under the same CID conditions gave the spectrum shown in Figure 3B, and the fragment ions observed are indicated in Scheme 2A and B. The major fragment ions include those derived from 1,5X cross-ring cleavages that, along with the minor Y and Z ion series, provide useful sequence information. The 0,2X2 ion is the only other cross-ring fragment present that incorporates the reducing end of the sugar, although this ion does not give useful linkage information. However, several important cross-ring fragments from the nonreducing end are observed. These include 0,4A , 1,3A , and 3,5A that provide linkage information about core 5 5 5 mannose, although 0,4A5 and 1,3A5 are isobaric due to equivalent antennae. Furthermore, a minor signal for 1,3A4 indicates that R-mannose is substituted at C-2 or C-3. Other ions in the spectrum include those derived from multiple cleavages, which involve loss of fucose, for example, at m/z 388.2 and 534.2. As observed in the MS/MS spectrum of the native nonfucosylated oligosaccharide, a doublet of ions is observed below each Z-type fragment that is attributed to the elimination of substituents from around the hexose ring. In addition, the major ion labeled ion a is likely to be derived from a Z-type cleavage followed by elimination of fucose from C-3 of GlcNAc and its linking oxygen (see also Figure 4). This mechanism is likely because it has often been reported that there is a preferential loss of the group attached to the C-3

position of GlcNAc.7,12,19,24 However, this ion also corresponds to the loss of two hexose residues, one fucose and a water molecule from the molecular ion. Thus, unambiguous assignment of the CID spectrum of this native oligosaccharide is not possible. Interpretation of the fragment ion spectrum of the permethylated derivative (Figure 3C) of this compound is less ambiguous, and ions derived from the elimination of subtituents from the pyranose ring are more definitively assigned. For example, major signals at m/z 1821.6 and 1662.1 correspond to elimination of substituents on C-6 and C-3, respectively (see Figure 4). This latter signal corroborates the assignment given for ion a in the MS/ MS spectrum of the corresponding native saccharide. The cluster of signals below the B3 ion is also derived from elimination of substituents around the ring. In particular, the signals at m/z 763.4 and 646.2 correspond to the loss of N-acetyl and fucose (with its linking oxygen) from C-2 and C-3 of GlcNAc, respectively. Once again, the major 1,5X ions give useful sequence and branching information, because they are formed in each constituent monosaccharide. Minor signals for cross-ring fragment ions containing the nonreducing end are also present for this permethylated structure and are the same as those observed for its native counterpart. However, signals for 0,4A5 and 1,3A5 are now differentiated and give linkage information about the core 3,6mannose residue. Furthermore, a signal at m/z 503.1 for 3,5A3 is observed that was not detected for the native structure, which indicates that galactose is linked via C-4 of GlcNAc in the antennae. (24) Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 59645970.


2349

Figure 5. MALDI-TOF/TOF CID spectra of the [M + Na]+ ions of the high-mannose glycans (A) Man5GlcNAc2 and (B) Man7GlcNAc2 using DHB as the matrix. Ions labeled with an asterisk are derived from the elimination of substituents from around the pyranose ring. The insert in panel A shows the 0,4A2γ and 3,5A2 γ ions that are 1 Da higher in mass than Y1 and 1,5X1, respectively.

MS/MS of N-Linked Glycans Released from Avidin. The N-linked glycans from avidin have been characterized previously and are composed of ∼19 glycoforms of the high-mannose and hybrid type.25,26 The permethylated glycans from avidin were chosen for the present study because the fragmentation of these compounds is less ambiguous and, therefore, easier to interpret. Furthermore, the permethylated glycans gave more intense signals than their native counterparts and provided better sensitivity for MS/MS. The CID spectra of the permethylated high-mannose glycans Hex5HexNAc2 and Hex7HexNAc2 using DHB as a matrix are shown in Figure 5A and B, and the fragmentation producing the ions observed is shown in Scheme 3A and B, respectively. Only minor signals for fragment ions produced from glycosidic cleavages are present, while the 1,5X-type cross-ring cleavages are the most prominent in the spectrum. Both of these types of ions can be used to provide sequence and branching information. Two major ions which can be used to differentiate the substitution on the antennae of these two high-mannose sugars are the internal cleavage ions denoted ion b at m/z 839.4 and 1043.6 in Figure 5A and B, respectively. These ions are likely to be derived from the C-type fragment, formed by cleavage between GlcNAc at position (25) Brush, R. C.; White, H. B. Biochemistry 1982, 21, 5334-5341. (26) Oliver, R. W. A.; Green, B. N.; Harvey, D. J. Biochem. Soc. Trans. 1996, 24, 917-927.


