Assays Using a NIMS Chip: Loosely Bound but Highly Selective

Article pubs.acs.org/ac

Assays Using a NIMS Chip: Loosely Bound but Highly Selective J. Li and R. H. Lipson* Department of Chemistry, University of Victoria, P.O. Box 3065, Stn CSC, Victoria BC V8W 3V6, Canada S Supporting Information *

ABSTRACT: A fluorous-affinity nanostructure initiator mass spectrometry (NIMS) chip has been developed as an enzyme activity assay. As a proof-of-principle, an assay for detecting cysteine-containing peptide phosphorylation by protein kinase A (PKA) was designed using NIMS technology. The efficacy for an enzyme inhibition assay was characterized by deriving an IC50 value from the ratio of the substrate-to-product mass spectral signal intensities, using known inhibitors of PKA and Abl kinase activity. Lastly, the potential use of a NIMS chip as a multiple screening enzyme inhibition platform was explored.

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laborious and costly protocols associated with substrate labeling. Unlike fluorescence-based assays, mass detection avoids sample autofluorescence and/or light scattering, thereby reducing the number of false positives and negatives during high-throughput screening for enzymatic inhibitors.15 Furthermore, unlike label-based methods, mass detection can be used to simultaneously monitor the concentrations of a range of compounds during an enzyme-catalyzed reaction including those of the substrate, product, possible cofactors, coenzymes, and any internal standards.16,17 Today, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) MS have emerged as powerful methodologies capable of directly measuring substrates and reaction products.2,18−20 Recently, the capabilities of MALDI-MS have been extended to include imaging (MALDIIMS).21,22 Desorption Ionization on Silicon (DIOS) MS has also been used to measure enzyme substrate kinetics by analyzing the remains of a reacting enzyme and substrate droplet as it traveled down the length of a porous silicon (pSi) microfluidic channel.21 Despite the power of MALDI MS it possesses a number of inherent limitations including the presence of “sweet spots”, areas on the solid solution surface where the MALDI ion signal intensities are particularly intense. These are due to the heterogeneity of the solid solution formed when the matrix and analyte are cocrystallized. Quantitative analyses are therefore extremely challenging because of the resultant shot-to-shot variability.23 Furthermore, low molecular weight substrates and products can be readily masked by strong matrix and matrix fragment ion signals. Similarly, a difficulty associated with high throughput enzyme inhibitor screening based on ESI MS is the need to desalt the sample which significantly slows down the analysis and increases its cost.14

nzymes play a crucial role regulating a wide range of life processes such as cell growth, migration, differentiation, and death.1,2 They are also widely used in industry to screen for drug targets3 and as biocatalysts in the manufacturing of biofuels.4 It is therefore becoming increasingly important to develop sensitive methods to elucidate enzyme catalytic mechanisms, to characterize enzyme activity, and to rapidly screen for inhibitory compounds.5 Most current enzyme assays make use of the spectroscopic differences between a substrate and a product during conversion. Typical analytical technologies include fluorescence spectrometry and ultraviolet (UV)visible absorption spectroscopy.6,7 Although these methods are rapid, simple, and suitable when using high-throughput microtiter plates and microarrays,8−10 they can suffer from limitations which include faulty outcomes caused by the introduction of a fluorescence group into the substrate to overcome the fact that most naturally occurring substrates do not readily exhibit any absorption or emission properties in the optical regime.11,12 Derivatization can also change the kinetic properties of the substrate and perturb the absorption and fluorescence spectra of enzymes, cofactors, and/or buffers in the reaction mixtures.13 Radioactivity assays of enzyme-catalyzed reactions rely on the use of substrate radioactive labels. Although the use of radioactively labeled substrates preserves the chemical integrity of the substrate, the synthesis of radioactive-labeled compounds can be quite laborious, and the handling and ultimately disposal of radioactively “hot” materials requires strict safety protocols.5 Mass spectrometry (MS) is a technique that is ideally suited for enzymatic activity assays and inhibitor potency screening because mass detection is a direct measurement, and product formation can be readily established by simply measuring the reactant-product mass differences resulting from the enzyme reaction. 14 The attractiveness of MS over UV−visible absorption spectrometry, fluorescence, and radioactivity detection stems from its ability to perform enzyme assays using label-free substrates, thereby circumventing the often © 2013 American Chemical Society

