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Quantitative Mass-Spectrometric Sequencing of Chitosan Oligomers Reveals Cleavage Sites of Chitosan Hydrolases Stefan Cord-Landwehr, Phillip Ihmor, Anna Niehues, Heinrich Luftmann, Bruno M. Moerschbacher, and Michael Mormann Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04183 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on February 13, 2017

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Analytical Chemistry

Quantitative Mass-Spectrometric Sequencing of Chitosan Oligomers Reveals Cleavage Sites of Chitosan Hydrolases Stefan Cord-Landwehr1, Phillip Ihmor1†, Anna Niehues1, Heinrich Luftmann², Bruno M. Moerschbacher1*, and Michael Mormann³ 1

Institute for Biology and Biotechnology of Plants, University of Münster, Schlossplatz 8, 48143 Münster, Germany

²Institute for Organic Chemistry, University of Münster, Corrensstraße 40, 48149 Münster, Germany ³Institute for Hygiene, University of Münster, Robert-Koch-Str. 41, 48149 Münster, Germany

Supporting Information Placeholder ABSTRACT: Partially acetylated chito-oligosaccharides (paCOS) have diverse bioactivities that turn them into promising compounds especially for medical and agricultural applications. These properties likely arise from different acetylation patterns, but determining the sequences of paCOS and producing paCOS with patterns of interest have proven difficult. We present a novel method for sequencing sub-microgram amounts of paCOS using quantitative mass spectrometry, allowing to rapidly analyze the substrate specificities of chitosan hydrolases that can be used to produce paCOS. The method involves four major steps: (i) acetylation of free amino groups in paCOS using a deuterated reagent; (ii) labeling the reducing end with an 18O-tag; (iii) quantifying paCOS using [13C2, 2H3]-labeled isotopologs as internal standards; and (iv) sequencing paCOS by tandem MS. Eventually, this method will aid in developing enzymes with cleavage patterns optimized for producing paCOS with defined patterns of acetylation and specific bioactivities.

INTRODUCTION Chitin, a linear biopolymer of β(1,4)-linked N-acetylglucosamine (GlcNAc; A) residues, is one of the most abundant renewable resources, and its partially deacetylated counterparts, chitosans, consisting of both GlcNAc (A) and glucosamine (GlcN; D) units, are among the most promising functional bio-polymers. As a result of their excellent material properties and versatile biological activities, chitosans can be used in many fields. These include medical applications for scar-free wound healing1,2 or anti-tumor1–5 and anti-inflammation therapy1–3, as well as agricultural utilization for protecting crops from diseases3,6,7 and as a substitute for antibiotics2 in animal feed. Chitosans have such a wide applicability because their structures vary, giving rise to different properties. They can differ in their degrees of polymerization (DP) and acetylation (DA), and in their pattern of acetylation (PA). These parameters deeply influence their physico-chemical properties and biological functionalities.8–10 However, molecular structure-function relationships and cellular modes of action of chitosans are not well understood today. While structural differences make chitosans widely useful, these differences also make it difficult to determine which activities correspond to which structural patterns.10 Powerful tools for analyzing and modifying chitosans are chitinase and chitosanase enzymes cutting chitosans at

specific sequence motifs.10 But for these enzymes to be useful, both for the development of fingerprinting analyses and for the production of defined partially acetylated chitosan oligosaccharides (paCOS), precise knowledge of their subsite specificities is required.5,6,11 Unfortunately, we know these for only a few chitosan hydrolases, mostly because so few analytical tools exist that allow detailed analysis of these enzymes’ polymeric substrates and oligomeric products, especially when only small amounts of these compounds are available.12 For example, the conventional analytical method, size-exclusion chromatography followed by nuclear magnetic resonance (SECNMR), requires many milligrams of sample, only separates oligomers according to their DP, and only gives average values of the DA and of the occurrence of GlcN or GlcNAc subunits at or near the reducing and nonreducing ends of the oligomers.13,14 Also, SEC-NMR is time consuming, but rapid analyses are needed e.g. for screening steps in evolutionary optimizations of chitosan modifying enzymes. To overcome these drawbacks, mass spectrometry (MS) has recently emerged as a powerful tool for rapid glycan analysis at sub-microgram sensitivities, but quantification is difficult when using MS.15–17 Several approaches have been developed to achieve quantitative glycan MS analyses. Methods involving chemical modification or derivatization with hydrophobic or ionic reagents are used for

