On-Chip Peptide Mass Spectrometry Imaging for Protein Kinase

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On-chip peptide mass spectrometry imaging for protein kinase inhibitor screening Young-Lai Cho, Young-Pil Kim, Jin Gyeong Son, Miyoung Son, and Tae Geol Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03557 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

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Analytical Chemistry

On-chip peptide mass spectrometry imaging for protein kinase inhibitor screening Young-Lai Cho,1,4,† Young-Pil Kim,2,† Jin Gyeong Son,1 Miyoung Son,1,5 and Tae Geol Lee1,3* 1

Center for Nano-Bio Measurement, World Class Laboratory, Korea Research Institute of Standards and Science, Daejeon 34113, Korea,

2

Department of Life Science and Institute of Nano Science and Technology, Hanyang University, Seoul 04763, Korea, 3

Department of Nanoscience, University of Science and Technology, Daejeon 34113, Korea, 4

5

Present address: Department of Chemistry, Dongguk University, Seoul 04620, Korea

Present address: Department of Research Development, Gumi Electronics & Information Technology Research Institute, Gumi 39171, Korea

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) †

These authors contributed equally to this work.

*

To whom correspondence should be addressed: Phone: +82-42-868-5129; +82-42-868-5032, E-mail:

[email protected]

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ABSTRACT

Protein kinases are enzymes that are important targets for drug discovery because of their involvement in regulating the essential cellular processes. For this reason, the changes in protein kinase activity induced by each drug candidate (the inhibitor in this case) need to be accurately determined. Here, an on-chip secondary ion mass spectrometry (SIMS) imaging technique of the peptides was developed for determining protein kinase activity and inhibitor screening without a matrix. In our method, cysteinetethered peptides adsorbed onto a gold surface produced changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides, which were quantitatively dependent on protein kinase activity. Using mass spectrometry imaging of multiple compartments on the gold surface in the presence of a peptide substrate, we screened 13,727 inhibitors, of which seven were initially found to have inhibitor efficiencies that surpassed 50%. Of these, we were able to identify a new breakpoint cluster region-abelson (BCR-ABL)T315I kinase inhibitor, henceforth referred to as KR135861. KR135861showed no cytotoxicity and was subsequently confirmed to be superior to imatinib, a commercial drug marketed as Gleevec®. Moreover, KR135861 exhibited a greater inhibitory effect on the BCR-ABLT315I tyrosine kinase, with an IC50 value as low as 1.3 µM. In in vitro experiments, KR135861 reduced the viability of both Ba/F3 cells expressing wild-type BCR-ABL and BCRABLT315I, in contrast to imatinib’s inhibitory effects only on Ba/F3 cells expressing wild-type BCRABL. Due to the surface sensitivity and selectivity of SIMS imaging, it is anticipated that our approach will make it easier to validate the small modifications of a substrate in relation to enzyme activity as well as for drug discovery. This mass spectrometry imaging analysis enables efficient screening for protein kinase inhibitors, thus permitting high-throughput drug screening with high accuracy, sensitivity, and specificity.

KEYWORDS. mass spectrometry imaging, SIMS, protein kinase, inhibitor, drug screening, imatinib


