High-Throughput Screening

High-Throughput Screening Assays on Solid Supports or Chips -> Array Technology

Antibody Arrays to Measure Protein Level in Cells • Problem with mRNA level corresponding to Protein level: • Protein Stability • Regulation of splicing • mRNA stability • Location of mRNA in the cell • Posttranslational modifications to make active protein • -> Protein Microarray system better -> in development • -> Antibodies recognizing specific regions of proteins • -> AB spotted onto microarrays -> to bind target protein • -> Protein extracts from 2 cells (compared) are labeled • -> signal intensity proportional to amount of active protein

Protein Microarrays to Screen for Protein-Protein Interactions -> Each protein from organism with a Tag (GST) expressed -> Purification by high-throughput affinity chromatography -> spotted onto glas plates -> protein of interest labeled and added -> possible to screen many proteins in a few experiments 6

Protein Microarrays to Screen for Protein-Protein Interactions -> Each protein from organism with a Tag (GST) expressed -> Purification by high-throughput affinity chromatography -> spotted onto glas plates -> protein of interest labeled and added -> possible to screen many proteins in a few experiments -> study interaction motif 7

Protein Microarrays for detection of Enzymatic Activity Previously enzyme activity could not be detected, since only the binding and not the catalytic activity were detected Detection of enzyme catalytic activity is possible e.g. by formation of colorimetric products Can be used to create substrate “fingerprint” profiles (JEAN-LOUIS REYMOND)for each enzyme  reveals type of chemical compounds accepted by enzyme (or type of enzyme) Using different assay conditions (resembling cytoplasm etc.) reveals complex biological pathways of the enzyme

Detection Methods Colorimetric methods widely used due to simplicity, reliability and sensitivity Fluorescence preferred over radioactive and chemiluminescentmethods Radioactivity: Dangerous Chemiluminescence: Limited range Fluorescence: High signal-to-noise ratio; compatible with standard microarray scanners Protein Microarrays for detection of Enzymatic Activity

Proteases, esterases, lipases: Utilize cleavage activity to obtain fluorescent probe Kinases: Fluorescently labeled antibody binds to phosphorylated probe Protein Microarrays for detection of Enzymatic Activity Coumarin

Protein Microarrays for detection of Enzymatic Activity - Fingerprint A substrate microarray for fingerprinting lipase activities. Aminofunctionalized glass slides were coated covalently with bovine serum albumin and the substrate using appropriate spacers based on active ester chemistry and reductive alkylation. The assay includes stepwise incubation and glass-washing cycles following the indicated sequence. The array is then recorded using a microarray scanner. Each spot is present in quadruplicate on the array.

Protein Microarrays for detection of Enzymatic Activity - Fingerprint Periodate activated fluorogenic substrates. (5) substrate for epoxide hydrolases. (8) Substrate for lipases and esterases. (9) reference commercial lipase substrate.

Protein Microarrays for detection of Enzymatic Activity - Fingerprint Enzyme fingerprints from a periodate activated substrate array. The substrates are derivatives of (5) and (8) with different substitution patterns and enzyme reactive functional groups as shown in the array reference at lower right (cpds numbers from WAHLER et al. 2002) The relative activity is color-coded in each array position according to the relative reaction rate (color intensity) and enantioselectivity (blue to green ratio) according to the reference scale at lower left. Below each array the enzyme abbreviation is given in three-letter code and the number indicates the maximum reaction rate of the most reactive substrate in the array in pM/sec.

Protein Microarrays for detection of Enzymatic Activity - Fingerprint Fingerprint analysis of chain length and cosolvent selectivity of lipases and esterases. All assays were run in microtiter plates. The array represent the activity data with substrates 11a-h and 12a-b on a series of lipases and esterases (code at each column heading) according to the reference color-scale shown below. Color intensity represents the relative reaction rate (value of the maximum observed reaction rate is given below each column) and the blue-to-green ratio indicates the relative reactivity in aqueous buffer vs. buffer + cosolvent.

Enzyme Inhibitor Identification Inhibitors or specific enzymes can be detected by using fluorescently labeled inhibitors Protein Microarrays for detection of Enzymatic Activity • Probes react in time- and concentration-dependent manner  obtain quantitative kinetic data for the enzyme activity • Can also be used to detect activators • Found inhibitors and activators can be used in the development of novel therapeutics

Reverse Phase Protein Array Coupling Laser Capture Microdissection (LCM) With High Throughput Protein Arrays Patient biopsy tissue cells are microdissected: 30,000 cells = 100 arrays LCM -> method for isolating specific cells of interest from microscopic regions of tissue that has been sectioned under the microscope Each patient sample is arrayed in a miniature dilution curve: Always in linear dynamic range of any antibody/ analyte pair Arrays probed with labeled amplified antibody: e.g. Ovarian cancer progression From one patient probed with Phospho-ERK antibody Phosphorylated extracellular signal-regulated kinase -> Cancer marker

Reverse Phase Protein Array Reverse phase proteinmicroarrays (RPPA) do not require labelling of cellular proteinlysates, and constitute a sensitive high throughput platformfor marker screening Reserve Phase Protein Microarrays are applied to: • Clinical Research – utilized in clinical trials for assessing response to therapy and demonstrating protein molecular changes to therapy. • Disease Prognostics - utilized for determining which patient is likely to respond to a given therapy. • Personalized drug treatment – monitoring response to therapy before, during and after treatment.