2 and the core 3,6-mannose residue, and elimination of the substituent at C-3 of core mannose (see Scheme 4). This internal cleavage has been observed previously in the MS/MS spectra of native high-mannose structures using a hybrid MALDI-sector TOF instrument.12 Importantly, the presence of the major signal at m/z 1043.6 in the MS/MS spectrum of Hex7HexNAc2 indicates that the C-3 position of core mannose is substituted with two mannose residues. The absence of a signal at m/z 839 suggests that the isobaric oligomannosidic structure with three mannose residues at the C-3 position of β-mannose, previously reported as an avidin glycoform,26 is not present at a detectable level. Cross-ring fragments from the nonreducing end of the oligosaccharides confirm these assignments. For example, a prominent cross-ring 3,5A fragment ion from the core mannose residue is present in both spectra and defines the antennae compositions of both oligosaccharides. In addition, 0,4A ions are detected that also define the substitutions at C-6 on core mannose. Unfortunately, the 1,3A ions, which indicate linkage at C-2, are absent. However, minor signals at m/z 301.1 and m/z 329.1 for 0,4A2 γ and 3,5A , respectively, are present the MS/MS spectrum of the Hex 2γ 5 HexNAc2 structure (see Figure 5A insert). These important A-type fragment ions define linkage at C-6 on mannose in the antennae. The CID spectrum of the putative bisected hybrid structure Hex5HexNAc4 using DHB matrix is shown in Figure 6, and the fragmentation proposed is shown in Scheme 5A. Features com-

Scheme 3. Glycosidic and Cross-Ring Cleavages Found in the CID Spectra of (A) Hex5HexNAc2 and (B) Hex7HexNAc2

mon to the high-mannose structures were also present in this case. For example, the major ion labeled c at m/z 1084.5 is derived from the C3 fragment ion with elimination of the substituents at the C-3 position of core mannose. Therefore, this ion indicates the presence of three mannose residues and one GlcNAc bound to core mannose, and the further loss of one GlcNAc residue from this ion to give the signal labeled d suggests the presence of the bisecting GlcNAc at C-4 of the core mannose. Similar fragmenta-

tion has also been observed in high-energy CID of native hybrid structures.12 Furthermore, the 1,5X3γ ion and the 0,4A3 and 3,5A3 cross-ring fragments define the three mannose residues and one GlcNAc linked to core β-mannose with the latter two A-type ions revealing linkage via C-6 and C-4, respectively. Interestingly, other ions at m/z 737.4 (3,5A3) and 1389.7 (1,5X3R) indicate the presence of a minor amount of the isobaric nonbisected hybrid structure shown in Scheme 5B. This structure has not been previously Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

2351

Scheme 4. Scheme Showing Proposed Route for the Formation of Ion b in the MS/MS Spectra of High-Mannose Glycans from Avidina

a

R1 ) Me and R2 ) Man3 for Man5GlcNAc2. R1 ) Man and R2 ) Man4 for Hex7GlcNAc2

Figure 6. MALDI-TOF/TOF CID spectrum of the [M + Na]+ ion of the hybrid-type glycan Hex5HexNAc4 from avidin, using DHB as the matrix. Signals labeled with an asterisk are derived from internal cleavages due to elimination of substituents from around the pyranose ring.