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relative quantification of glycans. Alternatively, to measure unmodified oligosaccharides, multiple reaction monitoring (MRM) can be performed.18 However, both methodologies are associated with serious drawbacks.15–17,19 Unintended fragmentation of analyte ions by in-source decay observed both under electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) conditions may lead to distorted results. Furthermore, even minor structural differences of analytes often result in altered ionization efficiencies that can only partially be balanced by derivatization methods. Thus, major issues related to instrument responses and ionization efficiencies or fragmentation processes make absolute quantification problematic. These can be overcome by using internal standards labeled with stable isotopes, which allow for relative and absolute quantification of glycans.20 Labeling-based techniques involve differential methylation employing various methyl iodide isotopologs, reductive amination by 2H- or 13C-labeled reagents, or stable isotope-labeled carbonyl-reactive tandem mass tags.21–23 However, these approaches are not suitable for comprehensively characterizing paCOS obtained by enzymatic hydrolyses of polymeric chitosans because these result in highly heterogeneous mixtures of oligomers differing in DP and, more importantly, in DA and PA, which limits the chromatographic resolution of individual species. Furthermore, paCOS cannot be absolutely quantified using any of these methods due to the lack of a library of isotopically labeled internal standards. We, therefore, developed a labeling approach for MS that can absolutely quantify paCOS and unambiguously determine their sequences, even in complex mixtures. Thus, this method can also be used to probe the subsite specificities of chitosan hydrolases by quantitative sequencing of the oligomeric products obtained upon hydrolysis of a chitosan polymer. As a benchmark, we chose to examine the chitinase ChiB from Serratia marcescens 24, as this is the best-studied chitinase and therefore allows us to assess the accuracy of our method and compare it with existing data. EXPERIMENTAL SECTION Production of ChiB. E. coli Rosetta [DE3] pET22b: ChiB-CStrepII was used to produce ChiB. Cultivation, harvesting, and enzyme purification were performed as described by Hamer et al.25 Substrate production and analysis. Chitosan DA0%, kindly provided by Dominique Gillet, Mahtani Chitosan PVT.LTD (Veraval, India), was N-acetylated to DA50% and DA60% according to the method by Lamarque et al.26 The DA was analyzed using 400 MHz 1H NMR, and the DP (1100 for both chitosan polymers) and polydispersity (1.57 for DA50% and 2.61 for DA60%) were analyzed using SEC-RI-MALLS.27 Enzymatic hydrolyses of chitosan polymers by ChiB. The chitosan polymer (DA50% or DA60%, 300 µg) and 0.6 µg ChiB were incubated in 300 µl of ammonium acetate buffer (pH 5.8; 80 mM) at 37 °C. The reaction was

