Quantitative Sequencing of Complex Mixtures of

Jun 27, 2007 - Center for Mass Spectrometry of Biopolymers, University of Potsdam, Karl-Liebknecht Strasse 24-25, Building 20,. 14476 Potsdam, Germany...
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Anal. Chem. 2007, 79, 5557-5566

Quantitative Sequencing of Complex Mixtures of Heterochitooligosaccharides by vMALDI-Linear Ion Trap Mass Spectrometry Sophie Haebel,*,† Sven Bahrke,‡ and Martin G. Peter†,‡

Center for Mass Spectrometry of Biopolymers, University of Potsdam, Karl-Liebknecht Strasse 24-25, Building 20, 14476 Potsdam, Germany, and Institute for Chemistry, University of Potsdam, Karl-Liebknecht Strasse 24-25, Building 25, 14476 Potsdam, Germany

Heterochitooligosaccharides possess interesting biological properties. Isobaric mixtures of such linear heterochitooligosaccharides can be obtained by chemical or enzymatic degradation of chitosan. However, the separation of such mixtures is a challenging analytical problem which is so far unresolved. It is shown that these isobaric mixtures can be sequenced and quantified simultaneously using standard derivatization and multistage tandem mass spectrometric techniques. A linear ion trap mass spectrometer equipped with a vacuum matrix-assisted laser desorption ionization (vMALDI) source is used to perform MS2 as well as MS3 experiments. The biopolymer chitin is mainly found in the exoskeleton of crustaceans, insects, and the cell walls of fungi.1 Chitin is composed of 2-acetamido-2-deoxy-D-glucose (GlcNAc or A for “acetylated”) and 2-amino-2-deoxy-D-glucose (GlcN or D for “deacetylated”) linked via β-1,4 glycosidic bonds. It is insoluble in mineral acids and common organic solvents. Deacetylation of chitin gives the acid-soluble aminoglucan chitosan. Oligosaccharides prepared by depolymerization of either chitin or chitosan are called chitooligosaccharides. We have to distinguish homooligomers from heterooligomers. Homooligomers exclusively consist of either glucosamine (D) or N-acetyl glucosamine (A). Heterooligomers are composed of both types of monomer units A and D (formula DxAy; x number of D units, y number of A units; the sum of x and y equals the DP ) degree of polymerization). Heterochitooligomers differ in their total numbers of monomer units, for example, D2A1 (three monomer units, DP 3) and D2A2 (four monomer units, DP 4). Heterochitohomologs have the same DP but differ in the relative numbers of A and D units () FA), for example, D2A1 (two D units, one A unit) and D1A2 (one D unit, two A units), both DP3. With respect to the sequence of A and D units we are able to distinguish numerous isomeric forms of heterochitohomologs which are called heterochitoisobars. For example, DDA, DAD, and ADD show the sequences of the three possible isobars of D2A1. (The reducing end is noted on the right.) * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +49 331 977 2512. † Center for Mass Spectrometry of Biopolymers. ‡ Institute for Chemistry. (1) Peter, M. G. In Biopolymers; Steinbu ¨ chel, A., Ed.; Wiley-VCH: Weinheim, Germany, 2002; Vol. 6, pp 481-574. 10.1021/ac062254u CCC: $37.00 Published on Web 06/27/2007

© 2007 American Chemical Society

The number of isobars for a given homolog DxAy is calculated by the formula (x + y)!/(x!y!). The number of isobars of the homolog D2A2 (DP 4) is 6. For D4A4 (DP 8) the number of isobars increases to 70. Chitooligosaccharides were found to play important roles in various biological processes (for an overview see ref 1). In vertebrates, chitooligosaccharides stimulate the immune system through activation of macrophages2 and induction of a chemotactic migration of polymorphonuclear cells.3 The interaction of N-acetyl glucosamine homooligomers (DP g 4) with the chi-lectin HCgp39 was studied.4 Dissociation constants in the micromolar range and induction of a significant conformational change of the protein emphasize the important role that chitooligosaccharides play in inflammatory and thus immunological processes. In many plants chitooligosaccharides act as growth regulators or elicitors.5 Oligomers and homologs show different biological activities. It was reported that N-acetylglucosamine homooligomers (DP g 7) elicit peroxidase in wheat leaves, heterochitooligomers elicit both peroxidase and phenylalanine-ammonia-lyase, whereas glucosamine oligomers were not active at all.6 Crystallographic structure analyses of the binding domains of several chi-lectins and chitinases,4,7-9 suggest that heterochitoisobars should exhibit different affinities toward receptors. These examples of biological activities of chitooligosaccharides emphasize the importance of a detailed analysis of complex mixtures of heterochitooligomers, homologs, and especially isobars. The chitooligosaccharides used in this study were obtained by enzymatic depolymerization of chitosan, using a family (2) Muzzarelli, R. A. A. Carbohydr. Polym. 1993, 20, 7-16. (3) Usami, Y.; Minami, S.; Okamoto, Y.; Matsuhashi, A.; Shigemasa, Y. Carbohydr. Polym. 1997, 32, 115-122. (4) Houston, D. R.; Recklies, A. D.; Krupa, J. C.; van Aalten, D. M. F. J. Biol. Chem. 2003, 278, 30206-30212. (5) Ernst, B., Hart, G. W., Sinay¨ , P., Eds. Carbohydrates in Chemistry and Biology; Part II, Vol. 4: Lectins and Saccharide Biology; Wiley-VCH: Weinheim, Germany, 2002. (6) Vander, P.; Vårum, K. M.; Domard, A.; El Gueddari, N. E.; Moerschbacher, B. M. Plant Physiol. 1998, 118, 1353-1359. (7) van Aalten, D. M. F.; Komander, D.; Synstad, B.; Gaseides, S.; Peter, M. G.; Eijsink, V. G. H. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8979-8984. (8) Houston, D. R.; Shiomi, K.; Arai, N.; Omura, S.; Peter, M. G.; Turberg, A.; Synstad, B.; Eijsink, V. G. H.; van Aalten, D. M. F. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9127-9132. (9) Rao, F. V.; Houston, D. R.; Boot, R. G.; Aerts, J.; Sakuda, S.; van Aalten, D. M. F. J. Biol. Chem. 2003, 278, 20110-20116.

