Monosaccharide Identification as a First Step toward de Novo

Mar 31, 2015 - (11) Common analytical techniques that are currently used for carbohydrate analysis include HPAEC (high-performance anion-exchange chro...
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Monosaccharide Identification as a First Step toward de Novo Carbohydrate Sequencing: Mass Spectrometry Strategy for the Identification and Differentiation of Diastereomeric and Enantiomeric Pentose Isomers Gabe Nagy and Nicola L. B. Pohl* Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States S Supporting Information *

ABSTRACT: De novo carbohydrate sequencing, including monosaccharide identification, largely remains a tremendous analytical challenge. A first step in the complete structural determination of any large polysaccharide is an accurate and robust method for analysis of the constituent monosaccharides. Herein, the first mass spectrometry-based method for the complete identification and absolute configuration determination of all 12 pentose isomers, including the D and L enantiomers for arabinose, lyxose, ribose, xylose, ribulose, and xylulose, is reported. As compared to earlier work to distinguish hexose isomers, the chiral separation of the pentose isomers was significantly more challenging. Specifically, the 12 pentoses are much more structurally similar to one another, with only the axial or equatorial orientation of two hydroxyl groups differentiating among these isomers in their five-membered ring furanose structure and smaller energetic differences between pentose conformations than between hexose conformations. Despite such inherently minimal energetic differences between the 12 pentoses, two unique fixed ligand kinetic method combinations were discovered to achieve chiral discrimination for this set of isomers. This assay can be readily applied to the identification of any isolated pentose monosaccharide using only microgram quantities and a commercial instrument and complements the method to distinguish hexose isomers. A workflow that incorporates this mass spectrometry-based method and thereby could achieve complete de novo identification of all monosaccharide building blocks in an oligo- or polysaccharide is proposed.

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N-glycans.11 Common analytical techniques that are currently used for carbohydrate analysis include HPAEC (high-performance anion-exchange chromatography), GC/MS (gas chromatography−mass spectrometry), HPLC (high performance liquid chromatography), and NMR (nuclear magnetic resonance).11−20 NMR is readily ruled out for such a task by complications of greater sample requirements, the absence of consistently diagnostic coupling constants, and the overlap of proton signals.16−20 Separations methods such as HPAEC, which can already not discriminate enantiomers,16 and GC/MS and HPLC, which already give convoluted chromatograms of α/β and furanose/pyranose forms,21−29 would need to reliably separate hundreds of sugars with consistent retention times regardless of the other sample components, a daunting challenge. Such separations are also hampered by the relative lack of sensitivity of most detectors to unmodified carbohydrates as compared to other analytes like amino or nucleic acids.21−29

eliable methodologies for the analysis of the constituent monomer building blocks are the first step in determining the structure of a biomolecule.1−6 Unfortunately, in the determination of carbohydrate biomolecules, no single analytical technique can even identify all monosaccharides in a given isomeric set.1−6 This lack of robust analytical tools has hindered the understanding of the biological roles of carbohydrates and the development of applications, such as vaccines and other therapeutics, to such an extent that in 2012 the United States National Academy of Sciences called for the “development of technology over the next 10 years to purify, identify, and determine the structures of all the important glycoproteins, glycolipids and polysaccharides in any biological sample”.7 Unfortunately, known carbohydrate analytical methods suffer significant limitations when applied to monosaccharide identification as a first step in a de novo carbohydrate sequencing problem, such as when encountering a new exopolysaccharide from a bacterial source,8−10 in which the possible sugar building block set cannot be severely limited at the onset as it can for the structural determination and monosaccharide composition analysis, for example, in human © 2015 American Chemical Society

Received: February 25, 2015 Accepted: March 31, 2015 Published: March 31, 2015 4566