identified as a glycan component on avidin, although N-glycans with similar antennae have been observed on other hen egg white proteins, such as ovalbumin.27,28 CONCLUSION Previous studies on peptides have shown that the “cool” matrix DHB partially suppresses MALDI-induced decomposition, as compared with the “hotter” matrix CHCA.17,20 The present study shows that this is also the case for MS/MS of oligosaccharides, in which the precursor ions [M + Na]+ are of much lower abundance (relative to the fragment ions) using CHCA than when (27) Da Silva, M. L. C.; Stubbs, H. J.; Tamura, T.; Rice, K. G. Arch. Biochem. Biophys. 1995, 318, 465-475. (28) Harvey, D. J.; Wing, D. R.; Kuster, B.; Wilson, B. H. J. Am. Soc. Mass Spectrom. 2000, 11, 564-571.


using DHB. Glycosidic cleavages, normally observed during metastable fragmentation in PSD experiments, are the major fragment ion signals observed using the CHCA matrix. DHB, however, gives a far richer display of fragment ions, many of which are cross-ring fragments that are probably formed by chargeremote processes. In addition, C or C-2 and Y or Y-2 ions are all produced in abundance for most glycosidic cleavages in the native synthetic oligosaccharides. The Y-type ions are less abundant for the permethylated derivatives, possibly due to the lack of free hydroxyl groups, as previous studies have shown that the hydroxylic hydrogen atoms migrate during glycosidic cleavages.24 Other major signals in all oligosaccharides studied include “satellite” signals below each Y- and Z-type ion that are due to the elimination of substituents from around the pyranose ring. These ions are also observed together with B- and C-type ions

Scheme 5. Glycosidic and Cross-Ring Cleavages Found in the High-Energy CID Spectrum of (A) the Bisected Hybrid-Type Structure and (B) the Isobaric Nonbisected Hybrid Glycan from Avidin

and give major signals for fragments that have eliminated the substituent linked to the C-3 position. This observation is not surprising, as it has often been reported that there is a preferential loss of the group attached to the C-3 position of sugars.7,19,24,29-31 (29) Garozzo, D.; Impallomeni, G.; Montaudo, G.; Spina, E. Rapid Commun. Mass Spectrom. 1992, 6, 550-552. (30) Egge, H.; Peter-Katalinic, J. Mass Spectrom. Rev. 1987, 6, 331-393. (31) Domon, B.; Mueller, D. R.; Richter, W. J. Biomed. Environ. Mass Spectrom. 1990, 19, 390-392.

These “internal” fragment ions provide important information about the compositions of the antennae in the avidin N-glycans. The major cross-ring fragments detected are the 1,5X cleavages, which are found in all constituent monosaccharide residues and enable the branching patterns to be defined. These are especially useful for the permethylated derivatives in which glycosidic Y ions are of low abundance. The linkage-revealing cross-ring fragments give smaller signals. The most important of these are the A-type Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

2353

cleavages that incorporate the nonreducing end of the oligosaccharide chain. In particular, the 3,5A, 0,4A, and 1,3A are present that define the linkages in the antennae, as reported previously for high-mannose glycans.32 These A-type cross-ring fragments are especially abundant around the core 3,6-mannose sugar where the 3,5A and 0,4A ions clearly distinguish the substituents on the 6-arm in all glycans analyzed. These fragments are important for the characterization of the avidin glycans, where the isomeric (32) Reinhold, V. N.; Reinhold, B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784.

2354


bisecting and nonbisecting glycoforms of the hybrid-type glycan Hex5HexNAc4 are differentiated. ACKNOWLEDGMENT The authors thank the BBSRC for funding E.S. and L.G., Dietmar Waidelich and Martin Hornshaw from ABI for their technical advice, and Prof. C. V. Robinson for her guidance and support. Received for review September 22, 2003. Accepted January 30, 2004. AC030333P

Fragmentation Characteristics of Neutral N-Linked Glycans Using a

Recommend Documents