Reverse Phase Protein Array Experimental Design Experimental Design

Tissue processing and microdissection 8.0 μm frozen sections of ovarian cancer tissues were placed on uncoated glass slides and stored at -80ºC prior to use. Ovarian tumor epithelial cells or other relevant cell populations were microdissected with a Pixcell II Laser Capture Microdissection system (Arturus). Approximately 5,000 LCM shots (20,000-25,000 cells) were microdissected for each case and stored on microdissection caps at -80ºC until lysed. Cell lysis and cellular lysate arraying Microdissected cells were lysed directly from the microdissection caps into 50 μL of lysis buffer containing a 1:1 mixture of 2x Tris-Glycine SDS sample buffer (Invitrogen Life Technologies) and Tissue Protein Extraction Reagent (Pierce) plus 2.5% β-mercaptoethanol for 30 min at 75ºC. Positive control samples included A431 control and A431+EGF lysates (BD Pharmingen) at 1.0 mg/mL. Reference standard peptides specific for the pAkt and pERK antibodies (Cell Signaling Technology) were diluted in lysis buffer to 1.0 μg/mL. Immediately prior to arraying, lysates were loaded into a 384-well plate and serially diluted with lysis buffer into a 5-point dilution curve (ovarian samples and A431 controls) ranging from undiluted-1:16 or 12-point dilution curve (reference standard peptides) ranging from undiluted-1:16. Approximately 60 nL of each sample was spotted onto nitrocellulose-coated glass slides (Schleicher and Schuell Bioscience) with a GMS 417 microarrayer (Affymetrix). Slides were stored dessicated at -20ºC. For estimation of total protein amounts, selected arrays were stained with Sypro Ruby Protein Blot Stain (Molecular Probes) according to the manufacturer’s instructions and visualized on a Fluorchem™ imaging system (Alpha Innotech). One day prior to antibody staining, the lysate arrays were treated with Reblot antibody stripping solution (Chemicon) for 15 min at room temperature, washed 2 x 5 min in PBS, and then incubated overnight in blocking solution (1g I-block (Tropix), 0.1% Tween-20 in 500 mL PBS) at 4ºC with constant rocking. Protein microarray staining Blocked arrays were stained with antibodies on an automated slide stainer (Dako Cytomation) using the Catalyzed Signal Amplification System kit according to the manufacturer’s recommendation (CSA; Dako Cytomation). Briefly, endogenous biotin was blocked for 10 min using the biotin blocking kit, followed by application of protein block for 5 min; primary antibodies were diluted in antibody diluent and incubated on slides for 30 min and biotinylated secondary antibodies were incubated for 15 min. Signal amplification involved incubation with a streptavidin-biotin-peroxidase complex provided in the CSA kit for 15 min, and amplification reagent, (biotinyl-tyramide/hydrogen peroxide, streptavidin-peroxidase) for 15 min each. Development was completed using diaminobenzadine/hydrogen peroxide as the chromogen/substrate. Slides were allowed to air dry following development. Primary antibodies used in these studies were: Akt 1:100 (Cell Signaling Technology); phosphoAkt S473 1:50 (Cell Signaling Technology); phosphoAkt T308 1:50 (Cell Signaling Technology); extracellular signal-regulated kinase (ERK) 1/2 1:200 (Cell Signaling Technology); phosphoERK1/2 T202/Y204 1:1000 (Cell Signaling Technology Secondary antibody and dilution used was biotinylated goat anti-rabbit IgG (H+L) at a 1:5000 dilution (Vector Laboratories). Reverse Phase Protein Array

Cell Arrays are used to test Promoter activity -> Reporter assay -> used to test many promoters at the same time

Protein Microarrays for Proteomics

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique Automated Micro-Chip Robot loader

SELDI is an ionization method (variation of MALDI) in mass spectrometry that is used for the analysis of proteinmixtures. Comparison of protein levels between patients with and without a disease can be used for biomarker discovery. SELDI technology was developed by T. William Hutchens at Baylor College of Medicine in 1993.[4] The technology was commercialized by Ciphergen Biosystems in 1997 as the ProteinChip system. It is now produced and marketed by Bio-Rad Laboratories.

High-Throughput Screening