Received: April 15, 2013 Accepted: June 17, 2013 Published: June 17, 2013 6860

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from Bachem Americas, Inc. (California, USA). Bradykinin (RPPGFSPFR, HPLC grade, ≥98% purity, BK), dithiothreitol (DTT), C-methylcalix[4] resorcinarene, calix[4]arene, and ammonium bicarbonate were obtained commercially from Sigma-Aldrich Chemical Co. (St. Louis, MO). Imatinib mesylate (a crystalline solid, purity, ≥98%) and staurosporine (a solution in ethyl acetate, purity, ≥98%) were purchased from Cayman Chemical (Ann Arbor, MI). Hydrogen peroxide (30% solution) was purchased from EMD Chemicals Inc. (Gibbstown, NJ). Methanol, ethanol, acetonitrile, trifluoroacetic acid (TFA), sulfuric acid, acetic acid, and hydrofluoric acid (HF, 48%) were purchased from Caledon Laboratories Ltd. (Georgetown, Ont., Canada). Distilled water was obtained from a Milli-RO water purification apparatus (Millipore Corporation, Bedford, MA, USA). Preparation of the Fluorous-Affinity NIMS Chip. The preparation of NIMS chips has been described elsewhere in detail,41,42 and only a brief description of the materials and experimental conditions used in this study is presented here. First, an appropriate size of silicon wafer was cut and soaked in Piranha solution (H2SO4/H2O2 = 2:1, V/V) for 30 min to clean off the surface. The wafer was then rinsed using deionized water and blown dry with UHP nitrogen gas. Second, the clean silicon was galvanostatically etched in a homemade Teflon electrochemical cell containing 25% HF ethanol solution, where a copper foil attached in the unpolished side of Si wafer served as the anode, and the counter electrode was a platinum wire immersed into the etching solution. The optimal etching current density and time for preparing the nanostructured Si surfaces used in this study were 45 mA/cm2 controlled by VersaSTAT 3 purchased from Princeton Applied Research (Oak Ridge, TN) and 30 min. The resultant porous silicon (pSi) was composed of ∼10−20 nm diameter pores. Third, 2 μL of initiator liquor compound (BisF17) was placed onto the pSi chip surface and left standing for 60 min. Any excess initiator was then blown off using a strong jet of UHP nitrogen leaving behind a thin film on the chip. Finally, 1 μL of RF17 methanol solution (10 μM) was spotted onto the NIMS chip. The fluorous-tagged molecules containing the maleimidefunctionalized group was held to the surface by the noncovalent fluorine−fluorine interactions between the fluorous tag and the perfluorous initiator molecules trapped within the nanostructured pSi. Experiments were performed that showed that a NIMS chip could be stored within a clean ambient environment for up to 6 months before using without exhibiting a significant decrease in analyte or assay ion intensity relative that observed using a freshly prepared NIMS platform. Enzyme Activity Assay and Inhibitor Screening. A substrate (KT, 200 fmol) was spotted onto a NIMS chip and then treated with 1 μL of PKA solution. The assays were performed according to the manufacturer’s protocol. In brief, reaction mixtures containing 200 μM ATP, 10 mM MgCl2, and 1 unit/μL PKA in Tris-HCl buffer (pH 7.5) were added to substrates in a humid chamber at 30 °C for 30 min. The chip surface was then rinsed using 40% methanol−water solution and analyzed by NIMS. Analysis of PKA inhibitor was performed in the same amount and reaction condition as PKA activity assay, except for using 2 μL of staurosporine having molarities ranging from 10−6 to 103 μM in intervals corresponding to one order of magnitude. In the same procedure as above, PKA inhibitor screening was achieved by adding 2 μL of 1 μM staurosporine, C-methylcalix[4]