DOI: 10.1021/acs.analchem.5b00760 Anal. Chem. 2015, 87, 4566−4571

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Analytical Chemistry Mass spectrometry (MS) is an attractive possibility for carbohydrate detection, especially because MS is a fast and high-throughput analysis method.14,15,19,30−32 Less obvious is that mass spectrometry could potentially also significantly simplify the separations/identification problem inherent to monosaccharide identification as a first step to complete carbohydrate characterization. Many carbohydrates, of course, share molecular weights, so mass spectrometry is not an obvious method for differentiating chiral compounds. However, Cooks’ kinetic method has shown some promise in distinguishing chiral compounds.33−39 This method relies on the measurement of dissociation rates of metal-bound analyte ligand cluster ions, where each unique MS/MS profile is dependent on the chirality of the analyte.33−39 Recently, we developed a variant of Cooks’ method for the first analytical, let alone mass spectrometric, method for the complete discrimination and absolute configuration identification of all 24 hexose monosaccharide isomers.40 This new methodology has distinct advantages over the aforementioned analytical techniques currently employed for monosaccharide identification. Specifically, it is able to match the sample requirements of HPAEC, HPLC, and GC/MS (tens of micrograms), while lowering that of NMR (milligram amounts).12,21,22,41,42 Furthermore, this mass spectrometry-based method allows for much easier data interpretation because only the relative abundances of two fragment ions are needed. Lastly, this methodology is both fast and simple, because each sample is analyzed within minutes using only a commercial instrument setup. However, the utility of these metal/ligand sets to distinguish other monosaccharides, especially those that cannot form a pyranose ring while maintaining a primary hydroxyl for metal binding, is not clear.43 Without this level of generality, the approach will do little to simplify the identification of monomer units in an unknown carbohydrate. Unfortunately, the six-membered pyranose ring is not as conformationally flexible as a five-membered furanose ring, and therefore, we did not expect binding differences between metal/ligand/pentose structures to necessarily be large enough to be diagnostic.43 Herein, we report efforts to find a protocol that can effectively distinguish among all 12 pentose isomers using only mass spectrometry and outline a vision for how this basic method can serve as the key first step for monosaccharide identification in a de novo carbohydrate sequencing workflow (Figure 1).

Figure 1. Proposed workflow for complete de novo identification of all monosaccharide building blocks in an oligo- or polysaccharide sample.

Figure 2. All 12 pentose isomers in their five-membered ring furanose forms.

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than the hexoses because of one less possible hydroxyl coordination site. The central challenge was to develop a set of ligands that takes advantage of the favored furanose structure to create the necessary chiral interactions for complete discrimination of all 12 pentose isomers with mass spectrometry. Fixed Ligand Kinetic Method. The fixed ligand kinetic method, developed by Cooks’ and co-workers, has emerged as an attractive technique in the field of chiral mass spectrometry.14,15,35,44−46 A trimeric ion complex, [MII(A)(ref)(FL− H)]+, is formed in the gas phase via electrospray ionization (ESI), where MII is a divalent metal cation, ref is a chiral reference, FL is a fixed ligand, and A is the analyte (Figure 3).14,15,35,44−46 This ion is subjected to collision-induced dissociation (CID), which results in two fragment ions, [MII(A)(FL−H)]+ and [MII(ref)(FL−H)]+.14,15,35,44−46 Rfixed relates these fragment ions to one another through their relative intensities (eq 1).14,15,35,44−46 Rchiral‑fixed defines the chiral selectivity between a specific pair of D/L enantiomers (eq 2), where a value further from one expresses greater enantiomeric separation.14,15,35,44−46 (For more detailed information on the

EXPERIMENTAL SECTION This work first aims to test whether similar ligand combinations that were successful in hexose identification could be applied to pentoses or if new ligand sets would be needed to develop a robust mass spectrometry-based assay to definitively identify isolated pentoses de novo. These 12 pentose isomers constitute six diastereomers, each of which is composed of two mirrorimage, D and L, enantiomers (Figure 2). Only the positioning of hydroxyl (OH) groups structurally differentiates these monosaccharides from one another; they all share a 150.0528 Da (Daltons) monoisotopic molecular weight (Figure 2). Specifically, these OH groups are positioned in either a fluxional pseudoaxial or pseudoequatorial position at carbon two (C2) and carbon three (C3). Since the 12 pentoses only vary in hydroxyl group orientation at two positions and are usually found in the more flexible five-membered-ring rather than sixmembered-ring conformation, minimal energetic differences are expected between isomers.43 The pentoses also have fewer possible chiral interactions with the metal/ligand combinations 4567

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Figure 3. Depiction of fixed ligand kinetic method with D-arabinose as the analyte (A), copper(II) as the divalent metal cation (MII), L-serine as the chiral reference (ref), and guanosine monophosphate as the fixed ligand (FL). Trimeric ion complex, [MII(A)(ref)(FL−H)]+, formation via electrospray ionization, and subsequent fragmentation via collision-induced dissociation into two diastereomeric fragment ions, [MII(A)(FL−H)]+ and [MII(ref)(FL−H)]+. Fragmentation pathways are from the neutral loss of a chiral reference molecule or neutral loss of an analyte molecule.