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stopped after 5, 20, 60, 180, or 1440 min by adding 300 µl 10% (v/v) NH4OH to the mixture and incubating for 5 min at 98 °C. The ammonium acetate buffer was removed in vacuo and the dried samples were dissolved in 300 µl H2O. Reducing end assay. A modified procedure based on the method reported by Horn et al.28 was used to determine the number of reducing ends in the oligomer mixtures derived from hydrolysis of the chitosan polymers. To this end, 40 µg of the products dissolved in 40 µl H2O were mixed with 40 µl 0.5 M NaOH, 20 µl 3-methyl-2benzothiazolinone hydrazone (3-MBTH; 3 mg/ml), and 20 µl dithiothreitol (DTT; 1 mg/ml). The solution was incubated for 15 min at 80 °C before 80 µl staining reagent were added. The staining reagent consisted of 0.5% (w/v) FeNH4(SO4)2, 0.5% (w/v) H3NSO3 and 0.25 M HCl, and the absorbance was measured at 620 nm in a microplate reader. The amounts of reducing ends were calculated using a standard curve produced with the Nacetylglucosamine monomer (50, 125, 250, 500, and 750 µM) (Sigma-Aldrich, St-Louis, USA). Product preparation for quantitative MS analysis. The [2H3]N-acetylation of the ChiB chitosan polymer hydrolysate was achieved using a modified version of the methods reported by Haebel et al.19 and Pohlentz and Egge.29 To this end, 2.5 µl of [2H6]acetic anhydride (SigmaAldrich, St-Louis, USA) was added to 30 µg of ChiBdigested chitosan polymer dissolved in 50 µl 1:1 50 mM NaHCO3:MeOH. Stepwise addition of the [2H6]acetic anhydride - which involved five steps each with 15 min incubation at 30 °C and 1200 rpm - resulted in complete acetylation of the free amino groups of the glucosamine units of the chitosan oligomers. Vacuum drying (1200 rpm at 30 °C for 1 h) was used to stop the reaction. Subsequently, the dry samples were dissolved in 70 µl H2O and freeze-dried to remove the remaining volatile compounds. Reaction products were monitored by use of LCMS. No evidence for the presence of oligosaccharides harboring unreacted free amino groups or O-acetylated was found in any samples examined. Syntheses of the double isotopically labeled internal standards (R*1-6). The methods of Haebel et al.19 and Pohlentz and Egge29 were modified to produce the double isotopically labeled internal standard R*1-6. As such, 2.5 µl of [2H6; 13C4]acetic anhydride (Sigma-Aldrich, St-Louis, USA) was added to 10 µg of chitosan oligomers with DP 1-6 (Carbosynth, Compton, UK) dissolved in 50 µl 1:1 50 mM NaHCO3:MeOH. Addition of the [2H6; 13 C4]acetic anhydride, which was done in five steps, each with 15 min incubation at 30 °C and 1200 rpm, resulted in the complete conversion of the glucosamine units to Nacetylglucosamine units with 2H and 13C carbon incorporated in the acetyl groups. Vacuum drying (1200 rpm at 30 °C for 1 h) was used to stop the reaction. Subsequently, the dried samples were dissolved in 70 µl H2O and freezedried to remove the remaining volatile compounds. Reaction products were monitored by LC-MS analysis. Again, no unreacted free amino groups or O-acetylation were detected in any samples.


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Product preparation for quantitative tandem MS analyses. Quantitative analyses of the patterns of the [2H3]N-acetylated chitin oligomers with LC-MS2 required differentiating between fragments harboring the reducing end and those comprising the non-reducing end. Thus, the reducing end of the chitin oligomers was labeled using a modified protocol of the method previously reported by Körner et al.30 As such, 10 µg of dry and [2H3]Nacetylated oligomers, products of the chitosan polymer digestion with ChiB, were dissolved in 5 µl H218O (eurisotop, Saint-Aubin, France) containing 0.1% formic acid and incubated for 6 h at 70 °C. The samples were dried in vacuo (1200 rpm at 30 °C for 15 min), dissolved in 10 µl H218O containing 0.1% formic acid, and incubated for 16 h at 70 °C. UHPLC-ESI-MSn method for quantitative analysis of isotopically labeled chitin and chitosan oligomers. The previously modified products from ChiB’s hydrolysis of the chitosan polymer were analyzed using the method described by Hamer et al.31 To this end, we used a Dionex Ultimate 3000RS UHPLC system (Thermo Scientific, Milford, USA) with an Acquity UPLC BEH Amide column (1.7 µm, 2.1 mm x 150 mm; Waters Corporation, Milford, MA, USA) combined with a VanGuard pre-column (1.7 µm, 2.1 mm x 5 mm; Waters Corporation, Milford, MA, USA) to perform a hydrophilic interaction liquid chromatography (HILIC) for separation of the chitin oligomers. The UHPLC system was coupled to an amaZon speed ESI-MSn detector (Bruker Daltonik, Bremen, Germany) and an evaporative light scattering detector (Model Sedex 90LT, Sedere, Alfortville, France). A flow rate of 0.4 ml/min and 35 °C oven temperature were used. To perform the quantitative analysis, we used 2 μl of the sample containing 1000-2000 ng of the partially isotopically labeled products from ChiB chitosan polymer hydrolysis and 75 ng each of the double isotopically labeled internal standards R*1-6. We performed an LC run over 30 min using the following gradient elution profile for sample separation: 0-3 min, isocratic 100% A (80:20 ACN:H2O with 10 mM NH4HCO2 and 0.1% (v/v) HCOOH); 3-23 min, linear from 0% to 20% (v/v) B (20:80 ACN:H2O with 10 mM NH4HCO2 and 0.1% (v/v) HCOOH); 23-25 min, linear from 20% to 75% (v/v) B; 25-26 min, isocratic 75% (v/v) B; column reequilibration: 26-27 min, linear from 20% (v/v) to 100% A; 27-30 min, isocratic 100% A. The LC-MS method was divided into six segments to achieve optimal ion transmission and detection for the chitin oligomers eluting at that time point of the analysis, where the target mass in the smart parameter settings of the trapControl software operating the amaZon speed instrument was set to the following m/z values in each segment: segment 1 (min 14), m/z 200; segment 2 (min 4-7.5), m/z 420; segment 3 (min 7.5-11.5), m/z 630; segment 4 (min 11.5-16), m/z 840; segment 5 (min 16-19), m/z 1040; segment 6 (min 19-25), m/z 1240. Compound stability and trap drive level were set to 100%. Mass spectra were acquired in positive mode and over a scan range of m/z 50 to m/z 2000, using the enhanced resolution scan mode. The spectra were then analyzed with Data Analysis 4.1 software (Bruker Dal-

