General Label-Free Mass Spectrometry-Based Assay To Identify

Jun 28, 2016 - Common glycosidase assays rely on the hydrolysis of non-natural labeled sugar substrates that thereby preclude obtaining information as...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/ac

General Label-Free Mass Spectrometry-Based Assay To Identify Glycosidase Substrate Competence Gabe Nagy,† Tianyuan Peng,† and Nicola L. B. Pohl* Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States S Supporting Information *

ABSTRACT: Common glycosidase assays rely on the hydrolysis of non-natural labeled sugar substrates that thereby preclude obtaining information as to the specificity of the leaving group and therefore the most kinetically competent natural substrates. A β-mannosidase could be known to hydrolyze β-mannose, for example, but from what is presently hard to determine by any high-throughput means. Herein, the first chiral dopant-based mass spectrometric assay, with its foundation rooted in the Cooks’ fixed ligand kinetic method, is presented to screen label-free monosaccharide-containing substrates for their kinetic competency with a given glycosidase as a step to name these enzymes not just for the sugar that is removed but also for the leaving group that is produced. This work also presents the first information about the substrate specificity of two specific hyperthermophilic enzymes and the first test of some native, unlabeled substrates (α-1-4 mannobiose and β-1-galactosylphingosine) with mesophilic enzymes.

E

the leaving group on the enzyme−substrate specificity or the kinetic competency of various substrates remain challenging. Mass spectrometry alone can of course identify whether a labelfree substrate has been cleaved or not but gives no information in isolation as to how competent that substrate might be, in a biological setting for example, compared to other possibilities to gain clues as to their natural function.18−24 The most competent glycosidic portion of the glycosidase reaction can be characterized on the basis of what monosaccharide substrate is cleaved in a specifically designed mass-differentiated substrate library.25 This assay can thereby name an unknown enzyme as, for example, an α-mannosidase; however, it also relies on a special tag and therefore cannot discover the best, or most kinetically competent, natural substrate of a given glycosidase. Ideally, a general assay that uses substrates in their “native” state, in a label-free manner, could be developed that would also measure the relative kinetic competency of various substrates in a fast and high-throughput manner with limited sample sizes. Herein, we report the first mass spectrometrybased label-free glycosidase assay that is able to distinguish among leaving groups (R, Figure 1) for their kinetic competency, not just for whether or not a particular substrate is cleaved, and demonstrate its use in discovering the competency of new substrates for several enzymes. Of the common analytical techniques available to develop a general glycosidase assay, all possess notable shortcomings. Of chromatographic methods, both high-performance liquid chromatography (HPLC) and high-performance anion-exchange chromatography (HPAEC) have several drawbacks,

nzymes called glycosidases that cleave glycosidic linkages are important reagents for a range of industrial processes and for structure elucidation in addition to being crucial components of living systems.1−17 The name of a glycosidase, those ubiquitous destroyers of sugar linkages, usually shrouds one party of the divorce in anonymity. When a mannosidase is involved, mannose is clearly on one side, but its former mate remains a mystery. Unfortunately, current methods to assay the biochemical function of a glycosidase, and thereby name it, are not designed to readily reveal both members of the breakup. Current assays often require some form of substrate derivatization, usually through colorimetric or fluorogenic substrates to detect hydrolysis products such as R = pnitrophenolate (Figure 1) rather than the natural leaving group; alternatively, surface plasmon resonance spectroscopy and surface enhanced resonance Raman scattering require 8hydroxyquinolinyl azo dye derivatives.1,15−17 Assays of glycosidases with natural substrates to determine the effect of

Figure 1. General proposed workflow for this glycosidase assay with a comparison to common glycosidase assays, where R = p-nitrophenolate. © 2016 American Chemical Society