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INTRODUCTION

Analyzing enzyme activity in its biological environment can yield valuable information on the physiological and biological functions of the enzyme as well as relevant substrate-enzyme interactions.1,2 In recent years, studies on enzyme activity have driven demand for primary screening methods of identifying specific inhibitors.3.4 Among the enzymes, protein kinases have been important targets for drug discovery because of their involvement in regulating the essential cellular processes,5,6 and to date, 28 kinase inhibitors have been approved for clinical use.7 However, because most assays used to determine kinase activity are based on chromogenic and fluorogenic methods,8-10 they suffer from intrinsic problems, such as the need to use an exogenous sensing module, which can affect enzyme activity, create chemical instability of the enzyme labile bond, and lead to the inevitability of falsepositive (or false-negative) signals from naturally occurring interferences. One promising alternative has been mass spectrometry (MS) methods because they are label-free and are capable of analyzing the target substrates of enzymes with high selectivity. Much attention has been paid to the matrix-assisted laser desorption/ionization (MALDI),11,12 self-assembled monolayers with MALDI (SAMDI),13 and surface-enhanced LDI (SELDI)14 techniques to analyze enzyme activity. However, MS-based methods have been hindered by multiple factors, including matrix ion interference and saturation in the low mass range. To resolve these issues, surface-assisted LDI (SALDI) methods using structured solid support materials such as porous silicon,15,16 carbon nanomaterials,17,18 metal nanomaterials,19,20 and clathrate nanostructures21 have been developed for use without matrix molecules. Of these methods, the desorption ionization on silicon MS (DIOS-MS) method has been most commonly used to monitor enzyme activity,22 but this too has been hindered by issues with reproducibility and quantitative analyses. Hence, to fully exploit the advantages of the MS techniques for screening protein kinase inhibitors, a more efficient and straightforward method is necessary. As a matrix-free approach, time-of-flight secondary ion MS (TOF-SIMS) analyzes secondary ions sputtered from a surface that is bombarded with primary ions to detect the changes in the relative peak ACS Paragon Plus Environment

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intensities of the phosphorylated and unphosphorylated substrate peptides. It has drawn considerable attention due to its high surface sensitivity, chemical specificity and inherent reproducibility.23-25 Due to the difficulty of obtaining peptide signals using the conventional SIMS technique, we previously reported on a SIMS-based analysis of protein kinase activity where we amplified the mass-to-charge signals of the peptides conjugated to gold nanoparticles (AuNPs) on the SAM.26,27 Despite the benefit of directly measuring the kinase activity on the chip surface without labeling, however, this method26,27 − even with amplification − still produced insufficient signals that led to a prolonged imaging process. As a result, this method was limited in fast drug screening. Herein, we describe a class of mass spectrometry fingerprinting and imaging to determine enzyme activity using cysteine-tethered peptide substrates on a gold chip, focusing in particular on establishing a SIMS imaging-based assay to screen for large compound libraries. In this method, a strong chemisorption between the sulfhydryl group at the terminus of the peptide and the gold facilitates the preferential enrichment of the cysteine-tethered peptides in the assay solution and allows the drug screening to be easier and faster than our previous method,26,27 as this method does not require surface modifications prior to the enzyme reaction. Once the enzyme reaction and peptide enrichment using the gold surface are completed, the SIMS imaging process generally takes approximately 10 min to screen 100 inhibitors on a single plate, so compared to other kinase assays based on MALDI,28,29 this matrixfree method can reduce analysis time. Although non-specific binding on the bare gold generally impedes the enzyme reaction,27 we found that non-specific signals on the gold chip could be easily reduced with the addition of a simple rinsing step in this method. This prepared the bare gold chip to be useful for SIMS imaging. Accordingly, the strong molecular ions of the peptides enabled the direct SIMS imaging of the active enzyme-induced peptide modification with a high degree of accuracy, compared to the fluorogenic5 or previous method.26 To our knowledge, there have been no attempts to utilize the SIMS imaging technique for drug screening.


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Chronic myeloid leukemia (CML) is a devastating type of cancer that begins in the bone marrow.30 To determine which chemicals have an inhibitory effect on enzyme activity, we designed an assay to identify the molecules that could act against breakpoint cluster region–abelson (BCR-ABL) tyrosine kinase, an enzyme associated with CML. In 1993, Imatinib (Gleevec®) was developed as an ABLselective tyrosine kinase inhibitor, but drug resistance during imatinib treatment was found to be mostly related to point mutations occurring within the kinase domain of BCR-ABL.30 Among all mutations in the ABL kinase domain, the T315I mutation accounts for 15% of the mutations; imatinib strongly inhibits phosphorylation of tyrosine in wild type BCR-ABL but does not act on BCR-ABL with T315I mutations.31 For these reasons, there is an urgent need to develop novel BCR-ABLT315I inhibitors.

In

this work, we identified the inhibitors and quantified the inhibition efficiency of each chemical in a large chemical library (13,727 molecules) using the simple ratiometric determination26 of coupled molecular ion MS images from the cysteine-terminated peptide.