Figure 4. Free energy diagram depiction of fixed ligand kinetic method showing energetic differences that need to be created between diastereomeric fragment ions to achieve chiral discrimination. Here, D/ L-lyxose is the analyte, nickel(II) is the divalent metal cation, L-aspartic acid is the chiral reference, and guanosine monophosphate is the fixed ligand.

fixed ligand kinetic method, please see the Supporting Information.) R fixed

[MII(A)(FL−H)]+ = [MII(ref)(FL−H)]+

R chiral‐fixed =

any further purification (>95% purity). HPLC grade H2O and HPLC grade methanol were used to create the stock solutions in 50:50 (v/v) amounts. Concentrations for each sample were 50 μM for the metal cation and 200 μM for the analyte, fixed ligand, and chiral reference. Formic acid was added to the room temperature 5′GMP-Na2 solution 2 weeks prior to use.44 The ion trap portion of an LTQ Orbitrap XL from Thermo Scientific (San Jose, CA, USA) with an electrospray ionization (ESI) source in positive ion mode was used for all experiments. Please consult the Supporting Information for detailed experimental conditions used for mass spectrometry, as well as a discussion of the conversion of normalized collision energy (%NCE) into units of voltage.

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[MII(AD)(FL−H)]+ /[MII(ref)(FL−H)]+ [MII(AL)(FL−H)]+ /[MII(ref)(FL−H)]+ (2)

Mass Spectrometry-Based Assay Methodology. While the fixed ligand kinetic method has shown great ability to separate enantiomers through Rchiral‑fixed from eq 2, there have been few attempts to chirally discriminate an entire analyte set.47−50 In order to create a high-throughput assay, the entire analyte set, in this study all 12 pentose monosaccharides, are required. From eq 1, it can be seen that each specific pentose isomer will yield a unique Rfixed term (RD‑arabinose, RL‑arabinose, RD‑lyxose, RL‑lyxose, RD‑ribose, RL‑ribose, RD‑xylose, RL‑xylose, RD‑ribulose, RL‑ribulose, RD‑xylulose, and RL‑xylulose). On the basis of these Rfixed values, a new Rfixed range term can be created that represents averaged triplicate trials with standard deviation error bars. The hypothesis is that, if all 12 pentose isomers can have nonoverlapping Rfixed ranges, then monosaccharide discrimination and absolute configuration determination is achieved. In order to accomplish this, ligand combinations must be found that can create significant enough energetic differences (Δ(ΔG)) among these pentose isomers (eq 3 and Figure 4), where R is the gas constant and Teff is the average effective temperature of the activated complexes. ln(R fixed) =

Δ(ΔG) RTeff

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RESULTS AND DISCUSSION In order to create a complete pentose monosaccharide mass spectrometry-based assay, suitable ligand combinations must be selected.17,35,40,44−46,50,51 Our recent hexose study showed that only fixed ligand combinations could create the necessary energetic differences to chirally discriminate an entire monosaccharide analyte set.40 Although boron readily binds to monosaccharides in cis-diols, it was found to be unhelpful in achieving chiral discrimination.35,52−55 A successful ligand combination has two key criteria: the initial trimeric ion complex, [MII(A)(ref)(FL−H)]+, must be formed in high enough abundance for subsequent fragmentation to be performed. Both of the desired fragment ions, [MII(A)(FL− H)]+ and [MII(ref)(FL−H)]+, must be formed via CID and be in high enough relative intensity for reproducible experiments. On the basis of earlier findings,40 the fixed ligand combinations of NiII/L-Asp/5′CMP, Ni II/L-Asp/5′GMP, MnII/L-Asp/L-Phe−Gly, and CuII/L-Ser/5′GMP were assayed first. Work with the fixed ligand combinations of MnII/L-Asp/LPhe−Gly and NiII/L-Asp/5′CMP showed that the initial trimeric ion complexes did indeed form. However, upon fragmentation, the desired diastereomeric fragment ions were either not generated at all or not in high enough abundance for accurate, quantitative, and reproducible results to be obtained. This was a very surprising result because our previous hexose

