Microfluidics and microarray based approaches to biological analysis 3

Chapter CHAPTER MICROARRAY-BASED SCREENING OF KINASE ACTIVITY 3.1 Introduction 3.1.1 Protein Phosphorylation Phosphorylation of proteins by kinases is one of the most important mechanisms for regulation of cell function.1,2 It has been estimated that more than one third of all proteins can be modified by phosphorylation in mammalian cells, and that more than % of the genes in the human genome encode protein kinases.3 A major challenge in the signal transduction field has been to define sequence, structural and mechanistic features responsible for the substrate selectivity, regulation and cellular function of individual protein kinases. Additionally, there is considerable interest in identifying potent and selective inhibitors for each of these enzymes since these can be used as potential therapeutic agents in the treatment of cancer, heart disease and immunerelated conditions.4 However, few kinases have yet been identified and fully characterized. One of the most important features of protein kinases is their substrate specificity, which to a large extent is determined by the primary sequence around the phosphorylation site of their targeting proteins. As a result, a number of methods have been developed to identify potential kinase substrates, including combinatorial synthesis of peptide libraries on membrane using the SPOT technology,5 one-beadone-compound peptide libraries,6 positional-scanning combinatorial libraries,7 and peptide libraries using affinity-column selection.8 65 Chapter 3.1.2 Microarray-Based High Throughput Kinase Study More recently, peptide-based microarrays have also been developed for screening of kinase activity.9,10,11,12 Compared to the SPOT technology, the much higher density of spots allowed in a peptide array makes it possible for simultaneous screenings of tens of thousands of kinase substrates on a 3” x 1” glass surface. Zhu et al analyzed the activity of 119 of the 122 yeast kinases using microwells made of PDMS.10 They covalently attached the substrates to the microwells and incubated them with the kinase and radioactively-labeled ATP. After phosphorylation had taken place, the kinases and ATP were washed away and the microwells scanned. Most of the other array-based kinase assays rely on standard microscope slides as support. Schreiber et al, in a proof-of-concept experiment, arrayed three kinase substrates onto glass slides and incubated them with different kinase solutions.9 Incorporation of radioactively labeled ATP was also used for detection. A very similar approach was also used by Falsey et al.11 Surface plasmon resonance was also recently reported for detection of kinase activity.12 3.1.3 Issues to Be Addressed Peptide-based microarray is one of the most promising technologies for the high throughput screening of protein kinase activity. Because of the high density of enzyme substrates arrayed on a microscope slide, one can potentially screen for a wide range of substrates and kinase activity as well as antigen-antibody or ligand-receptor interaction and so on.13,14 However, the development of these technologies is still in its infancy since some issues remain to be addressed. For efficient kinase assay to be performed, the kinase substrates need to be arrayed in a site-specific fashion so that efficient and optimum phosphorylation reaction can take place. Most peptide and protein arrays 66 Chapter reported so far use either non-specific, covalent immobilization of molecules to the slide or not very stable site – specific immobilization strategies. NHS-functionalized slides and epoxide functionalized slides were respectively used by Zhu et al and Schreiber et al to immobilize kinase substrates via their amine groups, resulting in random immobilization of proteins onto the glass surface.9,10 New strategies were developed by Falsey et al as well as Housman et al for site-specific immobilization of kinase substrates.11,12 However, in the first case, this resulted in an oxime bond which is relatively unstable, and the five-membered-ring thiazolidine may present immobilized peptides in an unfavorably restricted orientation to interact with their targeting proteins. In the second case, the developed strategy required the conjugation of peptides with an unnatural cyclopentadiene moiety, making it synthetically challenging and not easily accessible. Another issue to be addressed is the detection of substrate phosphorylation. To date, most of the peptide arrays developed for kinase assay require the use of radioactive 32 P for detection of substrate phosphorylation, presenting a serious threat to human health.9,10 Furthermore, the long exposure time (usually hours to days) needed for sensitive detection of 32 P upon substrate phosphorylation does not lend itself to high-throughput applications. 