EXPERIMENTAL METHODS Reagents. BCR-ABLT315I tyrosine kinase was purchased from Millipore Corporation (Billerica, MA, USA). BCR-ABL tyrosine kinase and kinase assay buffer were purchased from New England Biolabs. The substrate peptides were synthesized by Peptron, Inc. Ethylenediaminetetra acetic acid (EDTA), adenosine triphosphate (ATP), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma. Imatinib mesylate was obtained from Biovision. A chemical library of 13,727 molecules was provided by Korea Chemical Bank. The peptide substrate, Ac-IYAAPKKGGGGC was synthesized by Peptron Inc. (Daejeon, Korea).

Au-Coated Si Wafer Preparation. Gold substrates purchased from KMAC Inc. (Korea) were prepared by sputter deposition of a 20-Å-thick film of Ti and a 400-Å-thick film of gold onto a silicon wafer. The gold substrates were cut into 3 cm × 3 cm pieces and cleaned in a super-piranha solution (1:10:6) 61% HNO3:30% H2O2:95% H2SO4 (v/v/v).32 Sulfuric acid was slowly added until the solution ACS Paragon Plus Environment

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came to a boil with yellow fumes. (Caution: super-piranha solution reacts violently with most organic materials and must be handled with extreme care.) After 1 min, the gold substrates were rinsed thoroughly with deionized water for 3 min. This substrate cleaning method was previously demonstrated to be superior to the piranha cleaning method for removing heavy organic contamination, as it allows the production of reproducible self-assembled monolayers (SAMs) with a constant ratio of molecular ion peaks in TOF-SIMS.32

Protein Kinase Assay. Kinase reaction was performed in a reaction mixture (10 µL) containing 10 nM recombinant BCR-ABLT315I tyrosine kinase, 3 µM peptide substrate (Ac-IYAAPKKGGGGC), 500 µM ATP, and inhibitor in 1 × kinase buffer at 30°C for 2 h. The 1 × kinase buffer was supplied by a manufacturer (Cell Signaling Technology, USA) and contained 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, and 10 mM MgCl2 in 25 mM Tris-HCl (pH 7.5). For the initial inhibitor screening, the inhibitor (each chemical candidate from a 13,727 compound library or imatinib) was dissolved in dimethylsulfoxide (DMSO) and added to the reaction mixture for a final concentration of 5 µM. The different concentrations of the effective inhibitor were further validated after screening. To terminate the kinase reaction, 1 µL of 100 mM EDTA was added to the reaction mixture to produce a final volume of 11 µL. For multiple inhibitor analysis on the chip surface, a 10 × 10 grid was outlined with a hydrophobic barrier pen (liquid blocker, Sigma-Aldrich, USA) onto the clean gold chip (3 cm × 3 cm) for a total of 100 squares so that each square corresponded to approximately 2 mm × 2 mm. There was no contamination from the hydrophobic pen. Using a micropipette, 1 µL aliquot of each reaction was dropped into each square on the Au. The gold chip was left for 30 min at room temperature to ensure peptide immobilization through the formation of a self-assembled monolayer, and then thoroughly washed with deionized water, dried under a stream of N2 and subjected to SIMS analysis. To show reliability of our sampling method, SIMS images from a 1 µL drop of cysteine-tethered peptide solution with deionized water (DI)-washing and without DI-washing are shown in Figs. S1a and b, ACS Paragon Plus Environment

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respectively. As Figs. S1a and b show, in our sampling method the peptides are evenly distributed. However, without the DI washing step, the peptides migrated to the edges of the spot to produce a coffee ring shape. To analyze the 13,727 chemical candidates, a total of approximately 150 Au chips were used. One plate image analysis took 10 min. A single gold chip contained 96 inhibitors with 4 internal reference areas; these included two positive control sets (peptide and kinase without inhibitor) and four negative control sets (blank and only peptide (two areas)). Inhibitor selection was based on whether they exceeded 50% inhibitory efficiency.