tonik, Bremen, Germany). To quantitatively analyze the pattern of acetylation of the paCOS, 2 µl 18O-labeled samples (500-1000 µg/ml) were injected into the system. The isolation width of the ESI-MS was set to an m/z window of 0.8, the group length to an m/z window of 2, the fragmentation energy to 100% with enhanced fragmentation starting from 80% to 120% in CID (collision-induced dissociation), and a dissociation delay of 20 ms. RESULTS AND DISCUSSION Principle and workflow development. A mixture of oligomers consisting of either the fully acetylated GlcNAc or the fully deacetylated GlcN units ranging in DP from monomers to hexamers (500 ng of each oligomer) was separated by HILIC UHPLC and detected using ESI-MS.31 The LC-MS base peak chromatogram reveals significant differences in the response factors depending on both DP and DA (cf. Figure 1). Quantification of paCOS, thus, would require the availability of individual standards for each oligomer. The influence of DA on the MS response factor can be removed as shown by Haebel et al. by Nacetylation of the paCOS using [2H6]acetic anhydride,19 and we solved the problem of DP influence by using a series of double isotopically labeled standards prepared by N-acetylation of GlcN oligomers of DP 1 to 6 with [13C4,2H6] acetic anhydride (cf. Figure 2). In the first step, converting all paCOS into fully acetylated chitin oligomers evens out the different ionization efficiencies arising from different DA and PA. Then, adding the internal standards co-eluting in HILIC allowed us to account for varying ionization efficiencies resulting from different DP and distorted analyte ion abundances caused by unintended fragmentation processes. Also, occasional O– instead of N-acetylation would lead to a change in elution behavior during HILIC, which however was never observed. Moreover, this approach takes into account heterogeneous ionization patterns resulting from formation of different charge states and adduct formation. These pre-treatments of the paCOS allow the mass spectrometric quantification even of minor components in a mixture of isomeric (i.e. paCOS with identical DP and DA, but differing in PA) (see Figure S1 and Table S1). However, small interferences that might affect the accuracy of paCOS quantification, viz. isotope effects influencing chromatographic separation and gas-phase ion chemistry or rearrangement processes preceding fragmentation of gaseous analyte ions have to be taken into account. These cannot be excluded entirely, however, they can be considered as negligibly small. A secondary isotope effect which is frequently encountered when isotopologs are separated by liquid chromatography32 was not observed in any of the experiments performed, i.e. oligomers of same DP co-elute under HILIC LC conditions independent of the number of isotopic substitutions (cf. Figure 2). Furthermore, data shown in Figure 2 indicate that the ionization efficiency of the analyte molecules is not influenced by incorporation of heavier stable isotopes. Thus, an intermolecular secondary isotope effect on ionization processes can be regarded as very small.