Received: April 7, 2016 Accepted: June 16, 2016 Published: June 28, 2016 7183

DOI: 10.1021/acs.analchem.6b01360 Anal. Chem. 2016, 88, 7183−7190

Article

Analytical Chemistry

competency and would a suitable ligand combination be found to do so? From the heart of the kinetic method, an Rrelative‑fixed term can be defined (eq 2), which is the ratio of two unique Rfixed values for two unique analytes. Eq 2 can be solved for ΔΔG (in kcal/mol), which is the energetic difference created by a ligand combination for two unique analytes, where Teff is the average effective temperature of the activated complexes (assumed to be 850 K based on previous work58) and where R is the gas constant. It is important to point out that Teff is related to the internal energy of the dissociating ions and not necessarily a “true” thermodynamic temperature. Previous studies have reported variability in this Teff based on the species analyzed,59,60 and an exhaustive investigation of the “true” Teff of complexes presented here is beyond the scope of this study and would be a suitable future direction. However, the 850 K used in all calculations is simply a numerical constant that does not change the relative order of the kinetic competency of a substrate for a given glycosidase or any magnitude of comparison between various enzyme−substrate pairs. Nonetheless, rather than a report of pseudoenergetic values, a relative order scale (0− 100%) is provided based on the Rfixed differences between product and control to compare the kinetic competency of various enzyme−substrate pairs. To be able to apply the kinetic method for this assay, the Rrelative‑fixed must be defined as the ratio of the product of glycosidase hydrolysis (Rproduct‑fixed) and its control, no glycosidase hydrolysis (Rcontrol‑fixed). However, in order to be able to elicit a mass spectrometric response, through an Rrelative‑fixed term (once again, the ratio of Rproduct‑fixed over Rcontrol‑fixed), to measure and screen the kinetic competency of the natural product, R-group portion, of the substrate, a new term known as the chiral dopant must be applied. A chiral dopant would act as an internal standard and control and therefore should behave similarly to the sugar analyte of interest. This raises the question as to whether a suitable chiral dopant and fixed ligand combination could be discovered to enable such an assay.

including lengthy column regeneration times, reliance on reproducible retention times, expensive chiral columns to resolve some diastereomers and all enantiomers, and the need for preanalysis derivatization, which may be incomplete.26−28 Nuclear magnetic resonance (NMR) requires milligram quantities of sample, is very time-consuming, and can result in false positives and inaccurate structural characterization, especially when very small amounts of material are available.29−31 Even recovery procedures of monosaccharides released from natural products via desalting are unreliable, often resulting in zero monosaccharide being recovered because common recovery methods are designed for large hydrophobic biomolecules such as peptides and proteins but not for the small hydrophilic molecules that are monosaccharides.32−37 Although mass spectrometry is commonly considered an achiral technique,38−43 a variant of the Cooks’ fixed ligand kinetic method (please see the Supporting Information) was recently developed to allow individual discrimination between all possible regioisomeric and enantiomeric pentoses and hexoses44,45 (rather than just a small subsection of those monosaccharides).46−49 These results make mass spectrometry a potentially attractive choice for new assay development. The next question was whether such an approach could be viable to monitor monosaccharides cleaved by glycosidases from a range of substrates to assay the kinetic competency of various possible leaving groups. Whereas D-glucose has been widely studied with glucosidases,50−52 often depending on specific enzymes for their analysis, label-free glycosidase assays that can screen substrates with D-galactose and D-mannose for their leaving groups, two other very important carbohydrates,53−55 with galactosidases and mannosidases, respectively, are still lacking. These two enzyme types then became our test cases for developing a general glycosidase assay. The fixed ligand kinetic method can differentiate isomeric analytes from one another based on their dissociation rates when forming gas-phase noncovalent diastereomeric complexes with other chiral ligands. Briefly described, a singly charged trimeric ion complex of the form [(MII)(A)(ref)(FL−H)]+ is formed via electrospray ionization (ESI), where MII is a divalent metal cation, A is the analyte of interest, ref is a chiral reference molecule, and FL is a fixed ligand molecule. Upon fragmentation of this trimer via collision-induced dissociation (CID), two unimolecular dissociation pathways yield two fragment ions, [(MII)(A)(FL−H)]+ and [(MII)(ref)(FL−H)]+, from the neutral loss of either “ref” or “A”, respectively. A branching ratio term, Rfixed, relates the ratio of the relative intensities of these two fragment ions for a given, individual, analyte (eq 1).46,48,49,55−57 For the proposed general glycosidase assay, the analyte isomer would be the monosaccharide released from the enzymatic hydrolysis of a natural product substrate with a named glycosidase. R fixed =