Determining Phosphorylation Efficiency and IC50. The phosphorylation efficiency of the kinase reaction was calculated using the following equation, as previously reported17: Ep = AMP / (AM + AMP) × 100, where Ep is the phosphorylation efficiency (%) and AM and AMP are the peak areas of [MH-SHCOOH]+ and [MH-SH-COOH+HPO3]+, respectively. The peak area was normalized to total ion count and was calculated within the limits of the full width half-maximum. To determine the half maximal inhibitory concentration (IC50) value, the phosphorylation efficiency was plotted as a function of the inhibitor concentration and fitted to a 4-parameter logistic equation using the non-linear regression procedure in SigmaPlot (version 10.0, SYSTAT software).17

TOF-SIMS Analysis. TOF-SIMS was performed using a TOF-SIMS V instrument (ION-TOF GmbH, Germany) with 25 keV Bi1+ primary ions. The primary ion source was operated with an average current of 0.4 pA in the high-current bunched mode, a pulse width of 15.2 ns (0.6 ns after bunching), and a repetition rate of 5 kHz. TOF-SIMS spectra were acquired from a 200 µm × 200 µm area at the center of the sample, and the primary ion dose was maintained below 5 × 1011 ions cm−2. Positive ion spectra were mass calibrated using the CH3+, C3H5+ and Au3+ peaks. The mass resolution was higher than 5000 at m/z > 500 in the positive modes. The TOF-SIMS images were obtained in the positive mode at 128 ×


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128 pixels with an area of 3 cm × 3 cm and a primary ion influence of 1 × 1012 ions cm−2. The mass spectrometry images of interest were collected from the raw data below m/z of 1500.

Cell Culture. Mouse pro-B cell line Ba/F3 cells were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI Medium 1640 with 10% FBS, 2 mM L-glutamine, 100 U mL-1 penicillin, and 100 U mL-1 streptomycin at 37°C in 95% air/5% CO2. To test the cytotoxicities of KR135861 and imatinib, the cells were treated with each drug at various concentrations. The cell viabilities were evaluated 48 h after the addition of each drug using (1-(4,5-dimethylthiazol-2-yl)-3,5diphenylformazan or thiazolyl blue formazan, MTT) according to a previously reported protocol.33 To determine the cell viabilities, 100 µL of a MTT solution (5 mg mL−1, in phosphate buffered saline) was added to each well, incubated for 2 h at 37°C in a cell incubator, and solubilized with DMSO. The absorbance of the converted dye was measured at a wavelength of 570 nm with a spectrophotometer.

RESULTS AND DISCUSSION Cysteine-tethered peptides, substrates of protein kinase, were used for mass fingerprinting and imaging to identify protein kinase activity and inhibition as illustrated in Fig. 1. In our TOF-SIMS analysis, quasi-molecular secondary ions (i.e., [MH−SH−COOH]+) rather than molecular secondary ions (i.e., [MH]+) from the cysteine-tethered peptide were more readily observed due to the loss of the sulfhydryl (−SH) and carboxyl (−COOH) groups in cysteine at the C-terminus. This was triggered by the preferential adsorption of cysteine onto Au, as previously reported.17 To screen the protein kinase inhibitors, matrix-free TOF-SIMS analysis was performed by dropping onto bare gold each kinasetreated peptide solution that was included in the chemical library (Fig. 1b), and then identifying the kinase activity by the changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides (Fig. 1c). By scanning the entire chip surface with mass spectrometry imaging analysis, protein kinase activity for every reaction could be simultaneously