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Reagents. All pentoses (D/L-arabinose, D/L-lyxose, D/Lribose, D/L-xylose, D/L-ribulose, and D/L-xylulose), chiral reference molecules (L-serine and L-aspartic acid), fixed ligand molecules (L-phenylalanyl−glycine (L-Phe−Gly), cytidine monophosphate (5′CMP), and guanosine monophosphate disodium salt (5′GMP-Na2)), and metal salts (CuCl2, NiCl2, and MnCl2) were purchased from Sigma-Aldrich (Milwaukee, WI, USA) and CarboSynth (Berkshire, UK) and used without 4568

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Analytical Chemistry study showed that these two fixed ligand combinations both worked well to generate Rfixed values. It is hypothesized that this lack of product ion formation must be caused by a lack of metal-monosaccharide binding, perhaps from the favored furanose conformational preference and the fewer number of hydroxyl groups in the pentoses. Fortunately, the two fixed ligand combinations of NiII/L-Asp/ 5′GMP and CuII/L-Ser/5′GMP did form the initial trimeric ion complex in high enough abundance, as well as producing the desired fragment ions in high enough relative intensity to collect reproducible, quantitative data. (Please see the Supporting Information for an MS1 spectrum with CuII/LSer/5′GMP.) Figure 5 illustrates MS2 spectra with the CuII/L-

the breaking of noncovalent bonds. If the same optimized mass spectrometric conditions are applied for the two fixed ligand combinations of NiII/L-Asp/5′GMP and CuII/L-Ser/5′GMP, all 12 pentose isomers can be discriminated from one another on the basis of their respective Rfixed ranges. Figure 6 illustrates a 2D plot with the natural logarithm of Rfixed with CuII/L-Ser/ 5′GMP on the x-axis and the natural logarithm of Rfixed with NiII/L-Asp/5′GMP on the y-axis. The data is shown in a natural logarithm manner so as to fit all the Rfixed values on a single figure. (Please see the Supporting Information for raw Rfixed values.) The plot clearly shows how an unknown pentose could be identified using only two fixed ligand combinations. From these results, as compared to our recent hexose work, it becomes clear that the pentose isomers form much less stable complexes in the gas phase. This is evidenced by the fact that some of the fixed ligand combinations attempted here either do not form the product ions of interest or their relative intensities are far too low to collect reproducible and quantitative data. As previously reported, it is hypothesized that the central divalent metal cation will adopt a coordination number of four and a square planar conformation.18,39,56 From this, it becomes evident that the pentose monosaccharide and metal cation binding interaction will occur at two hydroxyl groups (as seen in Figures 2 and 3). Since the only structural differences among these 12 isomers is their pseudoaxial or pseudoequatorial hydroxyl orientation at C2 and C3, this positioning clearly plays a role in their chiral interactions, although the specific interactions involved are uncertain. This mass spectrometry-based assay for complete pentose isomer discrimination can be easily implemented into the analysis workflow of any isolated pentose isomer, as long as the sample is cleaned of salts through dialysis and lyophilization.57,58 In order to achieve a complete characterization of all possible monosaccharide constituents (hexoses, pentoses, amino-sugars, deoxy sugars, etc.) in a more complex polysaccharide-containing biological sample, we envision other sample preparation steps in addition to our mass spectrometry-based identification technique. The first step

Figure 5. MS/MS spectra with the fixed ligand combinations of CuII/ L-Ser/5′GMP, for the pentose analytes of D-arabinose, D-ribose, and D/ L-lyxose.

Ser/5′GMP fixed ligand combination with four different pentose isomers. From Figure 5, it can be seen that the desired fragment ions formed in high relative abundance, as well as how the Rfixed values are calculated from eq 1. There exists some secondary fragmentation in certain isomers, perhaps caused by

Figure 6. Two-dimensional natural logarithm-natural logarithm plot of the Rfixed ranges with CuII/L-Ser/5′GMP on the x-axis and NiII/L-Asp/5′GMP on the y-axis. Optimized conditions for each fixed ligand combination are at 15% NCE and 30 ms activation time. All results represent triplicate data with the error bars represented by one standard deviation. 4569