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18 chitinase from Penicillium sp.10 Oligomers were analyzed by GPC,10-12 homologs by IEC,13 or amino phase separation.14 Matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) MS was employed extensively for the analysis of both species.15 Sequences of heterochitoisobars have been studied by exohydrolysis with N-acetylglucosaminidases and glucosaminidases, followed by reacetylation and chromatographic identification of the N-acetyl glucosamine homooligomers.13,16-18 Analysis of the frequencies of diads and triads by NMR spectroscopy afforded information about the reducing and nonreducing end sugars and the variations in the nearest neighbors.19,20 MALDI-TOF post source decay (PSD) MS was used for the analysis of mixtures of heterochitoisobars affording qualitative information about the sequences present.12 Now we report about a mass spectrometric method employing standard derivatization and multistage tandem mass spectrometric techniques to obtain rapidly quantitative sequence information about complex mixtures of heterochitoisobars up to DP 8. EXPERIMENTAL SECTION Materials. If not stated otherwise all chemicals were bought from Sigma-Aldrich (Taufkirchen, Germany). Activated carbon Empore disks were purchased from 3M, Minneapolis, MN. Two samples (sample 1 and 2) of heterochitooligomers were provided by Genis ehf., Reykjavik, Iceland. They had been prepared by depolymerization of chitosan (sample 1, FA ) 0.6; sample 2, FA ) 0.5) employing a family 18 chitinase from Penicillium sp. (MALDITOF mass spectra of both samples are shown in Supporting Information Figure S-1). Fractionation of Heterochitooligomers. For fractionation, 2.0 g of sample 1 and 2, respectively, were dissolved in 180 mL of 0.05 M ammonium acetate buffer of pH 4.2. The resulting solution was filtered sequentially through a 0.8 µm and a 0.2 µm cellulose acetate membrane (Schleicher and Schuell). Afterwards sample 1 was filtered through a 3 kDa cutoff ultrafiltration membrane (Millipore), and sample 2 was additionally filtered through a 0.5 kDa cutoff membrane. The filtrate was lyophilized. Both samples were subjected in batches of 300 mg to gel permeation chromatography (GPC) on Biogel P4, fine grade (BioRad, Mu¨nchen, Germany): column dimension, 5 cm i.d. × 200 cm; mobile phase, 0.05 M ammonium acetate buffer, adjusted with 0.23 M acetic acid to pH 4.2; flow rate, 1 mL‚min-1; detector, Shimadzu RID 6A. (10) Bahrke, S.; Einarsson, J. M.; Gislason, J.; Haebel, S.; Letzel, M. C.; PeterKatalinic´, J.; Peter, M. G. Biomacromolecules 2002, 3, 696-704. (11) Sørbotten, A.; Horn, S. J.; Eijsink, V. G. H.; Vårum, K. M. FEBS J. 2005, 272, 538-549. (12) Domard, A.; Cartier, N. Int. J. Biol. Macromol. 1989, 11, 297-302. (13) Mitsutomi, M.; Ueda, M.; Arai, M.; Ando, A.; Watanabe, T. In Chitin Enzymology; Muzzarelli, R. A. A., Ed.; Edizioni Atec: Grottammare, Italy, 1996; Vol. 2, pp 273-284. (14) Takahashi, Y. Adv. Chitin Sci. 1997, 2, 372-377. (15) Akiyama, K.; Kawazu, K.; Kobayashi, A. Carbohydr. Res. 1995, 279, 151160. (16) Letzel, M. C.; Synstad, B.; Eijsink, V. G. H.; Peter-Katalinic´, J.; Peter, M. G. Adv. Chitin Sci. 2000, 4, 545-552. (17) Zhang, H.; Du, Y. G.; Yu, X. J.; Mitsutomi, M.; Aiba, S. Carbohydr. Res. 1999, 320, 257-260. (18) Mitsutomi, M.; Isono, M.; Uchiyama, A.; Nikaidou, N.; Ikegami, T.; Watanabe, T. Biosci. Biotechnol. Biochem. 1998, 62, 2107-2114. (19) Vårum, K. M.; Anthonsen, M. W.; Grasdalen, H.; Smidsrød, O. Carbohydr. Res. 1991, 217, 19-27. (20) Vårum, K. M.; Anthonsen, M. W.; Grasdalen, H.; Smidsrød, O. Carbohydr. Res. 1991, 211, 17-23.