The desorption/ionization technique of nanostructure initiator mass spectrometry (NIMS), developed by the Siuzdak group in 2007, which is an extension of DIOS24 has a number of features which can overcome the limitations noted above for MALDI and ESI MS. NIMS uses ‘initiator’ molecules trapped within a nanostructured material to assist the release and ionization of intact molecules adsorbed on that material’s surface upon laser or ion irradiation.25 The advantages of NIMS over MALDI MS and other surface desorption/ionization techniques include higher analyte sensitivity and simpler sample preparation; the latter due to the fact that NIMS is ″matrixfree″ in that the analyte is not cocrystallized with the initiator. The sensitivity and throughput of an enzyme assay can be markedly improved by transforming conventional solutionbased approaches to those involving an immobilized phase;26 for example, the evaluation of ligand−receptor interactions and enzymatic activities using peptide chips.27−31 Many enzyme processes, including kinase32 and protease33,34 activities, can be readily studied using peptides as model substrates. Biochips based on fluorous-affinity interactions have emerged as a new technique for studying biological systems.35,36 This approach takes advantage of a fluorous tag immobilized on the surface of a biochip to capture analytes of interest by fluorine− fluorine interactions.36−38 When combined with mass spectrometry, it can, through on-site enrichment, provide rapid and sensitive analyses of targeted species.39,40 Recently, we reported that polypeptides containing cysteine residues could be captured and held to a NIMS chip using a fluorous affinity tag of 3-(perfluorooctyl)-propyl-1-maleimide, which is a combination of a long perfluorous carbon chain(−C8F17) with a chemically active function group (−CC−).41 This “soft” immobilization, which is accomplished by noncovalent fluorine−fluorine interactions, allows enzyme substrates and reaction products to be efficiently desorbed and subsequently ionized in a mass spectrometer. In this work a PKA activity assay for cysteine-containing peptide substrates is demonstrated for the first time. The enzymatic inhibition potency could be characterized by an IC50 value obtained by directly measuring the ratio of substrate-to-product concentrations against known inhibitors for PKA and Abl kinase. Finally the potential of a fluorous-affinity NIMS chip to multiply screen different compounds was evaluated.

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EXPERIMENTAL SECTION Materials and Chemicals. Single-side polished p-type silicon ⟨100⟩ wafers with boron dopant, 76 mm diameter, 381 ± 25 μm thickness, and ≤0.005 Ω-cm resistivity were purchased from Silicon Quest International (Santa Clara, CA). 1,3-Bis(Heptadecafluoro-1,1,2,2-tetrahydrodecyl)tetramethyldisiloxane (C24H20F34OSi2, BisF17) was acquired from Gelest (Morrisville, PA). 3-(Perfluorooctyl)-propyl-1maleimide (C15H8F17NO2; RF17) was obtained from Fluorous Technologies Inc. (Pittsburgh, PA). Protein kinase A (PKA) and Abl protein tyrosine kinase (Abl), PKA reaction buffer (including 50 mM Tris-HCl, 10 mM MgCl2 and pH 7.5 at 25 °C), and NEBuffer for Abl (including 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Brij 35, and pH 7.5 at 25 °C) were purchased from New England Biolabs Ltd. (Pickering, Ontario). Kemptide (LRRASLGC; KT) and its standard phosphorylated variant, LRRASpLGC (HPLC grade, ≥98% purity, custom synthesis), were acquired from GenScript USA Inc. (Piscataway, NJ). The polypeptide Dalargin (YAGFLR; Dal; HPLC grade, >98% purity) was purchased 6861

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Figure 1. Schematic diagram for a NIMS-based PKA assay: 1. Cysteine-containing peptides are captured by an affinity tag immobilized on a NIMS chip. 2. The PKA enzyme is added onto the NIMS chip. 3. Phosphorylated peptides are produced by the enzyme reaction. 4. The substrate and product are detected by NIMS.

efficiently isolated and enriched from extremely complex samples such as a bovine serum albumin (BSA) protein digest.41 Specifically, a cysteine-containing peptide substrate could be anchored to the surface of a fluorous-affinity NIMS chip by a simple and highly selective Michael addition reaction between thiol and maleimide groups.43 In this work, the strategy used takes advantage of surfaceimmobilized substrates to perform an enzyme activity assay to provide a quantitative evaluation of enzyme inhibition. The workflow designed for this experiment is shown in the Figure 1. First, a crude mixture containing a particular substrate or a substrate mixture containing many types of species is loaded onto a NIMS chip with a specific fluorous affinity tag. Next, PKA buffer solution is added onto the loaded fluorous-affinity NIMS chip where the dissolved enzyme molecules diffuse to the sites at which the substrates are bound. Protein kinases catalyze the transfer of a phosphate group (PO43‑ ≡ “P”; net mass change in a neutral species of +80 Da) to tyrosine, serine, or threonine residues of a substrate.29 After the enzyme catalytic reactions are completed, the chip is eluted with a fluorophobic solvent such as 40% methanol−water solution with 0.1% TFA to remove any residual nonfluorous compounds not bound to the fluorous affinity tag as well as salts and other contaminants. This step does not affect the bound substrates or resultant products containing the fluorous tag. Finally, the NIMS chip is subjected to mass spectrometry analysis. Enzymatic Activity Assay of PKA with FluorousAffinity NIMS Chip. The feasibility of a PKA assay using this methodology is demonstrated here by examining KT or a mixture of three peptides: Dal, KT, and BK, where only the KT substrate contains a cysteine residue. Figure 2a shows a single major peak at m/z 1433 corresponding to the protonated product of KT with the