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Sequencing of paCOS to reveal their PA then requires labeling of the reducing end followed by MS2 analysis. Labeling the reducing end by incorporation of 18O allows for unequivocal discrimination of fragment ions comprising either the reducing or the non-reducing end. LC-MS² was used to determine the PA of the paCOS obtained after ChiB hydrolysis of the chitosan polymers. The reducing ends of the already partially deuterated products were labeled with an 18O oxygen tag. We analyzed different sequences in the paCOS by combining the relative amounts and the composition of the different Y- and Bfragment ions in the MS2 spectra. Figure 3 depicts an example for unequivocal discrimination of isomeric A2D3 oligomers and determination of their relative amounts within the mixture; thus, we were able to quantify the paCOS having different patterns of acetylation. Here, the free amino moieties were labeled with [2H6]acetic anhydride causing glucosamine (D) to convert into [2H3]Nacetylglucosamine (R), i.e. giving A2R3, which allowed for an unbiased quantitative sequencing of the oligosaccharide. Labeling of the reducing end with 18O allowed an unambiguous identification of those fragments making up the reducing end of the monosaccharide building block. The positive-ion LC-MS² spectrum of the protonated precursors [A2R3 + H]+ exhibits the formation of complementary B- and Y-type ions (nomenclature according to Domon and Costello33). The almost exclusive formation of oxonium-type B4-ions, composed of one light and three heavy N-acetylglucosamine building blocks, indicates that the reducing end can only be occupied by an A unit. Then, evaluation of the relative abundances of isotopologs Y2-ions at m/z 427.1 and 430.2 revealed that AA and RA (listed with the reducing end to the right) appear within the pentasaccharide sequence at a ratio of 60:40. The Y3-ions, comprising either one or two heavy building blocks - i.e., A2R1 or A1R2 with an m/z of 633.2 or 636.2, respectively - give rise to signals in a relative ratio of 65% to 35%. The only possible sequence for A1R2 is RRA (A must be at the reducing end) whereas for A2R1, both ARA and RAA are feasible. Since 60% of all precursor ions show the sequence AA at the reducing end, it becomes obvious that only 5% of precursor ions account for the structural motif ARA. Fragment ions containing A2R2 constitute 75% of all observed Y4-ions, and the heavier congener harboring A1R3 gives rise to a signal of 25% relative intensity. The latter finding indicates that 25% of all [A2R3 + H]+ ions probed have the sequence ARRRA. Hence, combining these with the previous results yields a relative amount of each oligomer sequence: 25% are ARRRA, 10% RARRA, 5% RRARA, and 60% RRRAA. Fully N-acetylated chitin oligosaccharides lack functional groups with high proton affinities that would lead to formation of fixed charges. It is likely to assume that initial protonation occurs at the oxygen of the N-acetyl groups and the energy required for mobilization of the proton is readily available under low-energy CID conditions.34 As a result cleavage of glycosidic bonds will proceed via an acid catalyzed fragmentation pathway as described recently by Gao et al.35 and direct participation of

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deuterium in charge-directed glycosidic bond rupture can be excluded. This is further substantiated by the absence of any hydrogen/deuterium scrambling processes. Consequently, only intramolecular secondary kinetic isotope effects altering fragmentation frequencies can be expected. However, these are typically very low especially since the internal energy of the majority of activated precursor ions is well above the dissociation threshold.36,37 These considerations are further corroborated by the experimental finding that the fragmentation patterns, i.e. the relative abundances of individual fragment ion types obtained by collisional activation of unlabeled chitin oligosaccharides are identical to those obtained from different isotopologs probed. Rearrangement processes impeding a precise quantitative determination of paCOS were not observed. Fragment ions pointing to an unintended loss of internal residues leading to false sugar sequence ions as previously reported for fragmentation of protonated oligosaccharides could not be detected.38,39 Migration of N-acetyl groups towards neighboring hydroxyl groups is unlikely as a result of the higher stability of the first mentioned moieties and is hampered by the trans configuration of the vicinal accepting site, i.e. the O−H group located at C3. Overall, this method allows for the absolute quantification of paCOS even in complex mixtures and also provides information on the relative amounts of isomeric oligosaccharides differing in PA. Probing the Processivity of ChiB. To validate our approach, we probed ChiB’s processivity, which is a measure of how many cuts ChiB makes before dissociating from the polymer substrate, thus influencing the DP of resulting oligomers, and subsite specificities by comprehensively characterizing the structure of the paCOS products resulting from ChiB hydrolysis of chitosan polymers. Using a reducing-end assay, we monitored the hydrolysis process and determined the endpoint of the reaction (Figure S2). The structure, i.e. DP, DA, and PA, and relative abundancies of the paCOS in the product mixture were determined using the combined approaches outlined above. Next, the processivity of ChiB was investigated by determining the dimer-to-trimer ratio obtained during hydrolysis of the chitosan polymer (cf. Figure 4).40,41 The AA/AD ratio was calculated with the molar percentage of the AA and AD in the sample, determined by the quantitative sequencing of ChiB chitosan polymer hydrolysate. The HILIC-MS Base Peak Chromatogram of [2H3]Nacetylated hydrolysate obtained by incubating the chitosan polymer DA60% with ChiB, mixed with [13C2, 2H3] labeled internal standard is depicted in Figure 4A. At the beginning of the reaction, we found a high dimer-totrimer ratio, indicating that ChiB initially degrades chitosan polymers processively. The trimer concentration then increased throughout the course of the reaction, indicating that the intermediately released paCOS undergo non-processive hydrolysis (cf. Figure 4B).40 We found