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Fractions of 20 mL were collected, appropriately combined, concentrated to a small volume, and finally lyophilized three times up to constant mass. The overall yield was 34% for sample 1 and 54% for sample 2. Preparation of Heterochitohomologs. Only GPC fractions of sample 1 containing several homologs were further separated by high-performance cation-exchange chromatography (HPCEC) to obtain pure homologs. An amount of 4 mg of lyophilized fractions from GPC was dissolved in 200 µL of aqueous hydrochloride of pH 3.0. The solution was filtrated through a 0.45 µm syringe filter with Nylon membrane (Nalgene). The homologs were separated by HPCEC on Resource 30S (Amersham Pharmacia Biotech, Sweden): bed volume, 1 mL; mobile phase, aqueous hydrochloride of pH 3.0 (A), 1 M aqueous sodium chloride solution of pH 3.0 (B); elution profile, 0-5 min 100% A, 5-45 min 100% to 50% A, 45-46 min 50% to 0% A, 46-55 min 0% A, 55-56 min 0% to 100% A, 56-80 min 100% A; flow rate, 1 mL‚min-1; detector, Jasco UV-MD-910. Fractions were collected manually, dialyzed against water (2 L, 4 days) employing Floatalyzers (SpectraPor), concentrated to a small volume, and finally lyophilized. All fractions were analyzed by MALDI-TOF MS for the purity of homologs. Deutero-N-acetylation of chitooligosaccharides with deutero acetic acid anhydride (Ac2O-d6, degree of deuteration 97%) was performed as follows: A 1 mg/mL solution of the chitooligosaccharide was prepared. A volume of 5 µL (5 µg) of this solution was transferred into an Eppendorf tube and dried in a vacuum centrifuge. A volume of 5 µL of a mixture of Ac2O-d6 and methanol (v/v 4:6) was added, and after agitation in a vortexer (30 s), the reaction was left to proceed for 1 h at room temperature. The reaction mixture was then dried in a vacuum centrifuge. For all chitooligosaccharides tested, this treatment resulted in almost complete N-acetylation with some extent of O-acetylation. O-acetylation could be removed selectively by treatment with 10% aqueous NH3 for 15-30 min. Reductive amination of chitooligosaccharides with 2-aminoacridone (AMAC) was performed essentially as described by Okafo et al.21 Preparation of 3-(Acetylamino)-6-aminoacridine (AA-Ac). AA-Ac was prepared according to Charlwood et al.22 by selective acetylation of 3,6-diaminoacridine (proflavine). Reductive Amination of Chitooligosaccharides with AAAc. An amount of 1.5 mg of AA-Ac was dissolved in 17 µL of a (70/30) mixture of DMSO and acetic acid. The final labeling reagent was made by adding 1 mg of Na(CN)BH3 and mixing until it was dissolved. A volume of 2 µL of the reagent was added to 5 µg of the dry chitooligosaccharide, agitated for 30 s, and incubated in the dark for 3 h at 70 °C. The reaction mixture was dried in a vacuum centrifuge. The samples were either analyzed immediately or stored in the dark at -20 °C. Purification of the AA-Ac Chitooligosaccharide Derivatives. The AA-Ac chitooligosaccharide derivative is dissolved in 100 µL of H2O. The purification is performed on small custommade activated carbon columns. Briefly, approximately 2 mm3 of 3M Empore activated carbon material was cut out of a disk and (21) Okafo, G.; Langridge, J.; North, S.; Organ, A.; West, A.; Morris, M.; Camilleri, P. Anal. Chem. 1997, 69, 4985-4993. (22) Charlwood, J.; Birrel, H.; Gribble, A.; Burdes, V.; Tolson, D.; Camilleri, P. Anal. Chem. 2000, 72, 1453-1461.

Figure 2. Mixture of D3A4 isobars was used to demonstrate the reproducibility of the relative peak intensities of fragments with a given DP and varying collision energies (CE). The energies were chosen in the range between low (13%) and total fragmentation (100%) of the parent ion. The relative values were calculated based on peak heights and in one case (to ensure that similar results are obtained) from peak areas. Apparently, a slightly better reproducibility can be obtained from peak areas (see the standard deviation, sd). However, since the Thermo software does not provide access to peak areas, intensities were used throughout this work.