3.2 Results and Discussion 3.2.1 Fluorescent Antibody-Based Detection of Kinase Substrate Phosphorylation All peptide arrays developed to date for kinase assay require the use of radioactive 32P for detection of substrate phosphorylation, presenting a potential risk to human health. Furthermore, the long exposure time needed for sensitive detection of 67 32 P upon Chapter substrate phosphorylation does not lend itself to high-throughput applications. Fluorescently labeled antibodies have been reported, in a microarray format, for detection of protein/protein, protein/peptide and protein/small molecule interactions9 as well as cell assay.11 The use of poly- and monoclonal antibodies directed against phosphoamino acids have been widely used to detect phosphorylated proteins in gel electrophoresis.15 This method is extremely sensitive since antibodies can detect as little as a few fmol of phosphorylated epitopes.16 In addition, due to the highly specific nature of antibody-antigen recognition, little or no cross-reactivity of one phosphoamino acid antibody (e.g. anti-phosphotyrosine) to other phosphoamino acids or non-phosphorylated amino acids was observed.17 3.2.1.1 Qualitative Fluorescent Antibody-based Detection of Phosphorylated Amino Acids and Peptides In order for peptide arrays to gain wider popularity for kinase screenings, it is imperative to develop an alternative detection method that poses less health risks than 32 P, yet provides similar sensitivity for efficient detection of kinase activity in a microarray format. In order to determine whether FITC-labeled anti-phosphoamino acids could be used to detect phosphorylation of kinase substrates in a peptide array, we first tested their use for detection of phosphorylated amino acids and peptides. Both phosphorylated and non-phosphorylated tyrosine amino acids were spotted on an amine functionalized glass slide, and detected using FITC-labeled anti- phosphotyrosine and anti-phosphoserine. As shown in Figure 3.1, only the FITClabeled anti-phosphotyrosine was able to detect the phosphotyrosine immobilized on the slide. Neither binding of anti-phosphoserine to phosphotyrosine, nor that of antiphosphotyrosine to non-phosphorylated tyrosine, was observed. We also attempted to 68 Chapter detect the protected phosphotyrosine with both FITC-labeled antibodies and no binding was observed. a) Tyr P-Tyr b) Tyr P-Tyr Figure 3.1. Fluorescent antibody-based detection of phosphorylated amino acids. Phosphorylated (p-Tyr) and non-phosphorylated tyrosine (Tyr) were arrayed onto amine-functionalized slides and probed with (a) FITC-labeled anti-phosphotyrosine and (b) and anti-phosphoserine The same experiment was then repeated with phosphorylated peptides instead of amino acids. Both phosphorylated and non-phosphorylated peptide substrates of p60 tyrosine kinase (YIYGSFK) were synthesized with an additional CGG N-terminal linker for immobilization purpose. Both peptides were arrayed onto glass slides functionalized with glyoxylic acid.11 After blocking with BSA, the slides were probed for phosphorylation by incubation with FITC-labeled anti-phosphotyrosine and antiphosphoserine for an hour. Only anti-phosphotyrosine was able to detect the phosphorylated p60 substrate as shown in Figure 3.2, confirming the high specificity of this antibody-based detection of tyrosine phosphorylation. 69 Chapter a) p60 P-p60 b) p60 P-p60 Figure 3.2. Fluorescent antibody-based detection of phosphorylated peptides. Phosphorylated (P-p60) and non-phosphorylated (p60) substrates (CGG-YIYGSFK) of the p60 tyrosine kinase were arrayed onto slides functionalized with glyoxylic acid and probed with (a) FITC-labeled anti-phosphotyrosine and (b) FITC-labeled antiphosphoserine. 