[MII(A)(FL−H)]+ [MII(ref)(FL−H)]+

ln(R relative‐fixed) =

Δ(ΔG) RTeff

■

EXPERIMENTAL SECTION Materials. All monosaccharide standards, D-allose, Dgalactose, and D -mannose (>99%), α-1-2 galactobiose (>98%), α-1-4 galactobiose (>97%), α-1-3 galactobiose (>95%), α-1-2 mannobiose (>95%), α-1-3 mannobiose (>95%), α-1-4 mannobiose (>95%), α-1-6 mannobiose (>95%), β-1-4 mannobiose (>95%), β-1-4 galactobiose (>95%), and β-1-6 galactobiose (>95%), were purchased from Carbosynth (Berkshire, UK) without further purification. 1-β-D-Galactosylphingosine (>98%), 4-nitrophenyl-α-D-galactopyranoside (99%), and 4-nitrophenyl-α-D-mannopyranoside (>99%) from Sigma-Aldrich (Milwaukee, WI USA) were purchased and used without further purification. Commercial enzymes, β-galactosidase (from Escherichia coli with enzyme commission #3.2.1.23) and α-mannosidase (from Jack bean gum; Canavalia ensiformis with enzyme commission # 3.2.1.24), were purchased from Sigma-Aldrich (Milwaukee, WI USA). Metal salts, NiCl2, CuCl2, MnCl2, and ZnCl2, from SigmaAldrich (Milwaukee, WI USA) were purchased and used without further purification (>95%). Chiral references, L-serine, L-aspartic acid, L-threonine, and L-tryptophan, Sigma-Aldrich (Milwaukee, WI USA), were purchased and used without further purification (>95%). Fixed ligands, L-phenylalanyl glycine, cytidine 5′monophosphate sodium salt, and guanosine

(1)

(2)

In order to create a label-free mass spectrometry-based glycosidase assay to test the kinetic competency of monosaccharide-containing natural product substrates, several important questions arose. How could what is known about the fixed ligand kinetic method be used to interrogate kinetic 7184