Figure 1. MALDI-TOF spectra recorded to control the success of the derivatization reactions. Spectrum A shows a spectrum of the underivatized mixture of D4A3 isobars. Spectrum B shows the same sample after N-deutero-acetylation. Some minor over- and underacetylations are observed. Spectrum C shows the same sample after reaction of the reducing end with AA-Ac; it can be noted that in contrast to the previous spectra the sample preferentially ionizes by proton rather than Na+ adduct formation.

squeezed into the narrow part of an Eppendorf GELoader pipet tip. The column is conditioned with 40 µL of 80% acetonitrile/20% H2O followed by 40 µL of H2O. A volume of 20 µL of the derivative solution is applied to the column; the flow-through is colorless. The column is washed with 40 mL of H2O. Underivatized oligosaccharide is eluted with 20 µL of 20% acetonitrile/80% aqueous TFA (0.1%). The derivatized oligosaccharide is eluted with 40 µL of 40% acetonitrile/60% aqueous TFA (0.1%). Mass Spectrometry. An aliquot of the purified AA-Ac chitooligosaccharide derivative (0.5 µL) was mixed on the target with 0.5 µL of DHB matrix solution (2,5-dihydroxybenzoic acid; 15 mg/ mL in 30% aqueous methanol), and the drop was dried under a gentle stream of air. MALDI-TOF mass spectra were recorded on a Bruker Reflex II (Bruker Daltonik, Bremen, Germany) in the positive ion mode. All spectra were measured in the reflector mode using external calibration (angiotensin II).

MALDI ion trap mass spectra were recorded on a Finnigan vMALDI-LTQ mass spectrometer (Thermo Electron Corporation). All experiments were performed by careful selection of the monoisotopic peak of the tagged, per-N-acetylated mixture of chitooligosaccharides. Since a single isolation did not provide a clean selection of the monoisotopic peak, the isolation was performed in two steps by using a window of 1.5 mass units, carefully adjusting the center of the windows so that in the first step the 13C isotopes are completely removed and in the second step the lower mass peaks, which arise from incomplete deuteration. MS2 spectra could be recorded in the normal as well as in the zoom mode which provides much higher resolution. Due to its higher sensitivity, the normal mode was preferred for MS3 spectra. To obtain the best possible signal-to-noise ratio for the fragment peaks, the collision energy was adapted to obtain complete or almost complete fragmentation of the parent ion. Although higher collision energies should not affect the fragment ion spectrum, care was taken not to exceed this minimum value. Cation-Exchange Chromatography. A MONO S column (GE Healthcare) was used on a SMART System (GE Healthcare). An amount of 20 µg of D2A3 was loaded to the column and eluted with a gradient of 0.6 M NaCl in 20 mM formic acid of pH 3.8. UV detection was performed at 214 nm. RESULTS AND DISCUSSION The aim of the present work was to determine the relative amounts and sequences of the components of an isobaric mixture of heterooligosaccharides (DxAy) by multistage (sequential) tandem mass spectrometry (MSn). Underivatized heterochitooligosaccharides fragment almost exclusively by cleavage of the glycosidic bond yielding Y- and Z-type ions if the charge is retained on the reducing end or B- and C-type ions if the charge is retained on the nonreducing end, respectively. Due to overlapping m/z values for ions of identical monosaccharide compositions, we cannot distinguish between a fragmentation of native heterochitooligosaccharides from the reducing or nonreducing end. Thus, Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

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Figure 3. MS2 spectrum of the protonated D3A4-T parent ion at m/z 1684. A clean isolation of the monoisotopic peak was performed prior to fragmentation. Nevertheless, the fragment ions exhibit an isotopic pattern due to the fact that some incompletely deuterated, 13C-containing parent ions were coisolated with the fully deuterated monoisotopic species. This can be clearly seen in the insets representing enlarged views of the respective Y fragment cluster.

for sequencing, the introduction of a tag at the reducing end of the molecule is essential to distinguish B- from Z-type and C- from Y-type ions. In initial experiments, AMAC was used for this purpose. However, since the mass increment introduced by AMAC is equal to 194 Da, an m/z overlap between B and Y ions may occur. The results presented here were obtained by using the AA-Ac tag introduced by Charlwood et al.22 This tag has the additional advantage of strongly retaining the charge (in this case a proton) on the reducing end of the molecule so that fragmentation gives rise to almost exclusively Y ions which all carry the AA-Ac tag (T). This simplifies the spectra and increases the signalto-noise ratio of the ions which are relevant for the sequencing. The quantification of different sequence components of an isobaric mixture is based on relative peak areas (or intensities) of Y fragments of identical DP but different monosaccharide composition (FA) in the MSn spectra. The fragment Y3, for example, could theoretically appear at four masses: A3-T, D1A2T, D2A1-T, and D3-T. Therefore, to obtain reliable results which reflect the real composition of the sample, peak intensities must be highly reproducible and the fragmentation probability of a given glycosidic bond must be independent of the extent of fragmentation (the collision energy) as well as of the type of monosaccharides (GlcN vs GlcNAc) involved in the bond. Earlier experiments have shown that this is unfortunately not the case. Indeed, the lower the collision energy, the stronger the fragmentation probability is influenced by the monosaccharides involved in the glycosidic bond. This became clear when comparing MS2 spectra of the same tagged chitooligoisobar mixture either as (M + 2H)2+ species (ESI) or M + Na+ species (MALDI). The 5560 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