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analyzed. This was done by imaging either a new secondary ion peak or a high-mass secondary ion peak, in addition to the original peptide secondary ion peak, such as phosphorylation of the peptide (+80 Da, changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides by a mass equivalent of phosphoric acid (HPO3)) induced by the protein kinase, as shown in Fig. 1c. In general, in cases where a particular chemical inhibits the protein kinase and impedes changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides, the peptide retains its original mass-to-charge. Using our method, this particular chemical was easily identified as a specific inhibitor by using mass spectrometry imaging analysis. In our previous works,26,27 SIMS-based drug screening required surface modification, including SAM formation, and immobilization of the AuNPs and peptides prior to the enzyme reaction. In the present work, screening for enzyme activity and inhibitors calls for the enzyme reaction to be first performed in a reaction mixture of enzymes, cysteine-tethered peptides and inhibitor in a reaction well (as has been traditionally done in conventional drug screening). This assay solution was directly dropped onto a gold chip and then rinsed in deionized water to reduce non-specific signals on the gold chip. Since the signal enhancement of the peptides on bare gold is generally much higher than that of the AuNP-modified surface,27 the gold chip could then be directly used for signal amplification, without surface modification. We employed a specific peptide substrate for the BCR-ABLT315I tyrosine kinase because the constitutively active form of the protein kinase, an oncoprotein, is implicated in the pathogenesis of chronic myeloid leukemia (CML).34 As shown in Fig. 2a, the secondary ion changes in the relative peak intensities of the phosphorylated and unphosphorylated cysteine-tethered peptides (PepABL; M=AcIYAAPKKGGGGC, MW=1162.6) showed distinct peaks (i.e., m/z 1085.6 (unphosphorylated peak, [MH-SH-COOH]+) and m/z 1165.6 (phosphorylated peak, [MH-SH-COOH+HPO3]+)) corresponding to before and after the protein kinase reaction. On the other hand, the cysteine-deficient peptides (PepABL’: M’=Ac-IYAAPKKGGGG, MW=1062.5) exhibited no strong peaks in the SIMS spectrum (Fig. 2b), suggesting that the cysteine-mediated adsorption on Au was necessary for the SIMS analysis. Moreover, phosphorylation efficiency in relation to protein kinase activity was easily quantified by using the areas ACS Paragon Plus Environment

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of the phosphorylated and unphosphorylated mass peaks (see Experimental Methods) as a function of protein kinase concentration (Fig. 3). These results support our method of quantitatively evaluating protein kinase activity and inhibition. We further verified the utility of SIMS imaging for the kinase assay in a multiplex manner by performing a dual kinase reaction using two kinases (BCR-ABL and PKA) and two peptide substrates (PepABL and PepPKA (Ac-LRRASLGGGGC)). After either one or two peptides were reacted with a mixture of BCR-ABL tyrosine and PKA kinases, SIMS images from four different mass-to-charge ratios were used to distinguish the respective phosphorylation of a combination of peptides (Fig. 4), which provided compelling evidence that each peptide reacted specifically with the respective kinase without cross reaction. The utility of mass spectrometry imaging for drug screening was tested by identifying the specific inhibitor of a target mutant form of a protein kinase (BCR-ABLT315I) from among a library containing 13,727 compounds as the kinase interacted with the cysteine-tethered peptide (PepABL). Previously, specific inhibitors of BCR-ABL tyrosine kinase such as imatinib mesylate (IM, STI-571 or Gleevec®) were identified, marking the first BCR-ABL tyrosine kinase inhibitor to be used in the treatment of chronic myeloid leukemia (CML).35 Subsequent studies have been undertaken to overcome imatinibresistance in advanced treatments for CML.36 After the kinase reaction in a buffer solution, the assay solution containing each inhibitor was dropped onto a 10 × 10 line-arrayed Au plate, and the SIMS fingerprinting images were then used to identify the kinase inhibitor via a straightforward comparison between the unphosphorylated peptide mass-to-charge (m/z 1085.6) and the phosphopeptide mass-tocharge (m/z 1165.6), as shown in the images in Fig. 5a. There was no significant image or peak at m/z 1165.6 for the control negative spots (A4B8 and A9B8), which contained the peptide substrate without protein kinase, whereas a control positive spot (A4B7 and A9B7) for the kinase reaction in the absence of inhibitors yielded a strong SI mass spectrometry image at m/z 1165.6, with a decrease in image intensity at m/z 1085.6. However, the SI image at m/z 1085.6 from one spot (A8B4) showed no decrease


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in the presence of one compound, KR135861 (Fig. 5b). This strongly indicates that a potential inhibitor of BCR-ABLT315I tyrosine kinase had been easily detected using the SIMS image. Of a total of 13,727 inhibitors, 7 inhibitors were initially screened on the basis of whether they exceeded 50% inhibitory efficiency (Table S1, 7 inhibitors are marked in red) and a final candidate (KR135861, Chemical ID#:135861 in Table S1 and Table S2) was identified after ruling out concentration-independent or cytotoxic inhibitors. Our final candidate in Fig 5c, 7-((4λ2-morpholin-2yl)-N-(4-chloro-3-(trifluoromethyl)phenyl)-6-methoxyquinazolinn-4-amine,