3.2.1.2 Quantitative Fluorescent Antibody-Based Detection of Peptide Phosphorylation Fluorescent antibodies allows for the rapid screening of phosphorylated amino acids and peptides. However, substrate phosphorylation levels must be correlated with measured fluorescence intensity. Increasing ratio of phosphorylated to nonphosphorylated p60 kinase substrates were mixed with a combined constant concentration, and were arrayed onto thioester-functionalized slides (see paragraph 3.2.2.2). After a 3-hour incubation, ensuring the total binding of the substrates, the slides were probed with the FITC-labeled anti-phosphotyrosine antibody for hr. The intensity of the spots increased as the ratio of phosphorylated substrate to nonphosphorylated p60 substrate increased (Figure 3.3). The fluorescence of any fluorescent substrate is directly proportional to its concentration, and a graph of fluorescence intensity versus the ratio of phosphorylated p60 substrate (Figure 3.4) showed a linear correlation for a p60 ratio ranging from to 100 %, demonstrating the feasibility for on-chip quantitation of phosphorylated peptides using this method. 70 Chapter Phosp60/p60 ratio 1/100 1/100 25/75 25/75 50/50 50/50 75/25 75/25 100/0 Fluorescence intensity Figure 3.3. Fluorescence of increasing ratios of phosphorylated to non-phosphorylated p60 substrate. Increasing ratios of phosphorylated/non-phosphorylated GGC p60 peptides, with a combined constant concentration, were arrayed onto thioesterfunctionalized slides, incubated for hours, and probed with the FITC-labeled antiphosphotyrosine antibody for h. Fluorescence intensity vs amount of phosphorylated substrate 25000 20000 15000 10000 5000 0 0.5 1.5 2.5 3.5 p60 substrate concentration (mM) Figure 3.4. Fluorescence intensity vs amount of phosphorylated substrate. Increasing ratios of phosphorylated/non-phosphorylated GGC p60 peptides, with a combined constant concentration, were arrayed onto thioester-functionalized slides incubated for hours, and probed with the FITC-labeled anti-phosphotyrosine antibody for hr. 3.2.1.3 Fluorescent Antibody-Based Detection of Kinase Activity Current array-based kinase assays are time consuming due to the long time required for detection. A detection relying on fluorescent antibodies is much faster, and a very fast screening of kinase activity can be performed by first incubating the slides with kinase, and subsequently with the fluorescent antibodies (Scheme 3.1). 71 Chapter PO4 Kinase PO4 Scheme 3.1. Very fast screening of kinase activity. Glass slides were arrayed with kinase substrates and after substrate phosphorylation with kinase, the phosphorylation level are rapidly detected with fluorescent antibodies. The two substrates of the serine protein kinase PKA and tyrosine kinase p60, ALRRASLG and YIYGSFK respectively, were synthesized with an additional GGC linker. Both peptides were spotted onto the same slides functionalized with glyoxylic acid. Following blocking with BSA, the slides were first incubated for an hour with the corresponding kinase (p60 tyrosine kinase for Figure 3.5 a) and PKA kinase for Figures 3.5 b)), and then detected with FITC-labeled anti-phosphotyrosine and antiphosphoserine. Only anti-phosphotyrosine was able to detect the tyrosine kinase activity of p60. Similarly, only anti-phosphoserine was able to detect the serine kinase activity of PKA. The complete absence of cross-detection between the two FITClabeled antibodies demonstrates the high specificity of the antibodies against their corresponding phosphorylated amino acids/peptides. 72 Chapter PKA a) p60 PKA p60 b) Figure 3.5. Detection of kinase activity with FITC-labeled antibodies. PKA (left panel) and p60 (right panel) substrates were arrayed onto glyoxylic acid-functionalized slides. Slide a) was incubated with p60 kinase and slide b) with PKA. Slides were probed with both FITC-labeled anti-phosphotyrosine and FITC-labeled antiphosphoserine, incubation of slide a) with only FITC-labeled anti-phosphotyrosine is shown and incubation of slide b) with only and FITC-labeled anti-phosphoserine is shown. The concentration- and time-dependent detection of peptide phosphorylation on chip were then studied. Decreasing concentrations (3 mM, mM, 0.3 mM and 0.1 mM) of GGC p60 substrate in PBS, pH 7.4, were arrayed onto a thioester-containing glass slide and incubated with p60 kinase for increasing periods of time (1, and 12 hrs). The slides were incubated with the FITC-labeled anti-phosphotyrosine for hour, washed, dried, scanned (Figure 3.6) and the fluorescence intensity of the spots was measured (Figure 3.7). 