relative peak intensities within the Y fragment cluster of a given DP were extremely different for both species of ions. Thus, the chitooligoisobar mixture was per-N-acetylated with Ac2O-d6 prior to the tagging reaction to transform all GlcN units to GlcNAc-d3. After this reaction both sides of the glycosidic bond are equivalent from a mass spectrometric point of view, but the fragment composition can still be derived from the measured mass. The procedure as well as the MALDI-TOF spectra acquired for controlling the success of the reaction are shown in Figure 1. In the mass spectra a “former” glucosamine will now be a deuterated N-acetyl glucosamine with a mass of 206 Da, whereas the original N-acetyl glucosamine has a mass of 203 Da (mass values are calculated for monosaccharides in the polymer chain taking into account the loss of water in the glycosidic bond formation). Under these conditions, the relative peak areas (and intensities) of the Y fragments of identical DP but different monosaccharide composition (i.e., m/z) are independent of the original monosaccharides on both sides of the glycosidic linkage as well as independent of the collision energy. Figure 2 illustrates that this is indeed the case and that a high reproducibility between different spectra is achieved, although significantly different collision energies were used. It is also shown that highly similar results are obtained by calculating the relative amounts from either peak areas or peak intensities. Quantitative Sequencing of the Mixture of D3A4 Heterochitoisobars. The new quantitative sequencing procedure for isobaric mixtures of heterochitooligosaccharides is illustrated in detail by means of a relatively simple mixture of DP7 and FA 0.57,

Figure 4. MS3 spectrum of the mixture of Y6 fragments of composition D3A3-T (m/z 1481) which were obtained by fragmentation of the D3A4-T precursor ion (see Figure 3). The inset lists the three possible components of the mixture. On the left of the inset is given the relative amount of these components among all possible Y6 fragments (the value was calculated from the corresponding MS2 spectrum). On the right is given the quantitative information which can be derived from the MS3 spectrum.

Figure 5. Schematic description of the quantitative sequencing procedure for a mixture of D3A4 isobars. Sequences that may be excluded due to the absence of a peak at the corresponding mass are printed in gray. Results which were obtained from the MS3 spectra of the two Y6 fragments at m/z 1481 (see Figure 4) and 1478 (result not shown) are printed in dark and light blue, respectively.

D3A4. Figure 5 illustrates schematically the conclusions which can be drawn from the MS2 spectrum in Figure 3 and the MS3 spectrum in Figure 4. The starting point is the smallest fragment in the MS2 spectrum which includes the tag at the reducing end (T). The peak at m/z 457 results from the protonated Y1 fragment A-T. The absence

of a peak at m/z 460 indicates that no fragment D-T is produced, i.e., all sequences in this isobaric mixture of D3A4 end with an A at the reducing end. For the Y2 fragment, again a single peak is observed corresponding to the fragment AA-T. Thus, all chitooligosaccharides in this mixture end with the sequence AA-T. The next higher fragment Y3 is a doublet resulting from a small peak Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

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Figure 6. MS2 spectrum of the protonated D4A4-T parent ion at m/z 1890. A clean isolation of the monoisotopic peak was performed prior to fragmentation.

at m/z 863 (AAA-T) and a large one at m/z 866 (DAA-T). From the relative intensities of the peaks, the relative amounts of the two fragments can be derived (AAA-T 4.4%; DAA-T 95.6%). For the next higher fragment Y4, three peaks could theoretically be present, D0A4-T (m/z 1066), D1A3-T (m/z 1069), and D2A2-T (m/z 1072). However, only two peaks are observed at m/z 1069 (81%) and 1072 (19%). The absence of a peak at m/z 1066 allows us to exclude all sequences ending with AAAA-T. The relative intensities of the other two peaks allow us to say that sequences ending with the motif DDAA-T represent 19% of the overall sequences, and by taking advantage of the information obtained from the Y3 fragment, the amount of sequences ending with DAAA-T and ADAA-T can be deduced to be 4.4% and 76.6%, respectively (see Figure 5). The next higher fragment Y5 is again present as a single peak at m/z 1275. As shown in Figure 5 this peculiarity allows us to deduce the relative amounts of all possible Y5 fragment sequences knowing the relative amounts of Y4 sequences. For the next fragment Y6, two monomer compositions are possible (D2A4-T and D3A3-T), and both are present since we observe a doublet of peaks at m/z 1478 and 1481. At this stage the information which can be gained from the MS2 spectrum is not anymore sufficient to deduce the relative amounts of the possible Y6 fragment sequences depicted in Figure 5. For each peak, three different Y6 fragment sequences are possible and only the relative amount of the sum of them is known from the MS2 spectrum. At this point an MS3 spectrum is needed to further unravel this isobaric mixture of chitooligosaccharides. Recording an MS3 spectrum of the Y6 MS2 fragment at m/z 1481 (D3A3-T) is equivalent to fragmenting the subset of intact D3A4-T sequences which can produce a Y6 fragment of composition D3A3-T (marked 5562 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