was

a

series

of

4-

substituted quinazoline derivatives which was shown to inhibit tyrosine kinase activity.37 Bosutinib (Bosulif, made by Pfizer, Inc.) is a representative quinazoline derivative drug which was approved by the FDA for the treatment of chronic, accelerated, or blast phase Philadelphia chromosome positive (Ph+) CML. As a tyrosine kinase inhibitor, our final candidate also showed promise in the treatment of CML. To address the efficacy of the inhibitor, KR135861 was confirmed to be a potential inhibitor, when compared with imatinib, a BCR-ABL tyrosine kinase inhibitor marketed as Gleevec® (Fig. 5d). When compared to the IC50 value (> 10 µM from the present result and a previous report38) of imatinib, our newly identified KR135861 exhibited a greater inhibitory effect on the BCR-ABLT315I tyrosine kinase, with an IC50 value as low as 1.3 µM. As shown in Fig. 6, KR135861 functioned as a competitive inhibitor of ATP binding to the enzyme, whereas the KM value for the kinase was independent of the concentration of the peptide substrate. This result indicates that our mass spectrometry imaging analysis is suited to screening for protein kinase inhibitors. To gain insight into the in vitro efficacy of KR135861 on leukemia cells, we tested three different cell lines: control mouse fibroblast cells (NIH3T3), mouse pro-B cells (Ba/F3, BCR-ABL+), and Ba/F3 mutant cells (BCR-ABLT315I+). KR135861 and imatinib exhibited no cytotoxic effects on the control NIH3T3 cells. Imatinib had stronger inhibition effects on the BCR-ABL+ cells than KR135861 (Fig. 7b), whereas KR135861 reduced the viability of both Ba/F3 cells expressing wild-type BCR-ABL and BCR-ABLT315I (Fig. 7b and c), suggesting that KR135861 alone has specific inhibitory effects on the BCR-ABLT3151 mutant cells. Because the point mutation (T315I) in BCR-ABL reduces the binding of ACS Paragon Plus Environment

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imatinib to the protein,17 KR135861 has the potential to overcome imatinib resistance. Although subsequent research for clinical use of this inhibitor is under exploration, this result shows that our mass spectrometry imaging-based analysis can serve as a means for screening and validating protein kinase inhibitors.

CONCLUSIONS Despite notable progress in MS in recent years, a simple, quantitative method of analyzing enzyme activity has been difficult to find. Here, we have reported a high-throughput peptide mass spectrometry imaging technique with reproducible ion signals on a chip surface and its application to protein kinase inhibitor screening. Through the matrix-free SIMS imaging of cysteine-tethered peptides on Au, we were able to quantitatively analyze the activity of the BCR-ABLT315I protein tyrosine kinase; moreover, a specific kinase inhibitor from a chemical library could be identified with considerable selectivity and sensitivity. Although the current work involves TOF-SIMS, an instrument that is not easily accessible, we anticipate an increased demand for the SIMS imaging technique to identify biomolecules as greater demands are placed on biochips. Our SIMS imaging technique, which measures subtle changes in the relative peak intensities of low-mass molecules within a biological milieu will offer a new means of drug screening in the biological and pharmaceutical fields.

ACKNOWLEDGMENTS The work was supported by the Development of Platform Technology for Innovative Medical Measurements Program (GP2016-0022) from the Korea Research Institute of Standards and Science, the Pioneer Research Program (NRF-2012-009541), the Bio & Medical Technology Development Program

(NRF-2015M3A9D7029894)

and

the

Global

Frontier

Project

(H-

GUARD_2013M3A6B2078962) of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning, and Basic Science Research Program (No. 2012R1A6A1029029) ACS Paragon Plus Environment

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through the NRF funded by the Ministry of Education. The chemical library used in this study was kindly provided by Korea Chemical Bank (http://www.chembank.org/) of Korea Research Institute of Chemical Technology.


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Figure 1. Scheme of protein kinase inhibitor screening of an inhibitor candidate from a chemical library using cysteine-tethered peptides and SIMS imaging.