73 Chapter mM mM 0.3 mM 0.1 mM Figure 3.6. Antibody-based fluorescence measurement of kinase activity: Decreasing concentrations (3 mM, mM, 0.3 mM and 0.1 mM) of GGC p60 substrate in PBS, pH 7.4, were arrayed on thioester slides and incubated with the p60 kinase for increasing period (1, and 12 hrs). The slides were incubated with the FITC-labeled antiphosphotyrosine for hour, washed, dried, scanned and the fluorescence intensity of the spots measured. The concentration-dependent kinase activity was confirmed by plotting the observed fluorescence intensity over the differing concentrations of the peptide spotted on the slide following the same incubation time (5 hrs) with the corresponding kinase (Figure 3.7). It was found that the fluorescence intensity was directly proportional to the concentration of the substrate, showing the feasibility for determination of concentration-dependent kinase activity. Using this antibody-based fluorescence detection, kinase phosphorylation was readily detected even with 0.1 mM – which, with a spot size of nL, corresponds to ~0.1 pmol of the peptide substrate. By increasing the scanning time or changing the dye used to label the antibody, a 10- to 100-fold lower detection limit should be obtainable. 74 Chapter with maleimide-functionalized slides,22 or any nucleophilic group (-NH2, -SH, -OH) may react with NHS9- or epoxy-functionalized slides.10 Formation of a stable peptide bond between the immobilized peptides and the glass surface via native chemical ligation is also in contrast with that developed by Falser et al where an unstable oxime bond or a rigid thiazolidine ring was formed.11 NH3+ O O O O Si NH C CH NH2 O NH Peptide S O Si C - S O O O O O C O CH C Peptide S O O O O O NH Si NH C Peptide O SH Scheme 3.5. Site-specific immobilization, via native chemical ligation, of N-terminal containing cysteine peptides onto thioester slides. A JAK peptide (KGTGYIKTG) and the phosphorylated p60 peptide (YIYGSFK) were synthesized with an N-terminal cysteine and two glycines as linker. These were dissolved in PBS, pH 7.4, and spotted onto the thioester slide. Following incubation for hours, the peptides were successfully detected with their corresponding fluorescently-labeled antibodies (Figure 3.10 a and b), further demonstrating the compatibility of this immobilization method with microarray-based kinase assay. 82 Chapter a) b) Figure 3.10. Site-specific immobilization of kinase substrates via native chemical ligation. N-terminal cysteine containing peptides were arrayed on thioester functionalized slides and probed with fluorescent antibodies. (a) Immobilized JAK peptide probed with Cy3-labeled anti-JAK. (b) Immobilized phosphorylated p60 peptide probed with FITC-labeled anti-phosphotyrosine. 3.2.2.2.4 Surface Study of Thioester Functionalized Slides Additional studies were conducted to further evaluate this novel thioester-containing glass surface. Cysteine-containing fluorescein1 (Figure 3.11) was dissolved in PBS, pH 7.4, serially diluted and arrayed onto thioester slides. Following overnight incubation, the slides were washed with PBS, water, dried and scanned. It was observed that the fluorescence intensity reaches saturation with mM cysteine fluorescein, indicating the equivalent loading capacity of thioester on the glass surface (Figure 3.12). HO O O CO2 H O H2N HS NH N H O Figure 3.11. Structure of cysteine-containing fluorescein This compound was synthesized by Dr Zhu, Chemistry Department, NUS 83 Chapter mM mM mM 0.3 mM 0.1mM Figure 3.12. Saturation studies of thioester slides using different spotting concentrations of cysteine-containing fluorescein. In order to evaluate the time needed for efficient immobilization of peptides, different concentrations of the cysteine-containing fluorescein in PBS (pH 7.4) were spotted and incubated for increasing periods of time (Figure 3.13). It was observed that the reaction took place within the first 30 minutes of incubation. After 3h of incubation, spots corresponding to lower concentrations were eventually observed and the intensity reached saturation, indicating the completion of the native chemical ligation reaction, hence the complete peptide immobilization. 30 hr hrs mM mM 0.3 mM Figure 3.13. Rate of on-chip chemical ligation reaction. Increasing amounts of cysteine-containing fluorescein were arrayed on thioester functionalized slides and incubated for increasing period of time. 84 Chapter 3.2.2.2.5 PEGylated Thioester Functionalized Slides BSA is commonly used as a blocking agent to remove non-specific binding of proteins to the glass surface. However, it cannot be used directly in peptide-based arrays, presumably because it obscures the molecules of interest.9 In order to solve this problem, slides functionalized with BSA containing reactive NHS were used to immobilize peptides.9 However, peptides randomly reacted with NHS on the slide, resulting in non-specific immobilization on the surface. PEG is known to remove nonspecific binding of proteins to glass and glass slides were derivatized glass slides with two types of PEG followed by treatments with other chemicals to give thioester surfaces containing a PEG layer. Two different functionalized PEG were used to functionalize the slides. In a first approach, slides were functionalized with an epoxide containing silane and amine PEG was subsequently used (Scheme 3.6). The thioester group was then obtained as previously described (Scheme 3.4). In a second approach, slides were functionalized with an amine silane, and NHS PEG was subsequently used (Scheme 3.7). This second protocol was much shorter, since the NHS PEG can be reacted with benzyl mercaptan to obtain the thioester functionalized slides. 