DOI: 10.1021/acs.analchem.6b01360 Anal. Chem. 2016, 88, 7183−7190

Article

Analytical Chemistry

16 h. Cell cultures were harvested by 30 min centrifugation at 4 °C, 5000 rpm. Further cell lysis was achieved by ultrasonication. The cell lysis mixture was incubated at 75 °C for 20 min to denature all the heat-labile proteins. According to the protocol recommended in the Ni-NTA fast kit, His-tagged proteins were purified and the protein concentration was determined by the Bradford method with bovine serum albumin as a standard. SDS-page gel analysis of purified protein PF1208 and JL-18 is shown in the Supporting Information. Hydrolysis Conditions. Enzymatic reactions with disaccharide substrates were carried out with four different enzymes: β-galactosidase (from Escherichia coli), α-mannosidase (from Canavalia ensiformis), α-galactosidase JL-18, and βmannosidase PF1208 (from Pyrococcus f uriosus, previously characterized67). To set up the reaction, an aliquot of 100 μL of disaccharide stock solution (all prepared in 50/50 v/v water/ methanol), stock solution concentration of 10 mM for α-1-3 galactobiose, α-1-2 mannobiose, α-1-3 mannobiose, α-1-4 mannobiose, α-1-6 mannobiose; 2 mM for α-1-4 galactobiose, α-1-2 galactobiose; 4 mM for β-1-4 mannobiose; 30 mM for β1-4 galactobiose, β-1-6 galactobiose, was dried over speed vacuum. The dried disaccharide was resuspended by adding 45 μL of phosphate buffer (250 mM, pH 7.0). For enzymatic reaction samples, 5 μL of enzyme was added from enzyme stock solution (concentration: α-mannosidase: 2 unit/μL; βgalactosidase: 0.4 mg/mL; α-galactosidase JL-18:0.37 mg/mL; β-mannosidase PF1208:0.86 mg/mL). For control samples, 5 μL of enzyme buffer (250 mM NaCl, 50 mM Tris, pH 7.0) was added instead. The sample vials were reacted for 8 h at the optimal temperature of each enzyme (37 °C for β-galactosidase and α-mannosidase; 90 °C for α-galactosidase JL-18 and βmannosidase PF1208). One β-galactose containing natural product substrate, 1-β-D-galactosylsphingosine from bovine brain (abbreviated β-1-galactosylsphingosine or referred to as psychosine), was also used in this assay. β-1-Galactosylsphingosine was dissolved in 50/50 v/v chloroform/methanol and made into a stock solution of 40 mM. To set up the reaction, an aliquot of 50 μL of psychosine stock solution was added to a reaction vial along with 140 μL of phosphate buffer (250 mM, pH 7.0) and 10 μL of β-galactosidase enzyme stock solution (0.4 mg/mL). For control samples, 10 μL of enzyme buffer (250 mM NaCl, 50 mM Tris, pH 8.0) was added instead. The sample vials were reacted at 37 °C for 20 h before speed vacuum solvent reduction and desalting. Desalting. Sample desalting is achieved with the use of carbon graphite tips (10−200 μL). The procedure is as follows: attach the NuTip to a micropipette and aspirate and expel 200 μL of the binding solution (0.1% formic acid), wash 3 times with binding solution, and then aspirate and expel the sample solution 50−60 times to allow all of the monosaccharide to bind to the carbon media. To wash the bounded sample, aspirate and expel 200 μL of binding solution three times, discarding the expelled solution each time. To elute the bounded monosaccharide, aspirate and expel 20 μL of release solution (90% methanol, 0.1% formic acid) ten times to ensure complete release. The expelled solution was collected in a clean tube and dried over speed vacuum at 37 °C. Recovery ratio tests (please see the Supporting Information) were performed with p-nitrophenol labeled monosaccharide prior to testing analyte−reaction mixtures. It is presumed that the artificial pnitrophenol substrate used will result in a greater recovery ratio than a monosaccharide alone; the charged aromatic group likely