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Intensity

(a)

x103 1.6

[MH-SH-COOH]+

1.2 0.8 0.4 x103 1.2 0.9 0.6 0.3

[MH-SH-COOH+HPO3]+

1080

111 0 1100

1 140 1140

1170 1170

m/z

(b)

x103 1.2

[MH-SH-COOH]+

0.8

Intensity Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.4

x103 1.2

[MH-SH-COOH+HPO3]+

0.8 0.4 10 80 1080

1110 1100

1140

11 70 1170

m/z

Figure 2. Effect of cysteine residue tethered at the C-terminus of the peptide substrate in the SIMS analysis. (a) Spectra of cysteine-incorporated peptide (M=Ac-IYAAPKKGGGGC) before (top column) and after (bottom column) the BCR-ABLT315I tyrosine kinase reaction. The unphosphorylated and phosphorylated mass-to-charge regions are labeled [MH−SH−COOH]+ and [MH−SH−COOH+HPO3]+, respectively. (b) Spectra of cysteine-free peptide (M’=Ac-IYAAPKKGGGG) before (top column) and after (bottom column) the kinase reaction.


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Figure 3. (a) Positive TOF-SIMS spectra of the peptide (M=Ac-IYAAPKKGGGC) after the BCRABLT315I tyrosine kinase reaction. (b) Phosphorylation efficiency (%) for the peptide substrate was plotted as a function of kinase concentrations. The error bar represents the standard deviation (n = 3).


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Figure 4. Multiplex assay of two different kinases (PKA and BCR-ABL) using SIMS imaging and two peptide substrates (PepPKA and PepABL). Unphosphorylated (i and iii) and phosphorylated mass spectrometry images (ii and iv) are aligned under different reaction conditions. (i) [PPKAH−SH−COOH]+ (ii) [PPKAH−SH−COOH+HPO3]+ (iii) [PAblH−SH−COOH]+ (iv) [PAblH−SH−COOH+HPO3]+.


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Figure 5. SIMS imaging-based inhibitor screening for protein kinase (BCR-ABLT315I). (a) SIMS images at two mass-to-charge regions (m/z 1085.6 for unphosphorylated peptides and m/z 1165.6 for phosphorylated peptides) where phosphorylation inhibition was analyzed. A gold plate was composed of 10 × 10 spots, with each spot corresponding to a deposited assay solution composed of a different chemical compound from the chemical library. The yellow dots in the left panel (m/z 1085.6) and the red dots in the right panel (m/z 1165.6) represent strong intensity at the indicated mass-to-charges. The ACS Paragon Plus Environment

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rectangular boxes indicate the hitting area (red) or control positions (yellow). A4B6 and A9B6 are blank spots that contain nothing of substance. Two identical spots (A4B8 and A9B8) were used as the negative control (peptide without protein kinase), whereas A4B7 and A9B7 were used as the positive control (peptide with protein kinase in the absence of inhibitors). A8B4 (in the presence of the inhibitor, KR135861) was identified from the SIMS image. The scale bar is 1 mm. The inhibitors corresponding to the coordinates of each spot on the Au are listed in Table S1. (b) SIMS spectra containing blank, negative, and positive controls were obtained from different spots. (c) Chemical structure of KR135861, which was identified by SIMS imaging-based assay. (d) Inhibition assay of KR135861 and imatinib with BCR-ABLT315I tyrosine kinase. The error bar represents the standard deviation (n = 3).


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Figure 6. Inhibitory assay of KR135861 applied to BCR-ABLT315I kinase using SIMS analysis depending on the concentrations of the inhibitor, ATP, and substrate. Protein kinase activity between KR135861 and ATP concentrations (a and b); and between KR135861 and peptide substrate concentrations (c and d) were analyzed. The error bar represents the standard deviation (n = 3).


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(a) Cell viability (%)

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(b)

(c)

Imatinib

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120

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-

0.1 0.2 0.5

1.0 2.0 5.0 10.0

Inhibitor Concentration (µ µM)

Figure 7. Inhibitory effect and specificity of KR135861 and imatinib in different cell lines: (a) control mouse fibroblast cells (NIH3T3), (b) mouse pro-B cells (Ba/F3, BCR-ABL+); and (c) Ba/F3 mutant cells (BCR-ABLT315I+). The error bar represents the standard deviation (n = 3).