85 Chapter O O O + Si PEG H2N O O O O Si NH2 PBS, pH 7.4 N NH2 PEG O NaBO3 pH9, DMF 20 O O O O O Si O N PEG NH COOH TBTU/ DIEA/NHS,1:2:1, DMDMF F TBtu/DIEA/NHS, 1:2:1, O O O O Si N PEG NH CONHS SH DIEA hrs O O O O O Si N PEG NH C S Scheme 3.6. PEGylation of thioester slides with amine PEG O O O O O Si NH2 + NHS PEG NHS 0.1 M NaHCO3, pH 9, 30 O O O O O Si NH NHS PEG SH DIEA O O O O O Si NH PEG Scheme 3.7. PEGylation of thioester slides with NHS PEG 86 S hrs Chapter The phosphorylated p60 substrate containing a CGG linker was arrayed on the two PEG-COSR slides obtained with amine PEG and NHS PEG and probed with the FITC-labeled anti-phosphotyrosine without any BSA blocking. Very little non-specific binding was observed on both surfaces and the background noise was very low (Figure 3.14), presumably due to the PEG layer between immobilized peptides and the glass surface. In addition to minimizing non-specific binding, PEG also acts as a spacer between the slide and the immobilized peptides, which should facilitate phosphorylation by kinase. (a) (b) Figure 3.14. Site-specific immobilization of kinase substrates via native chemical ligation onto PEGylated thioester slides. Phosphorylated p60 peptide was spotted onto glass slides functionalized with (a) NHS PEG and (b) Amine PEG, and probed with FITC-labeled anti-phosphotyrosine. 3.2.3 Rapid Microarray-Based Kinase Assay Rapid fluorescent antibody-based detection was combined with the site-specific immobilization developed for rapid kinase assay. 87 Chapter 3.2.3.1 Avidin-Biotin Interaction The biotinylated, non-phosphorylated, p60 substrate containing an additional GG spacer (Biotin-GG-YIYGSFK) was synthesized and arrayed on avidin-functionalized slides. Upon incubation with p60 tyrosine kinase for an hour, the slide was probed with FITC-labeled anti-phosphotyrosine (Figure 3.15). Successful phosphorylation and detection of the p60 substrate further demonstrate the utility of this new method for site-specific immobilization of peptides on glass slides. Figure 3.15. Kinase assay of site-specifically immobilized p60 substrate via avidinbiotin interaction. Biotinylated p60 substrate was spotted onto avidin-functionalized slide, phosphorylated with p60 kinase and then probed with FITC-labeled antiphosphotyrosine. Note: no BSA blocking was used in this experiment. 3.2.3.2. Native Chemical Ligation The two non-phosphorylated CGG-containing substrates of tyrosine kinase p60 and serine kinase PKA (CGG-YIYGSFK and CGG-ALRRASLG respectively) were spotted onto the thioester-functionalized slide, incubated with their corresponding kinases, and detected successfully with FITC-labeled anti-phosphotyrosine and antiphosphoserine (Figure 3.16), further demonstrating the compatibility of the native chemical ligation immobilization strategy with microarray-based kinase assay. 88 Chapter (a) (b) Figure 3.16. Kinase assay of site-specifically immobilized p60 substrate via native chemical ligation. (a) Non-phosphorylated p60 peptide was immobilized, treated with p60 kinase and probed with FITC-labeled anti-phosphotyrosine. (b) Nonphosphorylated PKA peptide was immobilized, treated with PKA kinase and probed with FITC-labeled anti-phosphoserine. 3.2.3.3. Native Chemical Ligation with PEGylated Thioester Slides Glass slides were functionalized with NHS-PEG and with a thioester function. The non-phosphorylated CGG-containing p60 substrate was spotted onto the PEGylated thioester slide, incubated with p60 kinase and probed with FITC-labeled antiphosphotyrosine (Figure 3.17). A positive signal with little background noise was observed, confirming the additional advantage of the PEG slide. Figure 3.17. Kinase assay of site-specifically immobilized p60 substrate via native chemical ligation with PEGylated slides. Non-phosphorylated p60 peptide was spotted onto a NHS PEGylated slide, treated with p60 kinase and probed with FITC-labeled anti-phosphotyrosine. 