by a dark blue spot in Figure 5). On the basis of this MS3 spectrum the relative amounts of the components in this subset of sequences can be deduced analogously to the procedure described for the MS2 spectrum. The knowledge (from the MS2 spectrum in Figure 3) that the subset of D3A4-T sequences which can produce a Y6 fragment of composition D3A3-T amounts to 50.7% of all sequences subsequently allows for the calculation of the overall relative amounts. Briefly, from the MS2 spectrum we know that the three fragments DDDAAA-T, DDADAA-T, and DADDAA-T represent 50.7% of the Y6 fragments. From the MS3 spectrum shown in Figure 4, we can deduce that from this subset of sequences, 5.7% end with AAA-T and 94.3% end with DAA-T, which means that from the overall number of sequences 50.7% from 5.7%, i.e., 2.9%, end with AAA-T and 50.7% from 94.3%, i.e., 47.8%, end with DAA-T. In the same manner we can deduce that 17.6% (50.7% of 34.7%) end with DDAA-T and 33.1% (50.7% of 65.3%) end either with DAAA-T or with ADAA-T (see the inset in Figure 4). From these values, the relative amounts of the three fragments giving rise to the peak at m/z 1481 (marked with a dark blue spot in Figure 5) can be deduced. For each of the D3A3-T fragments there is a complementary fragment of composition D2A4-T (marked with a light blue spot in Figure 5) where the D at the nonreducing end is replaced by an A (see Figure 5). The relative amount of these fragments can be calculated knowing the relative amounts of the corresponding Y5 fragments. Since each MS3 spectrum gives information on another subset of sequences, the information obtained when more than one MS3 spectrum is recorded is, in general, redundant, and several values for the relative amount of a given sequence can be obtained. In Figure 5, the values obtained from the MS3 fragmentation of the

Figure 7. Schematic description of the quantitative sequencing procedure for a mixture of D4A4 isobars. Sequences which can be excluded are printed in gray. Results printed in black were derived from the MS2 spectrum (Figure 6). All other results are color-coded according to the MS3 spectra from which they were obtained (see Supporting Information Figures S-2, S-3, and S-4).

Figure 8. Relative amounts of isobaric components as determined for D4A4 are used to calculate the theoretical relative peak heights of the Yx peaks of varying m/z (composition). These values are compared to the relative peak heights as determined from the MS2 spectrum shown in Figure 6. Good agreement confirms the results of the quantitative sequencing procedure.

Y6 fragments at m/z 1478 (D2A4-T) and m/z 1481 (D3A3-T) are printed in light and dark blue, respectively. In principle both ways should give the same result, which means that the precision of the quantitative sequencing can be assessed on this basis. In conclusion it can be said that the mixture of D3A4 isobars is composed of six sequences from which three are rather abundant: DADADAA-T 45.4%, ADDADAA-T 31.2%, ADADDAA-T 17.5%. Three others are present in the range of a few percent. The precision of this measurement can be estimated to be around 2 percentage points, which is in close agreement to the reproducibility of the measurement illustrated in Figure 2.

The sequencing of this particular isobaric mixture shows a peculiarity which allows an appreciation of the methods reliability. Since the Y5 fragment is present as a single peak at m/z 1275, all sequences in the mixture of D3A4 isobars share the common D2A3-T fragment. Consequently, the MS3 spectrum of m/z 1275 must give the same relative amounts for the fragments DP1 to DP4 as the MS2 spectrum. The values derived from this MS3 spectrum are given in italics in Figure 5 and differ by not more than 1 percentage point from the values obtained employing the MS2 spectrum. Quantitative Sequencing of a Mixture of D4A4 Heterochitoisobars. Figure 7 summarizes the results obtained for the mixture of D4A4 isobars (MS2 spectrum shown in Figure 6). The sequencing of the mixture of D4A4 isobars is more complex than for the mixture of D3A4 isobars and requires the use of a minimum of four MS3 spectra. However, the principle is the same, i.e., every peak which is used for MS3 measurements represents a subset of the components in the mixture and the MS3 spectrum gives quantitative sequence information on this subset of sequences (in Figure 7 the determined relative amounts are color-coded according to the m/z value of the precursor fragment ion they were derived from). Although the information obtained from different MS3 spectra is redundant in many cases, it is advisable to use as many MS3 spectra as possible since good agreement of the results obtained via different “paths” increases the confidence in the results. In this case the seven MS3 spectra depicted in Supporting Information Figures S-2, S-3, and S-4 are used, and the results summarized in Figure 7. Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