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For TOC only


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Figure 1. Scheme of protein kinase inhibitor screening of an inhibitor candidate from a chemical library using cysteine-tethered peptides and SIMS imaging. 215x227mm (300 x 300 DPI)



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Figure 2. Effect of cysteine residue tethered at the C-terminus of the peptide substrate in the SIMS analysis. (a) Spectra of cysteine-incorporated peptide (M=Ac-IYAAPKKGGGGC) before (top column) and after (bottom column) the BCR-ABLT315I tyrosine kinase reaction. The unphosphorylated and phosphorylated mass-tocharge regions are labeled [MH-SH-COOH]+ and [MH-SH-COOH+HPO3]+, respectively. (b) Spectra of cysteine-free peptide (M’=Ac-IYAAPKKGGGG) before (top column) and after (bottom column) the kinase reaction. 108x107mm (300 x 300 DPI)


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Figure 3. (a) Positive TOF-SIMS spectra of the peptide (M=Ac-IYAAPKKGGGC) after the BCR-ABLT315I tyrosine kinase reaction. (b) Phosphorylation efficiency (%) for the peptide substrate was plotted as a function of kinase concentrations. The error bar represents the standard deviation (n = 3). 215x109mm (300 x 300 DPI)



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Figure 4. Multiplex assay of two different kinases (PKA and BCR-ABL) using SIMS imaging and two peptide substrates (PepPKA and PepABL). Unphosphorylated (i and iii) and phosphorylated mass spectrometry images (ii and iv) are aligned under different reaction conditions. (i) [PPKAH-SH-COOH]+ (ii) [PPKAH-SHCOOH+HPO3]+ (iii) [PAblH-SH-COOH]+ (iv) [PAblH-SH-COOH+HPO3]+ 107x60mm (300 x 300 DPI)


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Figure 5. SIMS imaging-based inhibitor screening for protein kinase (BCR-ABLT315I). (a) SIMS images at two mass-to-charge regions (m/z 1085.6 for unphosphorylated peptides and m/z 1165.6 for phosphorylated peptides) where phosphorylation inhibition was analyzed. A gold plate was composed of 10 × 10 spots, with each spot corresponding to a deposited assay solution composed of a different chemical compound from the chemical library. The yellow dots in the left panel (m/z 1085.6) and the red dots in the right panel (m/z 1165.6) represent strong intensity at the indicated mass-to-charges. The rectangular boxes indicate the hitting area (red) or control positions (yellow). A4B6 and A9B6 are blank spots that contain nothing of substance. Two identical spots (A4B8 and A9B8) were used as the negative control (peptide without protein kinase), whereas A4B7 and A9B7 were used as the positive control (peptide with protein kinase in the absence of inhibitors). A8B4 (in the presence of the inhibitor, KR135861) was identified from the SIMS image. The scale bar is 1 mm. The inhibitors corresponding to the coordinates of each spot on the Au are listed in Table S1. (b) SIMS spectra containing blank, negative, and positive controls were obtained from different spots. (c) Chemical structure of KR135861, which was identified by SIMS imaging-based assay. (d)



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Inhibition assay of KR135861 and imatinib with BCR-ABLT315I tyrosine kinase. The error bar represents the standard deviation (n = 3). 215x261mm (300 x 300 DPI)


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Figure 6. Inhibitory assay of KR135861 applied to BCR-ABLT315I kinase using SIMS analysis depending on the concentrations of the inhibitor, ATP, and substrate. Protein kinase activity between KR135861 and ATP concentrations (a and b); and between KR135861 and peptide substrate concentrations (c and d) were analyzed. The error bar represents the standard deviation (n = 3). 215x164mm (300 x 300 DPI)



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Figure 7. Inhibitory effect and specificity of KR135861 and imatinib in different cell lines: (a) control mouse fibroblast cells (NIH3T3), (b) mouse pro-B cells (Ba/F3, BCR-ABL+); and (c) Ba/F3 mutant cells (BCRABLT315I+). The error bar represents the standard deviation (n = 3). 215x74mm (300 x 300 DPI)


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On-Chip Peptide Mass Spectrometry Imaging for Protein Kinase

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