5′monophosphate sodium salt, were purchased and used without further purification from Sigma-Aldrich (Milwaukee, WI USA) (>95%). Traces of formic acid were added to the cytidine 5′monophosphate sodium salt and guanosine 5′monophosphate sodium salt stock solutions a week before being used.49 HPLC grade H2O, methanol, acetonitrile, and formic acid were used. Stock solutions were created in 50/50 (v/v) amounts. The pET21 vector, One Shot chemically competent E. coli cells, isopropyl-β-D-1-thiogalactopyranoside (IPTG), and SYBR safe DNA gel stain were obtained from Invitrogen (Grand Island, NY). The restriction enzymes and T4 DNA ligase used were from New England Biolab (Ipswich, MA). Plasmid DNA was purified on a mini prep spin kit from Qiagen (Holliston, MA). Protein mixtures were purified with Ni-NTA fast kit from Qiagen (Holliston, MA). Nutip hypercarbon tips and Nutip C4 tips were purchased from GlySci (Columbia, MD). All temperature-controlled enzyme reactions were performed with an Eppendorf Mastercycler gradient PCR instrument; temperatures stated are external. All absorbance readings were recorded with a Spectronic 20 Genesys instrument. Solvent reduction was done with a CentriVap benchtop Centrifugal vacuum concentrator. Gene Selection and Synthesis. The putative αgalactosidase gene was selected from the Carbohydrate-Active Enzyme (CAZy) database (http://www.cazy.org):61,62 putative α-galactosidase from Thermus thermophiles JL-18 (JL-18, 476aa, original sequence accession number: AFH40112). The selected gene was codon optimized for E. coli and synthesized by GeneScript (Piscataway, NJ). The β-mannosidase gene was selected from the Carbohydrate-Active Enzyme (CAZy) database: β-mannosidase from Pyrocuccus f uriosus DSM 3638 (PF1208,510aa, accession number: KR362604). The selected gene was codon optimized for E. coli and synthesized by GeneScript (Piscataway, NJ). It is important to note that αgalactosidase (JL-18) and β-mannosidase (PF1208) are hyperthermophilic enzymes, as compared to the β-galactosidase and α-mannosidase, which are commercially available enzymes that operate at 37 °C. Whole genome sequencing has been completed for both Pyrococcus f uriosus and Thermus thermophilus. This sequencing data identified more than 100 carbohydrate-active sequences (coded for both glycosidase and glycosyltransferase) in both species.63−66 Archaea and bacteria are a useful source of extremophiles, and enzymes from these organisms have potential uses in numerous catalytic processes.63−66 Thermostable enzymes have advantages over enzymes from mesophilic sources, such as increases in solubility, conformational flexibility at high temperatures, and ease of purification when expressed in systems like Escherichia coli.63−66 Protein Purification and Expression. Recombinant plasmids pET21a-JL18 and pET21a-PF1208 were sequenced with pET21a forward primer (5′-TTTTGAATTCGAAGGAGAT ATACATATGGAAGCGACCCTTCCCGT-3′) and reverse primer (5′-TTTT TTAAGCTTTTAGCGCCAGAGGACCACC-3′) prior to transformation into E. coli BL21α (DE3) cells. Cells were grown at 37 °C in 20 mL of 100 μg/mL ampicillin selected 2X YT medium for 16 h. A portion of seed culture (5 mL) was inoculated into 250 mL of ampicillin selective 2X YT medium with shaking at 250 rpm. After OD600 of 0.8 was reached, the cell culture was transferred to a 16 °C chamber. For pET21a-JL18 bearing cells, 150 μL of IPTG (1 mM) was added and the culture was incubated for an additional 7185

DOI: 10.1021/acs.analchem.6b01360 Anal. Chem. 2016, 88, 7183−7190

Article

Analytical Chemistry

Figure 2. Sample mass spectra. MS1 spectrum illustrating various ions seen in a precursor scan (top left). MS2 spectra for 100% D-galactose (top right), 100% D-allose (bottom left), and 100% D-mannose (bottom right), all with the MnII/L-Thr/L-Phe-Gly fixed ligand combination.