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Table 1. Overview of the Mixtures of Heterochitoisobars, the Components of Which Were Sequenced and Quantified Simultaneouslya DP5

DP6

D2A3

DADAA DDAAA ADDAA others

D2A4

57% 26% 15% 2%

(2%

DDAAAA DADAAA ADDAAA DAADAA ADADAA AADDAA

DP7 D3A3

4% 43% 12% 9% 19% 12%

(2%

DDADAA DADDAA

D4A3

51% 49%

DDDADAA DDADDAA DADDDAA ADDDDAA DDADADA DADDADA DADADDA others

(1%

D3A4

4% 42% 33% 11% 2% 3% 2% 3%

DADDAAA ADDDAAA DADADAA ADDADAA DAADDAA ADADDAA

(2%

2% 3% 45% 31% 2% 17%

(2%

DP8

DP9

D4A4

D4A5

DADDDAAA ADDDDAAA DDADADAA DADDADAA ADDDADAA DADADDAA ADDADDAA ADADDDAA others

3% 2% 18% 34% 6% 20% 11% 2% 4% (2%

DADADDAAA ADDADDAAA DADADADAA ADDADADAA ADADDADAA others

9% 8% 27% 14% 25% 17%

(5%

a The estimated precision of the determined relative amounts of sequences in the mixture is given in the last line (in percentage points). The estimation is based on the repetition of measurements and on results obtained from redundant MS3 measurements.

Figure 9. Separation of the mixture of D2A3 isobars by cation-exchange chromatography. The chromatogram is fitted with the sum of three double-Gaussian curves varying in intensity and retention time. The shape of the peaks, i.e., the parameters for the two Gaussian curves added to introduce peak asymmetry, was optimized first. Identical shapes were assumed for the peaks of the three components. Only the intensities and the retention times were optimized in a second step.

Once a result is obtained and the amount of each isobar present in the mixture is known, a simple check can be performed by calculating the theoretical MS2 spectrum (Y fragment intensities) from these values and comparing it to the measured one. This comparison is shown in Figure 8 and demonstrates that the values are in excellent agreement. Perspectives and Limitations. With the use of this procedure, a number of mixtures of linear heterooligosaccharides was sequenced and quantified. The chain length ranged between DP5 and DP9, and the results are summarized in Table 1. For sequencing and quantification the following general rules are followed: spectra with the best signal-to-noise ratio are used with priority to gain quantification and sequence information. Therefore, as much information as possible is taken from the MS2 spectrum before MS3 spectra are recorded, preferentially selecting the peaks with the highest intensity. This technique was very successfully applied to mixtures up to m/z 2000 (DP 8) which is also technically the limit of the linear ion trap standard mass range. Although the Thermo instrument which was used in the present study has an extended mass range 5564 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

of up to m/z 4000, it only functions at the expense of a lower sensitivity, which is of course detrimental to the precision of the peak height determination. Chemical limitations also become more severe at masses around 2000 Da. First, since the hexadeutero acetic acid anhydride possesses a deuteration degree of only 97%, the isotopic pattern of the deutero-N-acetylated oligosaccharide becomes broader for longer chains, and the overlap of the deuteration and 13C patterns results in a stronger contamination of the monoisotopic peak by incompletely deuterated, 13C-containing, species. This problem could be reduced using a higher degree of deuteration or more selective MS/MS isolation techniques like FTICR. Second, the tagging reaction becomes less effective for oligomers of higher DP, especially for the per-N-acetylated ones. Yet another, more fundamental problem is the increasing complexity of the mixtures. A mixture of D3A3 isobars could theoretically be composed of up to 20 components, and the mixture of D4A4 could contain up to 70 isobars of different sequences. Assuming equal amounts of isobars, each component would be present at 5% and 1.4%, respectively. For mixtures with randomly distributed acetylation pattern, the amount of all