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initial ratios of 5.1 and 8.2 for partially acetylated chitosans of DA50% and DA60%, respectively. Comparatively, for fully acetylated α- and β-chitin, Horn et al.41 reported dimer-to-trimer ratios of 11.4 and 12.6, respectively. These data imply that a polymer substrate with a higher DA results in a more processive hydrolysis, while a polymer substrate with more GlcN units encourages the enzyme to dissociate earlier from the substrate. This is supported by a more detailed analysis of the dimers produced (cf. Figure 4C). Initially, the ratio of the fully acetylated dimer GlcNAc-GlcNAc to the partially acetylated dimer GlcN-GlcNAc was high with both substrates, and it declined over the time of hydrolysis. As the AA/AD ratio is higher in both cases than expected from the DA of the substrate, it can be concluded that processivity is favored by the presence of acetylated residues in the substrate. Deciphering the subsite specificity of ChiB. By joining the MS1 and MS2 data, we confidently characterized the subsite specificities of ChiB (Figure 5). Hydrolytic enzymes cleave their substrate’s glycosidic bond between subsites −1 and +1. Thus, the sugar found at the reducing end of the product had been bound at subsite −1, whereas subsite +1 had harbored the non-reducing end residue. Correspondingly, the penultimate sugar units at both the reducing and non-reducing ends had been bound at subsites −2 and +2, respectively (see Figure S3). Analyzing the sugar moieties that were bound at the different subsites prior to cleavage allows to draw conclusions about the specificities of the respective subsites. The occurrence of an A- or D-unit at specific subsites of ChiB during the hydrolysis was calculated based on the amount and the architecture (DA, DP and PA) of each paCOS with a DP1 to DP6 produced by this enzyme. This can be achieved due to the combination of the results from the LC-MS1 measurement - determining the amount of the chitooligosaccharides according to their DA and DP - with the results of the LC-MS2 measurement - quantitative PA determination of oligosaccharides having the same DA and DP. Invariably, acetylated residues were found at the reducing end of all paCOS products, indicating an absolute specificity of the −1 subsite for GlcNAc, which is in agreement with data obtained for ChiB hydrolysis products using the conventional method employing 1H-NMR, and it agrees with the substrate-assisted cleavage mechanism of GH18 chitinases.42 The non-reducing end of the paCOS consisted less frequently of a GlcNAc residue than the DA of the substrate (30/35% GlcNAc using a substrate of chitosan DA50/60%), indicating the relatively highest GlcN tolerance for binding a non-acetylated GlcN residue at subsite +1. ChiB prefers acetylated subunits in subsites +2 and −2, because the frequency of GlcNAc bound at these subsites was initially higher than 80% and decreased with progressing hydrolysis, eventually almost reaching the DA of the substrates. Again, our data are in agreement with previous work performed using Monte-Carlo simulations in combination with SEC-NMR, which showed an initial preference for GlcNAc at subsite −2 and no preference at