fixed ligand combinations such as CuII/L-Ser/5′GMP, NiII/LAsp/5′CMP, NiII/L-Asp/5′GMP, and MnII/L-Asp/L-Phe-Gly.44 With this information known that D-allose presents a suitable chiral dopant, other metal/reference/fixed-ligand combinations were screened to determine which create the largest ΔΔG for D-galactose versus D-allose as well as D-mannose versus D-allose (Supporting Information). This screen showed that MnII/LThr/L-Phe-Gly along with the chiral dopant of D-allose presents the greatest energetic differences (∼30-fold difference in Rfixed for D-galactose/D-allose and ∼20-fold difference in Rfixed for Dmannose/D-allose). From this, calibration curves47,68−70 can be constructed that permit quantitation of the monosaccharide released via enzymatic hydrolysis than can be used to determine how much recovered monosaccharide is needed to apply this described assay (discussed further in the Supporting Information). Thus, with this suitable fixed ligand combination (MnII/L-Thr/L-Phe-Gly and D-allose), it becomes possible to screen the kinetic competency of the natural product, R-group portion, of a substrate, through eq 2 between the Rproduct‑fixed (glycosidase hydrolysis plus the chiral dopant) and Rcontrol‑fixed (no glycosidase hydrolysis plus the chiral dopant). Figure 2 illustrates sample mass spectra for chiral dopant selection. For all experiments, 20 nmol of D-allose was used as this provided a satisfactory signal-to-noise ratio for both product and control. It is important to mention that previous literature46 has demonstrated that dissociation rates (Rfixed) values are concentration independent; thus, other amounts of chiral dopant could indeed be used for future studies. It is observed that ∼100 nanograms of recovered monosaccharide is sufficient to yield reproducible and discriminative results to probe kinetic competency of various enzyme−substrate pairs (Figure S7). Method Validation. In order to test this proposed labelfree mass spectrometry-based assay, we began with a widely studied commercial enzyme, α-mannosidase (from Jack bean

results in better interactions and thus recovery with the Carbon Graphite NuTips. Mass Spectrometry Conditions. An LTQ Orbitrap XL (ion trap portion only) from Thermo Scientific (San Jose, CA, USA) with an ESI source operated in positive ion mode was used for all experiments. Data acquisition was performed with the Xcalibur software. Optimized conditions were: 5 kV spray voltage, 0 V capillary voltage, 150 °C capillary temperature, 40 V tube lens voltage, 20 units sheath gas flow rate, 0 units sweep gas flow rate, 10 units aux gas flow rate, 6.0 m/z isolation width, 16% normalized collision energy (%NCE) which is calculated to ∼22 mV collision energy from the instrument parameters, 30 ms activation time, wideband activation on, activation Q 0.250, and 50 scans each with 3 microscans per spectrum for standards, and 100 scans each with 3 microscans per spectrum for products and controls. Specifically, the concentrations of each component are as follows: 25 μM divalent metal cation and 100 μM each for fixed ligand, reference, and analyte.

■

RESULTS AND DISCUSSION Chiral Dopant Selection. A suitable fixed ligand combination can be defined as one that creates the largest energetic difference, ΔΔG, relative to the monosaccharide analyte released from glycosidase hydrolysis (D-galactose and Dmannose) in this assay. In lieu of reporting inaccurate energetic values based on the fact that Teff is not definitively known, only the magnitude of Rfixed will be used. In the cases reported herein, a promotive ligand combination and chiral dopant is one that creates the largest ΔΔG value for D-galactose versus the chiral dopant as well as for D-mannose versus the chiral dopant. This value can be simply expressed as the greatest degree of difference between RD‑Gal‑fixed versus Rchiral dopant‑fixed and RD‑Man‑fixed versus Rchiral dopant‑fixed. Previous work has shown that D-allose created the largest such energetic differences for 7186

DOI: 10.1021/acs.analchem.6b01360 Anal. Chem. 2016, 88, 7183−7190

Article

Analytical Chemistry gum; Canavalia ensiformis; enzyme commission # 3.2.1.24), and some of its preferred substrates.71−74 It was seen that both substrates yielded a mass spectrometric response, and it was calculated that α-1-2 mannobiose cleaved at a, ∼8 times faster rate than α-1-3 mannobiose. These results (presented in Table 1) match results previously reported that α-mannosidase from Table 1. Enzyme-Substrate Table for 11 Various Monosaccharide-Containing Natural Product Substrates and Their Respective Kinetic Competency (Relative Percentage Order of ΔRfixed (Rfixed‑product − Rfixed‑control) between Product and Control) for Four Different Glycosidases in This LabelFree Chiral Dopant Assaya

Figure 3. Sample MS/MS spectrum for product and control reactions for α-1-2 mannobiose with α-mannosidase. Precursor at 575 m/z is fragmented to form 456 m/z and 395 m/z. Rfixed is found by taking the relative intensity at 456 m/z divided by that at 395 m/z. Please note: α-1-2 mannobiose can also be commonly referred to as Manα-1-2Man.