89 Chapter 3.2.4 Summary Antibody-based fluorescence detection was developed as an efficient, sensitive and selective method for quantitative detection of kinase activity in a microarray format, eliminating the use of radioactive 32P, which poses a serious health threat to human. As a result, this detection method should be compatible with most fluorescence-based microarray applications, making it the method of choice for future high-throughput kinase screenings. Two new methods for site-specific attachment of N-terminally modified peptides were reported for usage in peptide arrays. Biotinylated peptides were spotted onto glass slides functionalized with avidin to achieve instantaneous immobilization. Avidin serves as both an immobilizing agent and an agent to minimize non-specific absorption of proteins on glass surface. Alternatively, peptides containing an N-terminal cysteine were chemoselectively immobilized onto thioester-funtionalized slides. Both methods show versatility in generating peptide-based microarrays suitable for high-throughput screenings of kinase activities, and potentially other enzymatic activities. The intercalation of various PEGs between the slide surface and the immobilized peptides minimized non-specific binding of proteins to the glass surface, rendering it possible to eliminate the blocking step using BSA. 90 Chapter 3.3 Materials and Methods 3.3.1 Peptide Synthesis2 The p60 and PKA and JAK substrates (YIYGSFK, ALRRASLG, and KGTGYIKTG respectively), containing appropriate N-terminal residues, were synthesized according to standard Fmoc-chemistry on Rink amide resin with a Pioneer™ automatic peptide synthesizer (Applied Biosystems, USA). O-Benzothiazol-1-yl-N, N, N’, N’Tetramethyluronium Tetrafluoroborate (TBTU), 1-Hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIEA) coupling chemistry was used. The phosphorylated substrates were synthesized with Fmoc-Tyr(PO(Obzl)OH)-OH and Fmoc-Ser(PO(OBzl)OH)-OH. An extended cycle (4 h coupling) was used to couple the phosphorylated serine and tyrosine to the peptides. At the end of synthesis, peptides were cleaved off the resin by using reagent R (90% TFA, 5% thioanizole, 3% ethanedithiol, % anisole) and precipitated with cold ether. Further purification of peptides was done by HPLC on a Waters™ 600 Station equipped with a Phenomenex™ C18 semi-preparative column. The identity of peptides was confirmed by Mass Spectrometry. 3.3.2 Slide Functionalization 3.3.2.1 Slide Cleaning Before any functionalization, glass slides (Sigma Aldrich, USA) were cleaned in piranha solution (H2SO4 : H2O2, : 3) for at least two hours. The slides were washed Most of the peptides were synthesized by Mahesh Uttamchandi 91 Chapter thoroughly with deionised water and rinsed with ethanol. Slides were usually kept in the piranha solution and washed when needed for functionalization. 3.3.2.2 Slide Silanization Two different protocols were used for the amine silanization of the slides. At first, slides were silanized in dry toluene: cleaned slides were soaked in aminopropyltriethoxisilane in dry toluene for hours under nitrogen atmosphere. The slides were subsequently rinsed with toluene, ethanol and dried. In a second protocol, slides were silanized in ethanol (Scheme 3.8 (a)) cleaned slides were soaked in % aminopropyltriethoxisilane in 95 % ethanol for 1h. The slides were subsequently rinsed times with ethanol and cured at 150 °C for at least hr. For functionalization with epoxide containing silane ((Scheme 3.8 (b)), the slides were covered with about 800 µL of a 1% solution of 3-glyicidoxypropyltrimethoxisilane (95 % ethanol, 16 mM acetic acid) for hr, washed times in 95 % and cured at 150 0C for hours. The slides were subsequently washed in 95 % ethanol and dried. 92 Chapter Si Si Si Si Si Si (MeO)3 O SiOH SiOH O SiOH Piranha solution H2SO4 / H2O2 7:3 SiOH O Si SiOH SiOH NH2 (MeO)3 95 % EtOH, 1h 150 deg, 2h 95 % EtOH, 1h 150 deg, 2h Si Si Si Si NH2 Si Si Si Si Si Si Si Si NH2 Si Si Si Si O Si O Scheme 3.8. Slide Silanization. (a) amine, (b) epoxide 3.3.2.3 Glyoxylic Acid Functionalization The derivatization of glass slides with glyoxyxlic acid was done according to published protocols with slight modifications.11 Amine slides were reacted with a solution of 10 mM protected glyoxylic acid (1 M glyoxylic acid, 0.1 mM HCl, M ethylene glycol in DMF, 100 ºC, 30 min), 50 mM TBTU (Advanced Chemtech, USA) and 50 mM HOBt (Advanced Chemtech, USA) in DMF for hours, then washed with DMF (2 x), DCM (2 x). The acetal protecting group was deprotected by placing the slides in a 10 mM HCl solution for hours, washed with H2O and air died. 3.3.2.4 Avidin Functionalization The resulting epoxy slides were reacted with a solution of mg/mL avidin (Pierce) in 10 mM NaHCO3 for 30 minutes, washed with water, air dried and the remaining 93 Chapter epoxides were reacted with a solution of mM aspartic acid in a 0.5 M NaHCO3 buffer (pH 9). 