subsite +1,43 but our data give additional information on the enzyme’s preference at subsite +2. Also, as our analysis reveals the frequency of the diads (AA, DA, AD, or DD) at positions −2 and −1 and at positions +1 and +2, we can deduce whether the occupancy of one position influences the probability at the neighboring position within the diad (e.g., an enzyme may accept a D-unit at either the +1 or the +2 position, but not at both positions simultaneously). This information would not easily be available by any other means. In addition to providing more information than SEC-NMR, our LC-MS method is especially advantageous over conventional SEC-NMR because it has nanogram sensitivity, easily separates oligomers according to DP and DA, and can reveal the PA even in mixtures of isobaric oligomers. Also, this is a one-pot method with no need for chromatography or any other step that could lead to losses during sample preparation. Thus, this sequencing method allows to quantify different oligomers as well as to determine the processivity and subsite specificities of chitosan hydrolases more easily, more rapidly, and more accurately. This rapid assessment of subsite specificity and processivity, that represent the key enzyme characteristics influencing the architecture (DP, DA, PA) and thereby the functionality of the generated paCOS, will eventually allow for the production of specific oligomers with a desired bioactivity. Furthermore, appropriate modifications will allow to use the technique described here to unravel the enzymatic specificity of other enzymes degrading other linear, partially substituted homopolysaccharides of biomedical importance (for further discussion cf. Supporting Information). CONCLUSION A novel method designed for probing processivity and cleavage sites of chitosan hydrolases based on quantitative mass-spectrometric sequencing of chitooligosaccharides was developed. Different ionization efficiencies of paCOS arising even in complex mixtures from different DA and PA were taken into account by conversion of paCOS products into fully acetylated chitin oligomers by employing a deuterated reagent for Nacetylation of free amino groups. Subsequently, the addition of [13C, 2H]-labelled isotopologs as internal standards allowed for absolute quantification of paCOS by use of HILIC LC-MS1, which provided exclusive formation of protonated paCOS oligomers simplifying data analysis significantly. Finally, labelling the reducing end with an 18 O-tag facilitated quantitative determination of isomeric paCOS by tandem MS. All derivatization procedures either used for conversion of paCOS to chitin oligosaccharides or the synthesis of the [13C, 2H]-labelled standards solely furnished the desired products and no further sample purification steps were necessary, thus minimizing undesired sample loss. In summary, the method described here allows for quantitative analysis and sequencing of partially acetylat-



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ed chitosan oligomers even in complex mixtures as e.g. obtained as products of enzymatic hydrolysis of chitosan polymers. This was demonstrated by bench-marking our technique against the current gold standard, SEC-NMR, on the best-studied chitosan hydrolase known today, ChiB from S. marcescens.

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can become the subject of biotechnological improvement or modification through evolutionary engineering to finetune enzymes’ substrate specificities and product patterns.

OUTLOOK The information obtained by this methodology will be crucial for elucidating molecular structure-function relationships of these oligomers concerning their biological activities, such as their anti-microbial, plant strengthening, or wound healing abilities.5,6 Moreover, the method will facilitate analyses of which cleavage sites chitinolytic enzymes prefer, thus promoting the design of tailor-made chitosan polymers that will yield defined oligomeric breakdown products with known bioactivities, as a prerequisite for the development of reliable applications in many fields including medicine and agriculture.11,44 In the future, this method could be critical for large-scale production of oligomer products through enzyme-based biorefinery processes, upon evolutionary engineering to fine-tune enzymes’ substrate specificities and product patterns. Such advancements have the potential to revolutionize the commercial availability of these promising compounds. With appropriate modifications, the technique described here will be applicable to other linear, partially substituted homopolymers. One physiologically and biotechnologically relevant example would be partially methyl-esterified homo-galacturonans in plant pectins. Cytochemical studies using monoclonal antibodies directed against different pectic fractions, such as homogalacturonans with low or high degree of methyl-esterification or with random or blockwise patterns of methylesterifications have shown an enormous complexity of pectins in plant cell walls45 which might also be related to disease resistance or susceptibility.46 The pattern of methyl-esterification has been shown to play a crucial role in the rheological properties of pectins which are of paramount importance for their application as food additives.47 Chemical48 and enzymatic49 fingerprinting protocols are available for pectin analysis but these are less powerful and require higher sample amounts compared to enzymatic / mass spectrometric fingerprinting developed here.

Figure 1. LC-MS base peak chromatogram for a mixture of pure GlcNAc (An, green) and pure GlcN (Dn, red) oligomers (DP1-DP6, 500 ng of each oligomer), indicating that analytes with different DPs and DAs exhibit significantly different ionization efficiencies in MS analyses. Peak splitting arises from different anomeric forms of the sugars.

Figure 2. LC-MS base peak chromatogram of equal amounts 2 13 2 of chitin (An) oligomers and the [ H3]-labeled and [ C2, H3]labeled isotopologs (Rn and R*n) with varying degree of polymerization (DP). The resulting LC-MS base peak chromatogram and the extracted mass spectra show that the species had identical ionization efficiencies, allowing the quantification of the analyte species. For monoisotopic masses and m/z values of the different oligomers, please refer to Tables S2 and S3).