Demonstration of New Enzyme−Substrate Pairs. Given the initially promising results with the Jack bean gum enzyme, we proceeded to study another commercial enzyme (β-galactosidase from Escherichia coli; enzyme commission #3.2.1.23) as well as hyperthermophilic enzymes (newly characterized α-galactosidase JL-18 from Thermus thermophilus; enzyme commission #3.2.1.22 and β-mannosidase PF1208 from Pyrococcus f uriosus; enzyme commission number 3.2.1.25) with this assay and to probe previously unstudied substrates and thereby yield new information on the kinetic competency of the natural product, R-group, portion of a monosaccharidecontaining substrate. To test this, 11 different substrates [1 (α1-2 galactobiose), 2, (α-1-4 galactobiose), 3 (α-1-3 galactobiose), 4 (β-1-galactosylphingosine), a key substrate related to the pathogenic mechanism of Krabbe disease,75 5 (β-1-4 galactobiose), 6 (β-1-6 galactobiose), 7 (α-1-2 mannobiose), 8 (α-1-4 mannobiose), 9 (α-1-3 mannobiose), 10 (α-1-6 mannobiose), and 11 (β-1-4 mannobiose)] were tested (Table 1). For raw data such as Rfixed values and calculations of total amounts of monosaccharide cleaved, please see the Supporting Information. Since the calculation of energetic values from eq 2 may be misleading, as previously mentioned, a relative order approach, on a scale of 0−100%, is taken that provides insight on which substrates are more or less kinetically competent for the characterized glycosidase. Furthermore, since the monosaccharide unit stays constant for all of these substrates, this assay can determine which R-groups are most conducive, or kinetically competent, for reaction with a given glycosidase. Table 1 illustrates the kinetic competency of each monosaccharide-containing natural product substrate for their respective glycosidases as a change in relative ΔR fixed (Rfixed‑product − Rfixed‑control) between product (glycosidase plus chiral dopant) and control (no glycosidase plus chiral dopant). To see how the values below were calculated from eqs 1 and 2 for raw Rfixed values of both product and control, please see Figures S5 and S6. Table 1 shows comparisons of the relative kinetic competency of monosaccharide-containing natural product substrates for a named glycosidase. Table 1 is arranged from high (top row) to low (bottom row) kinetic competency, where a high value indicates a greater/faster rate of cleavage for an enzyme−substrate pair. It is important to mention that each data point in Table 1 represents three independent ESI-direct infusion mass spectrometry experiments (±value error bars

a

Each data point consists of three independent mass spectrometry direct infusion experiments, where ± error values are from the three averaged trials where each consists of 100 mass spectrometry scans each with 3 microscans. Fixed ligand combination MnII/L-Thr/L-PheGly (25μM/100μM/100μM) and chiral dopant D-allose (20 nmol) were used for all experiments. Blue font denotes enzyme−substrate pairs not previously reported in the literature. Please see Figures S5 and S6 for raw values and to see how the values above were calculated.

Jack bean gum has primary substrate specificity for an α-1-2 mannose linkage, up to 15 times faster cleavage than other linkages,71 and the order of α-mannosyl linkage cleavage is as follows: α-1-2 > α-1-3 > α-1-6.72 No information has been available for an α-1-4 mannosyl linkage, as it was either not tested or unavailable.71,72 Clearly, the regiochemistry of the glycosidic linkage plays a tremendous role in determining the kinetic competency of a substrate and fully defining the substrate profile of a glycosidase. Such information is lost when only simple p-nitrophenol-linked monosaccharides are assayed. Previous studies73,74 have also demonstrated that a βgalactosidase from E. coli has activity toward a β-1-4 galactosyl linkage, which matches our results that a β-1-4 galactosyl linkage is hydrolyzed (presented in Table 1). However, previous literature also stated that the specificity of βgalactosidase from E. coli is based on specific recognition of the substrates, instead of better accessibility of one particular branch,74 thus inferring that its primary substrate is not well understood and is something that could be further explored with this mass-spectrometry-based assay. Figure 3 illustrates a sample tandem mass spectrum for both a product (glycosidase hydrolysis plus chiral dopant) and control (no glycosidase hydrolysis plus chiral dopant) to demonstrate how Rfixed is calculated. 7187