3.3.2.5 Thioester Functionalization Thioester-containing slides were prepared from amine slides as shown in Scheme 3.4. First, amine slides were placed in a solution of 180 mM succinic anhydride in DMF, pH (Na2B4O7) for 30 minutes and subsequently in boiling water for minutes. The slides were washed with ethanol, dried and the carboxylic acid was then activated with a solution of O-Benzothiazol-1-yl-N, N, N’, N’-Tetramethyluronium Tetrafluoroborate (TBTU), N, N-Diisopropylethylamine (DIEA) and N-Hydroxysuccinimide (NHS) (1:2:1) in DMF for hours and reacted overnight with a solution of 120 mM DIEA and 100 mM benzylmercaptan in DMF. 3.3.2.6 PEGylated Thioester Functionalization PEGylation of slides was done either with amine PEG (Scheme 3.6) or NHS PEG (Scheme 3.7). Amine slides were reacted with PEG-Succinimidyl Propionate (SPA-PEG; Shearwater, USA) for 30 minutes in 0.1 M NaHCO3, pH 9, and overnight with a solution of 120 mM DIEA and 100 mM benzylmercaptan in DMF. Alternatively, slides were prepared by first functionalization with epoxy groups, and then with a 100 mM solution of diamine-PEG (Shearwater, USA). The slides were subsequently placed in a solution of 180 mM succinic anhydride in DMF, pH (Na2B4O7) for 30 minutes and subsequently in boiling water for minutes. The carboxylic acid was then activated with a solution of TBTU/DIEA/NHS (1:2:1) in DMF for hours and reacted overnight with a solution of 120 mM DIEA and 100 mM benzylmercaptan in DMF. 94 Chapter 3.3.3 Array-Based Kinase Assay 3.3.3.1 Spotting The N-terminally cysteine-containing peptides and N-terminally biotinylated peptides were dissolved in PBS, pH 7.4, and arrayed using an ESI SMA arrayer (Ontario, Canada), with a spacing of 220 µm between the spots. After incubation, the slides were washed with PBS and distilled water and air-dried. 3.3.3.2 Antibody Labeling and Screening with Antibody FITC-labeled anti-phosphotyrosine and anti-phosphoserine antibodies, as well as the non-labeled anti-phosphotyrosine and anti-phosphoserine monoclonal antibodies were purchased from Sigma Aldrich (USA). Anti-phosphotyrosine, anti-phosphoserine and anti-JAK were labeled with Cy3-NHS and Cy5-NHS (Amersham Pharmacia, USA) according to the manufacturer’s protocols. The antibodies were reacted with the dye for one hour in 0.1 M NaHCO3, pH 9, and purified with a NAP5 column (Amersham Pharmacia, USA). The spotted slides were incubated with the labeled antibody (or mixture of antibodies) for hour, washed times, each time for 15 with PBST (PBS + 0.1 % Tween 20), dried and scanned with an ArrayWoRx microarray scanner (Applied Precision, USA). 3.3.3.3 Kinase Assay For kinase assay, the slides were incubated for various periods of time with the respective kinase solutions: (1) p60 assay (25 mM Tris, pH 7.4, 35 mM MgCl2, mM MnCl2, 0.5 mM EGTA, 100 µM ATP, 2U p60); and (2) PKA assay (25 mM Tris, pH 7.4, 15 mM MgCl2, mM DTT, mM EGTA, 100 µm ATP, 2U PKA). The slides 95 Chapter were subsequently washed with H2O, dried and incubated with the FITC-labeled antiphosphoamino acids. The slides were washed with H2O, scanned and the fluorescence intensity of the spots was measured with an ArrayWoRx™ scanner (Applied Precision, USA). 3.4 References Zhow, S.; Carraway, K. L.; Eck, M.; Cantley, L. C. Nature 1995, 373, 536 Pawson, T.; Scott, J. D. Science 1997, 278, 2075 Ahn, N. G.; Resing, K. A. Nat. Biotechnol. 2001, 19, 317 Levitski, A. Pharmacol. Ther. 1999, 82, 231 Dostmann, W. R. G.; Taylor, M. S.; Nickl, C. K.; Brayden, J. E.; Frank, R.; Tegge, W. J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14772 Lam, K. S.; Lebl, M.; Krchnak, V. Chem. Rev. 1997, 97, 411 Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84 Songyang, Z. et al. Nature 1995, 373, 536 MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760 10 Zhu, H.; Klemic, J. F.; Chang, S.; Bertone, P.; Casamayor, A.; Klemic, K. G.; Smith, D.; Gerstein, M.; Reed, M. A.; Snyder, M. Nat. Genet. 2000, 26, 283 11 Falsey, J. 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DMF 94 Chapter 3 3 .3. 3 Array -Based Kinase Assay 3. 3 .3. 