components is in the range of a few percent, and no significant values could be derived from such an experiment. However, the common production process of heterochitooligosaccharides (including deacetylation of chitin followed by enzymatic depolymerization of the resulting chitosan) apparently preferentially yields defined sequences. Nevertheless, the example of D4A4 shows that it contains eight sequences in the 0-3% range, and it is, of course, questionable how reliable this information is. It is possible that in some rare cases MS3 experiments will not be sufficient to quantify all components of a given mixture. In that case MS4 experiments can provide the necessary information to solve the problem. It was shown on several examples (m/z below 2000) that the linear ion trap instrument is capable of recording MS4 spectra of good quality from the samples investigated. From the samples listed in Table 1, only the DP9 mixture would actually require an MS4 measurement to precisely quantify all components of the mixture. However, in the extended mass range mode (m/z > 2000) the sensitivity of the instrument is too low to obtain MS4 spectra with sufficient signal-to-noise ratio to rely on the peak intensities. The problem can be circumvented by setting the amount of one of the components, whose maximum possible value was previously determined (by MS3) to be 4%, to either 0% or 4% and calculating the corresponding values for the other components. This of course yields a higher uncertainty (5%, see Table 1); nevertheless, it allows us to determine the principal components of the mixture and their approximate amounts. As shown in Table 1, very precise results could be obtained for mixtures up to DP8. Less precise, though acceptable, results could still be obtained for DP9 and could probably be obtained for even higher DP, but the difficulties are expected to increase rapidly. Chromatographic Separation of a Mixture of D2A3 Heterochitoisobars. The result for the mixture of D2A3 isobars is of special interest since it was possible to achieve an incomplete, however satisfactory, separation of this mixture on a cationexchange stationary phase (see Figure 9). The collected fractions were derivatized with AMAC, and MS2 spectra were recorded to identify the components in the fractions (results not shown). Peak 2 contains an almost pure isobar of sequence DADAA. Peak 3 contains a majority of DDAAA with some contamination of DADAA, and peak 1 contains mainly ADDAA with a minor contamination of ADADA (results not shown). This result allows a crude comparison of the quantitative values obtained by mass spectrometric sequencing/quantification and the peak areas of the chromatogram. Since the chromatographic peaks are not baseline separated, a simple integration of the peak area is not possible. It was therefore attempted to fit the chromatogram with Gaussian functions. A satisfactory result could be obtained by fitting each peak with the sum of two Gaussian functions to introduce the required peak asymmetry (see Figure 9). Additional complications occur since apparently sugar anomers are separated, as well. The small peak on the right edge of the chromatogram (peak 4) was shown to contain the same sequence as peak 2. The anomers of the other components are probably hidden by the larger peaks. Nevertheless, a reasonably good agreement between the values from the analysis of chromatographic peak areas and those obtained by mass spectrometry could be achieved.

CONCLUSIONS It could be demonstrated that derivatization techniques combined with vacuum MALDI (vMALDI) ion trap MSn measurements allow one to unravel complex mixtures of isobaric chitooligosaccharides elucidating the sequences and relative amounts of the components. The evaluation of the sequences of the mixtures of D2A3, D3A3, D2A4, D4A3, D3A4, D4A4, and D4A5 isobars shows that the partial hydrolysis of chitosan employing family 18 chitinases results in heterochitooligosaccharides characterized by a reducing end A unit, whereas the nonreducing end is either D or A. The result is explained by the cleaving specificity of family 18 chitinases preferably hydrolyzing -A-D- or -A-A- bonds, as the catalytic mechanism of these enzymes requires stringently the neighboring group assistance of the acetamido group of a nonreducing end A unit.23 The first step of the sequence analysis for the quantification of isobaric mixtures of heterochitooligosaccharides is the recording of MS2 spectra. The selection of the ion of interest in the mass spectrometer, previous to the first fragmentation step, offers the potential for the sequence analysis of heterochitoisobars from complex mixtures. The amount of each isobaric mixture might thus be much smaller than shown in the present case. In the MS2 experiment, all components of the isobaric mixture are fragmented simultaneously. In some cases this information already allows us to sequence and quantify the components of the mixture (for example, the mixture of D3A3 isobars, see Table 1). However, for the vast majority of mixtures of heterochitoisobars the information from the MS2 spectrum is not sufficient. In this case, additional information from one or several MS3 spectra (and in rare cases even MS4 spectra) is required to fully characterize the components of a complex mixture. Further structural analysis of a first generation fragment yields information on the subset of components that are capable of producing the fragment under investigation. Every fragment investigated, i.e., every different MS3 spectrum, generates information for a different subset of sequences. This combined data allows us to distinguish and quantify the components of the mixture assuming that the relative peak heights are equal to the relative amounts. This assumption can be made due to the generation of a mass spectrometric equivalence of all monosaccharides in the carbohydrate chain by per-deutero-N-acetylation in a simple and fast reaction. Derivatization of the reducing end with AA-Ac not only introduces a tag which allows us to distinguish Y from B ions, it also remarkably influences the fragmentation mechanism, strongly favoring the formation of Y ions including the tagged reducing end of the molecule. The almost exclusive formation of the sequencing relevant Y ions has two positive effects: First, it improves the signal-to-noise ratio since no signal is “wasted” on other ions; second, the spectra become very clear and can be interpreted in a straightforward manner. The vMALDI-linear ion trap mass spectrometer has proven to be ideally suited for this kind of investigation since it provides the necessary capacity of performing MSn experiments associated with high sensitivity and good front-end resolution. (23) Terwisscha van Scheltinga, A. C.; Armand, S.; Kalk, K. H.; Isogai, A.; Henrissat, B.; Dijkstra, B. W. Biochemistry 1995, 34, 15619-15623.

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These features make this new method a straightforward and reliable technique for the sequencing and quantification of isobaric components of heterochitooligosaccharide mixtures which can so far not be resolved by chromatographic techniques. ACKNOWLEDGMENT The first two authors contributed equally to this work.

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SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 28, 2006. Accepted May 10, 2007. AC062254U