DOI: 10.1021/acs.analchem.6b01360 Anal. Chem. 2016, 88, 7183−7190

Article

Analytical Chemistry

labeled substrates are unavailable, this assay would prove to be the most suitable. To aid in adoption of this assay by other laboratories, a workflow diagram is included in Figure S10. If a named glycosidase is of interest to test with various monosaccharidecontaining substrates unrelated to the mannose and galactosebased assays described here, the presented chiral dopant fixed ligand combinations might also be suitable, but that does not mean that additional ones may not provide improved chiral discrimination for other unique glycosidase−substrate pairs. Finally, this work also presents the first time that linkage specificity has been studied for the two thermophilic enzymes (a newly characterized α-galactosidase and a previously reported β-mannosidase).57 Whereas raffinose, containing an α-1-6 galactose-glucose linkage, lactose, containing an β-1-4 galactose-glucose linkage, and extensive studies with αmannosidases from Jack bean gum (Canavalia ensiformis) have received much attention, this study, to the best of our knowledge, demonstrates the first time that α-1-4 mannobiose was used as a substrate for an α-mannosidase from Canavalia ensiformis and that β-1-galactosylsphingosine was used as a substrate for a β-galactosidase from Escherichia coli. This approach is in contrast to previous glycosidase assays that have required labeling of the monosaccharide, such as with a pnitrophenolate or fluorescent derivatives, thereby obviously rendering them into non-native states that cannot indicate any kinetic competency for the original substrates.75−84

represent the triplicate average of 100 scans with 3 microscans for each spectrum). Here, various linkages were interrogated for their kinetic competency in monosaccharide-containing natural product substrates. It can be seen that not only the monosaccharide present in the natural product but also the R-group portion has a tremendous influence on the observed kinetic competency of a substrate for a named glycosidase. Certain substrates were not conducive to glycosidase hydrolysis at all, as denoted by no cleavage observed in Table 1; no relative change was observed between product and control reactions, within standard deviation. Even among cleaved substrates, however, differences in competency emerge that would be difficult to assess in a simple end-point assay.

■

CONCLUSIONS Herein, the first glycosidase assay for the screening of the leaving group portion of D-galactose- and D-mannosecontaining natural product substrates has been demonstrated with a mass spectrometry-based label-free chiral dopant approach. This assay has capabilities to quantitate the monosaccharide portion (presented in the Supporting Information) and provides a general solution to identify the kinetic competence of glycosidase substrates, as was demonstrated with four different glycosidases. This assay can be readily adopted to screen the kinetic competency of natural products that contain other analytes, such as peptides, nucleic acids, or other monosaccharides, in the same described procedure. This approach has the potential for the creation of a multiplex platform that could screen multiple natural products simultaneously with multiple enzymes when a full substrate profile is desired, especially if the desalting issues can be improved by the community, which could raise the recovery ratio of collected monosaccharide and improve ion abundances in the gas phase. From a biological perspective, a potential future direction would be to use this assay on an N- or O-linked oligosaccharide to determine the linkage of a terminal galactose, for example, based on the knowledge obtained from these known linkage disaccharides. Perhaps the greatest shortcoming with this assay is the poor recovery ratio associated with the desalting of monosaccharides. Even after countless aspirations, the recovery of monosaccharides remains