resorcinarene, and calix[4]arene, respectively. For Abl enzymatic inhibition assay, the mixture of Abl and imatinib was added to NEBuffer (including 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Brij 35, and pH 7.5) at 25 °C for 30 miuntes in the same method as PKA inhibitor analysis. Next NIMS chips containing the fluorous affinity tag were dosed by aliquots of a peptide mixture containing Dal (200 fmol), KT (200 fmol), and BK (200 fmol), which all dissolve in the 70% acetonitrile water solution with 1% DTT. The substrates were kept in a humidified 6-L water bath chamber (BIO-RAD) at 60 °C for 60 min, washed with 40% methanol and water solution with 0.1% TFA 3−5 times, and then dried under a stream of UHP nitrogen. NIMS Procedure. Each NIMS chip was anchored using electrically conducting double sided carbon tape onto the sample probe of a home-built MALDI mass spectrometer based on an Applied Biosystems/MDS Sciex LC/MS/MS (API-365) triple quadrupole (QqQ) mass spectrometer originally configured for electrospray ionization (ESI). The chips were irradiated with the output of a pulsed N2 laser (Laser Science, Inc. Model VSL-337ND-S) operating at a wavelength of 337 nm and a 10 Hz repetition rate. Each mass spectrum was generally collected from at least 5 different positions on the chip over ∼1000 laser shots, each pulse having 5 μJ of energy. The % intensity of the mass spectra peaks were obtained by normalizing to the base peak intensity in the spectrum which corresponds to 100%.

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RESULTS AND DISCUSSION Experimental Design. Our previous experiments using a fluorous-affinity NIMS chip showed that a desired subset of analytes (e.g., peptides containing cysteine residue) could be 6862

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Figure 2. a) The mass spectrum of the KT substrate plus fluorous tag before the addition of the PKA enzyme. b) The mass spectrum after adding PKA. The major peak corresponds to phosphorylated KT resulting from the reaction of the substrate with PKA. P denotes the phosphate group added to the substrate.

Figure 3. a) Mass spectrum of a mixture of peptides (Dal, KT, and BK) obtained using a fluorous-affinity NIMS chip before adding kinase PKA. b) Mass spectrum of the products formed from the mixture in a) after the addition of PKA followed by rinsing using a 40% methanol− water solution.

%MA = (%C with inhibitor/%C without inhibitor) × 100% (2)

fluorous affinity tag. After treatment with PKA according to the manufacturer’s protocol, the original peak corresponding to the substrate peptide disappeared, and a new peak was observed at m/z 1513 (Figure 2b) which corresponds to the phosphorylated peptide. The peak intensities suggest that fluorous affinity immobilization does not significantly interfere with the reactivity of either the enzyme or the substrate. Next, a mixture of Dal, KT, and BK was spotted into the chip surface as a model system to evaluate the utility of a fluorousaffinity NIMS chip as an enzymatic activity assay platform for complex biological samples. The phosphorylation reaction was allowed to proceed for 30 min in a water bath chamber maintained at 30 °C. The chip was then rinsed 3−5 times using a 40% methanol/water solution and then placed in the mass spectrometer for NIMS analysis. As expected (Figure 3), only the KT substrate was phosphorylated. This shows that the methodology developed here is suitable for the direct analysis of mixtures. Quantitative Analysis of Enzyme Inhibitor by Product/Substrate Ratio. A near-ideal quantitative strategy to directly measure enzyme activity and inhibition without internal standards involves simply monitoring the relative ratio of the substrate/product intensities by mass spectrometry.44−46 This approach was validated in this work for PKA by detecting the substrate peptide, KT, and its phosphorylated product using NIMS. The measured ion intensities of the substrates and products were used to calculate the percent conversion (% C) of a substrate to product and, subsequently, the percent maximal activity (% MA) in a manner similar to previous reports.44,47,48 % C is defined as the product ion intensity (Ip) divided by the sum of the substrate ion intensity (Is) and Ip multiplied by 100%:44 %C = [Ip/(Ip + Is)] × 100%