In a next step, making use of ring cleavages during mass spectrometric sequencing, the technique could even be extended to partially substituted neutral polysaccharides like cellulose and starch derivatives, to support the emerging field of regio-controlled polysaccharide substitution.50,51 Production of such novel compounds with potentially improved or even novel properties increasingly relies on enzymatic modifications, requiring the availability of well-defined polysaccharide modifying enzymes such as hydrolases, deacetylases, methyl-esterases, sulfatases, and the like. Our technique allows the detailed analysis of the products of such enzymes and, thus, an understanding of their regio-selectivities, which in turn


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Figure 3. Collision-induced dissociation (CID) tandem MS of + [A3R2+H] ions quantitatively assesses analyte acetylation patterns. Analytes (paCOS from ChiB hydrolysis) underwent 2 N-acetylation by exposure to [ H6]acetic anhydride, then 18 their reducing ends were labeled by use of [ O]water. The example pentamer A3R2 contains 3 GlcNAc units (A units; 2 green dots) and 2 heavy [ H3]GlcNAc (R units; blue dots). 18 The O-labeled reducing end is indicated by the yellow dot. Residues in the same box are derived from the same precursor or fragment ion. By combining the relative abundances of the different fragments (highlighted in brown), the composition and occurrence of each acetylation pattern was determined.

Figure 4. Determining the processivity and subsite specificity of ChiB. A) LC-MS base peak chromatogram for a paCOS mixture obtained by ChiB hydrolysis of chitosan (DA60%, 24 h incubation) followed by acetylation of the free amino 2 groups with [ H6]acetic anhydride, producing an oligomer mixture of DP1-DP6 containing both A and R units (A = 2 GlcNAc, R = [ H3]GlcNAc). Each mass shift of ~3 u (incorporation of one deuterated acetate residue, R) indicates the former presence of one D unit (D = GlcN). The paCOS mix13 2 ture was spiked with the [ C2, H3]-labeled internal standard R*. Insets depict representative extracted mass spectra of dimeric and pentameric paCOS ions. The use of a HILIC stationary phase in LC-MS experiments led to efficient removal of salt contaminants, which provided exclusive formation of protonated paCOS oligomers. B) Time dependence 2 of paCOS dimer-to-trimer ratios (DP2/DP3) for [ H3]Nacetylated ChiB hydrolysates of chitosan polymers having DA50% and DA60%. C) Time dependence of the ratio of fully acetylated dimer AA to partially acetylated dimer DA ChiB hydrolysates of chitosan polymers having DA50% and DA60%.



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The authors declare no competing financial interests.

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ACKNOWLEDGMENT We thank Prof. Martin Peter for critically reading the manuscript and Celeste Brennecka for editorial support. Financial support from the European Union ERA-IB project ‘‘ChitoBioEngineering’’ (EIB.10.042) is gratefully acknowledged.

REFERENCES

Figure 5. Determining the subsite specificity of ChiB at different times during hydrolysis of chitosan with DA50% and 1 2 DA60%. Data obtained from LC-MS and MS experiments were evaluated to determine which residues (A green ball or D red ball) occupied ChiB’s cleavage subsites, considering the DP, DA, and PA of the paCOS hydrolysis products. Knowing the quantities of each oligomer with different DP, DA, and PA produced, we determined the occurrence of the diads GlcNAc-GlcNAc (AA) GlcNAc-GlcN (AD), GlcNGlcNAc (DA), and GlcN-GlcN (DD) at the reducing ends of the oligomers (representing the subsites −2 and −1) and at the non-reducing ends (representing subsites +1 and +2) at different time points during hydrolysis. These data show how the preference for GlcNAc at different subsites changes over time, and therefore describes the enzyme’s initial and subsequent specificities.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Details of the quantification of paCOS including the linear range of the internal standards (Figure S1 and Table S1-S3), kinetics of the enzymatic hydrolysis (Figure S2) and link between a specific unit of the paCOS and a defined subsite position in the active site of the enzyme (Figure S3). Additional discussion of the possible extension of the shown method for other polysaccharides like pectin. (PDF)

AUTHOR INFORMATION Corresponding Author Prof. Dr. Bruno M. Moerschbacher, [email protected]

Present Addresses † Institute of Plant Biology, ETH Zürich, Universitätstraße 2, 8092 Zürich, Switzerland.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

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