1 Spotting The N-terminally cysteine-containing peptides and N-terminally biotinylated peptides were dissolved in PBS, pH 7.4, and arrayed using an ESI SMA arrayer (Ontario, Canada), with a spacing of 220 µm between the spots After incubation, the slides were washed with PBS and distilled water and air-dried 3. 3 .3. 2 Antibody Labeling and Screening... activities, and potentially other enzymatic activities The intercalation of various PEGs between the slide surface and the immobilized peptides minimized non-specific binding of proteins to the glass surface, rendering it possible to eliminate the blocking step using BSA 90 Chapter 3 3 .3 Materials and Methods 3. 3.1 Peptide Synthesis2 The p60 and PKA and JAK substrates (YIYGSFK, ALRRASLG, and KGTGYIKTG... was observed that the reaction took place within the first 30 minutes of incubation After 3h of incubation, spots corresponding to lower concentrations were eventually observed and the intensity reached saturation, indicating the completion of the native chemical ligation reaction, hence the complete peptide immobilization 30 min 1 hr 3 hrs 3 mM 1 mM 0 .3 mM Figure 3. 13 Rate of on-chip chemical ligation... Si (MeO )3 O SiOH SiOH O SiOH Piranha solution H2SO4 / H2O2 7 :3 SiOH O Si SiOH SiOH NH2 (MeO )3 95 % EtOH, 1h 150 deg, 2h 95 % EtOH, 1h 150 deg, 2h Si Si Si Si NH2 Si Si Si Si Si Si Si Si Si NH2 Si Si Si O Si O Scheme 3. 8 Slide Silanization (a) amine, (b) epoxide 3. 3.2 .3 Glyoxylic Acid Functionalization The derivatization of glass slides with glyoxyxlic acid was done according to published protocols with... thioester slides Phosphorylated p60 peptide was spotted onto glass slides functionalized with (a) NHS PEG and (b) Amine PEG, and probed with FITC-labeled anti-phosphotyrosine 3. 2 .3 Rapid Microarray- Based Kinase Assay Rapid fluorescent antibody -based detection was combined with the site-specific immobilization developed for rapid kinase assay 87 Chapter 3 3.2 .3. 1 Avidin-Biotin Interaction The biotinylated, non-phosphorylated,... NaHCO3 for 30 minutes, washed with water, air dried and the remaining 93 Chapter 3 epoxides were reacted with a solution of 2 mM aspartic acid in a 0.5 M NaHCO3 buffer (pH 9) 3. 3.2.5 Thioester Functionalization Thioester-containing slides were prepared from amine slides as shown in Scheme 3. 4 First, amine slides were placed in a solution of 180 mM succinic anhydride in DMF, pH 9 (Na2B4O7) for 30 minutes... functionalization 3. 3.2.2 Slide Silanization Two different protocols were used for the amine silanization of the slides At first, slides were silanized in dry toluene: cleaned slides were soaked in aminopropyltriethoxisilane in dry toluene for 2 hours under nitrogen atmosphere The slides were subsequently rinsed with toluene, ethanol and dried In a second protocol, slides were silanized in ethanol (Scheme 3. 8 (a))... anti-phosphotyrosine 89 Chapter 3 3.2.4 Summary Antibody -based fluorescence detection was developed as an efficient, sensitive and selective method for quantitative detection of kinase activity in a microarray format, eliminating the use of radioactive 32 P, which poses a serious health threat to human As a result, this detection method should be compatible with most fluorescence -based microarray applications,... NUS 83 Chapter 3 9 mM 3 mM 1 mM 0 .3 mM 0.1mM Figure 3. 12 Saturation studies of thioester slides using different spotting concentrations of cysteine-containing fluorescein In order to evaluate the time needed for efficient immobilization of peptides, different concentrations of the cysteine-containing fluorescein in PBS (pH 7.4) were spotted and incubated for increasing periods of time (Figure 3. 13) It... Spectrometry 3. 3.2 Slide Functionalization 3. 3.2.1 Slide Cleaning Before any functionalization, glass slides (Sigma Aldrich, USA) were cleaned in piranha solution (H2SO4 : H2O2, 7 : 3) for at least two hours The slides were washed 2 Most of the peptides were synthesized by Mahesh Uttamchandi 91 Chapter 3 thoroughly with deionised water and rinsed with ethanol Slides were usually kept in the piranha solution and . 3. 6) and the fluorescence intensity of the spots was measured (Figure 3. 7). 73 Chapter 3 3 mM 1 mM 0 .3 mM 0.1 mM 3 mM 1 mM 0 .3 mM 0.1 mM Figure 3. 6. Antibody -based. reaction, hence the complete peptide immobilization. 30 min 1 hr 3 hrs 3 mM 1 mM 0 .3 mM 30 min 1 hr 3 hrs 3 mM 1 mM 0 .3 mM Figure 3. 13. Rate of on-chip chemical ligation reaction. Increasing. synthesized by Dr Zhu, Chemistry Department, NUS 83 Chapter 3 9 mM 1 mM 3 mM 0 .3 mM 0.1mM 9 mM 1 mM 3 mM 0 .3 mM 0.1mM Figure 3. 12. Saturation studies of thioester slides using