The average background signal, which is taken as any product signals in the absence of the enzyme, was routinely subtracted from all C % measurements prior to calculating % MA.47 Validation of the quantitative kinase PKA enzyme assay in such a way it does not necessarily use internal standards was proved in NIMS detection experimentally (see the Supporting Information). The use of NIMS as an enzymatic inhibition assay was evaluated by determining the half maximal inhibitory concentration (IC50) values, which is a measure of the effectiveness of a compound to inhibit biological or biochemical function. IC50 values were calculated from a 10-point sigmoidal dose response curve obtained by plotting log[inhibitor] against % MA.14 In this experiment a known inhibitor against PKA, staurosporine (Stsp), was used.48 An IC50 value of 20 nM was obtained from a 10-point dose-dependent inhibition assay plot of log [Stsp] against %MA (Figure 4a). This agrees well with the IC50 values of 25−50 nM obtained by MALDI-MS studies and 20−60 nM using fluorescence detection.49 As an experiment to assess the generality of our approach, Imatinib, a second inhibitor against Abl used to treat certain types of cancer,50 was also examined. An IC50 value of 79 nM for Imatinib against Abl was obtained from Figure 4b, which also agrees with previous reports.29,51−53 These results demonstrate that fluorous-affinity NIMS can be effectively used for quantitative assays of kinase activities and its inhibition. A Potential Platform for Enzyme Inhibitor Screening. Rapid compound screening for enzyme inhibition using a fluorous-affinity NIMS chip was next investigated. Stsp and two known nonactive compounds, C-methylcalix[4]resorcinarene and calix[4]arene, were evaluated sequently as inhibitors of kinase PKA using a single NIMS chip spotted in a four dot array format. One of the areas containing kinase PKA and substrate but no inhibitor was used as a control. A comparison of the mass spectra from the other three areas with that of the control region (Figure 5) shows immediately that Stsp is

(1)

% MA is defined as the degree of enzyme inhibition to which the % C from the control reaction (no inhibitor) has been diminished in reactions that contain an inhibitor based on eq 2:47 6863

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Figure 5. Enzymatic inhibitor screening using a fluorous-affinity NIMS chip in a 4-spot format. a): NIMS spectrum of phosphorylated KT after addition of PKA without an inhibitor and other compounds (control); b): NIMS spectrum after adding a known inhibitor, staurosporine; c): NIMS spectrum after adding a noninhibitor calix[4]arene; d): NIMS spectrum after adding a noninhibitor Cmethylcalix[4]resorcinarene.

The advantages of the fluorous-affinity NIMS chip-based enzyme assays are manyfold. Sample preparation is relatively straightforward. The enzyme activity of complex biological samples can be directly characterized, and the methodology has the potential to be extended to multiple assays on a single platform. Unlike rigid covalent surface attachment strategies, NIMS chips can be washed and reused, and fluorous-phase noncovalent immobilization may enhance enzymatic activity by providing conformational flexibility.

Figure 4. a) A 10-point dose-dependent inhibition assay curve obtained by plotting the log [Stsp] against degree of enzyme inhibition, %MA. b) A similar assay curve of the Abl inhibitor imatinib.

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ASSOCIATED CONTENT

* Supporting Information

indeed an inhibitor of kinase PKA while the other two compounds are not. Given that fluorous-affinity immobilization has been successfully used to construct small molecular microarrays37 and that NIMS has used to characterize a peptide microarray,25 it is expected that the fluorous-affinity NIMS will also prove to be a useful platform for high throughput enzyme inhibitor screening for drug discovery.

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Text and Figure S-1. 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].

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Notes

CONCLUSIONS In this work, fluorous-affinity NIMS chips were evaluated as enzyme assays by directly detecting the products formed by the reaction of peptide substrates with the PKA enzyme, quantitatively analyzing enzymatic inhibition, and screening kinase inhibition in a multiple-area format. The utility of fluorous-affinity NIMS to quantitatively assess enzymatic inhibition was confirmed by determining the IC50 values of two inhibitors and comparing the results with those obtained by other methods. Finally, a fluorous-affinity NIMS chip spotted in an array-fashion suggests that the technology developed in the work could be scaled for combinatorial screening of enzymatic inhibition. It is easily envisaged that the screening for kinase inhibition could involve hundreds of sample spots on a single NIMS chip by making use of automated chip array production technologies such as robotic contact printing or inkjet printing.

The authors declare no competing financial interest.

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