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Furthermore, our method is very discriminative in that nonisobaric sugars (for example, hexoses and pentoses) will each form trimeric ion complexes that differ in their molecular weight. Mass selection of these complexes serves as a means to simultaneously identify individual nonisobaric monosaccharides in various isomer sets (hexoses and pentoses). We are currently in the process of developing a fraction collection chromatographic system that will serve as the premass spectrometry partial separation step. Work is also ongoing to apply this method to other monosaccharide analyte sets, including those that contain non-neutral sugars, to test the scope of this method and to take further steps toward complete monosaccharide identification.

commonly applied to a polysaccharide is acidic hydrolysis to cleave it into its monosaccharide subunits.21−25,27,29 The reported mass spectrometry identification technique dramatically simplifies any chromatography steps needed to neutralize the hydrolyzed sample and partially fractionate the resulting monosaccharides (as seen in Figure 1 in the introduction). This separation system does not need to be chirally selective, as our mass spectrometric method serves as the chiral detector. In a conventional chromatographic separation, there can exist up to four peaks for each sugar (α/β and furanose/pyranose) to significantly complicate its identification. The mass spectrometry-based technique herein does not distinguish among these interconverting isomers, however. Furthermore, any chromatography step does not need to be so good as to separate both enantiomers (D/L) of a given isomer set (for example, the pentoses) as well as varying monosaccharides from different nonisobaric isomer sets (for example, hexoses and pentoses). In our proposed pre-mass spectrometry separation method, there would only need to exist 12 peaks to fraction collect out all possible monosaccharide constituents in an unknown polysaccharide. These 12 peaks arise from the number of diastereomers in the largest monosaccharide isomer series (hexoses: 24 total isomers and 12 total diastereomers), with other monosaccharide isomers, such as the pentoses/deoxy sugars, etc., existing in these same chromatographic fractions. For example, if a given fraction consists of both D/L enantiomers for a given monosaccharide, eq 4 below can determine the enantiomeric excess (ee) of the two isomers based on the experimentally observed Rfixed value and the same Rchiral‑fixed, RD‑fixed, and RL‑fixed terms as seen in eqs 1 and 2. For a mixture of n isomers, it becomes a simple mathematics problem that requires n − 1 fixed ligand combinations to solve. Equation 5 illustrates this for a ternary mixture, where a, b, and c are the molar fractions for isomers A, B, and C, RA, RB, and RC are the respective Rfixed values for each individual isomer, and Rfixed is the experimentally observed value for all three isomers in a mixture. Both of these equations have been further discussed and applied in previous literature.17,35

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ASSOCIATED CONTENT

S Supporting Information *

More detailed information on the fixed ligand kinetic method and mass spectrometry conditions. All raw data tables of Rfixed values, shown with each fixed ligand combination. A sample MS1 spectrum. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 815-855-8300. Phone: 812855-0298. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge the Joan and Marvin Carmack Chair funds for partial support of this work and to thank Dr. Jonathan Karty and Dr. Jonathan Trinidad of the Indiana University Biological Mass Spectrometry facility, the METACyt initiative, and the Eli Lilly Foundation for the funds to purchase the instrument used in these experiments. We would also like to thank Prof. David Clemmer and his group (Indiana University) for stimulating discussions.

⎡ ln(R D‐fixed) + ln(R L‐fixed) ⎤ ⎡ ln(R chiral‐fixed) ⎤ ln(R fixed) = ⎢ ⎥+⎢ ⎥ × ee ⎣ ⎦ ⎣ ⎦ 2 2 (4)

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ln(R fixed) = a ln(RA ) + b ln(RB) + (1 − a − b)ln(R C) (5)

Not having to design a separations technique that could definitively separate and thereby identify every possible constituent monosaccharide of the hundreds possible is a major advantage to the current technique. Previous literature35 has also demonstrated that any mass spectrometer can be used to obtain Rfixed values. This clearly adds to the application of this methodology, in that any instrument (for example, Orbitrap, Ion Trap, QTOF, etc.) can be used for monosaccharide identification. In summary, if each fraction is collected with this proposed separation platform, our demonstrated mass spectrometry-based method can successfully be utilized as the chiral detector for definitive identification and absolute configuration determination of a monosaccharide isomer.

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CONCLUSIONS This work shows that mass spectrometry can serve as an unambiguous, chiral detector for monosaccharide identification. 4570

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