Custom Peptide Microarray Service

MULTIPLEX PROTEIN KINASE ASSAYS

Perform protein kinase reactions on arrays of comprehensive sets of substrates. Measure kinase activity and inhibitor effects. Obtain hundreds of reaction profiles and curves in parallel for parameters such as VMAX, KM, and KI.  Use of microarray titer plate format will increase your workflow by at least 40-fold that of a single microtiter plate.

LARGE SCALE HIGH RESOLUTION EPITOPE MAPPING

Perform large scale parallel epitope mapping from pathogens. Identify high affinity antibody binding peptides.  Screen sensitive antibody-detection peptides.  Detect autoantibodies/biomarkers from serum or biological samples, determine Kd (binding affinity) of hundreds of binding peptides, obtain titration curves for antibodies.

PATHWAY PHOSPHOPEPTIDE-BINDING PROTEIN STUDIES

Profile SH2-containing proteins in complex biological samples by an array of phosphopeptides which are carefully curated from literature.  Screen effective SH2 binding inhibitors and measure inhibitory activities.  Screen signature phosphopeptides for protein capture as a tool for signaling pathway characterization.

PEPTIDES AND PEPTIDOMIMETICS FOR PROTEIN CAPTURE

Screen biomarkers (proteins and peptide binding targets) from biological samples such as blood serum or cell lysates. Use novel protein capture peptides to detect and profile on the same chip platform a wide range of molecular biomarkers: signaling protein kinases, pathway phosphoprotein-binding and domain-binding proteins, immuno-responsive antibodies, metabolites and metabolic molecules, and other small molecules.

THERAPEUTIC SYNTHETIC PEPTIDE SCREENING

Rapidly design, synthesize, and screen diverse peptides, peptide analogs, and peptides modified with glyco-units against important therapeutic targets.

Cost Effective 1-Stop Proteomics Solution

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Innovative Microfluidic Array Platform

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Microfluidic Array Platform

These are not spotted arrays!  A proprietary µParaflo® microfluidic biochip is used and custom peptide sequences are synthesized on-chip.  The microfluidic technology produces a uniform distribution of the sample solutions on the array, ensures efficient sample-peptide contact and enhances binding reactions and stringency wash processes.  The microarray chip consists of thousands of three-dimensional chambers and is a closed system.  Under these conditions multiplex protein assays are carried out in a way much like in thousands of pico-liter tubes, enclosure keeps the proteins in a stable environment, in solution and protected from air– oxidation/contamination. The miniaturized system provides automation, sample/reagent-savings and simplicity in operation.

In situ Parallel Synthesis

In situ peptide synthesis using PGA (photogenerated acid) coupled with conventional t-boc chemistry and a programmable process means high probe quality, tight process control, and complete content flexibility.  Our advanced manufacturing process ensures highly uniform spots and high reproducibility from array to array and yet permits total customization of contents on each individual array.  In comparison, spotted microarrays tend to suffer from poor spot uniformity and large spot to spot and array to array variations, which lead to large data deviations.  The spotting process requires significant up-front investment for peptide libraries and spotting equipment and thus is inflexible for customization.

Complete Control of Peptide Microarray Quality

We ensure reliable and quantifiable results through the use of multiple negative and positive controls as well as reference peptides.  Multiple QC measures are implemented at various stages of array manufacturing and assay processes.  The evaluation of signal intensities of these control spots allows assessment of spot uniformity, cross-array spot-to-spot uniformity, and binding specificities and strength.

Comprehensive and Current Peptide Content

Each peptide of a predetermined sequence is synthesized on a long spacer.  The peptide sequences may be standard catalog content (from LC Sciences’ library containing database and/or literature validated peptides) or custom content (customer defined sequences).  We use the most updated information on proteins for peptide microarray design to deliver arrays of comprehensive, systematic and up to date content.  Sequence repeats are used on each array to allow statistical analysis of the data.

Multiple-Time Repetitive Assays on the Same Chip

Peptide microarrays can be used for time-, protein/antibody/enzyme concentration-, or co-factor/inhibitor-dependent measurements.  The array can be used multiple times, each time assay is carried out and assay signals are collected by fluorescence scan. These assays generate a very large set of data for proteomic assays and valuable information which would take weeks and months to produce using a conventional single protein/antibody/enzyme assay kit.

Pico-liter Scale Microarray Titer Plates

Peptide Concentration Variation On-Chip

Our unique synthesis chemistry makes it possible to vary the substrate density of reaction sites to create a gradient across the array. This array can generate the same amount of data equivalent to that of 40 conventional microtiter plates!  This form of miniaturized multiplex parallel protein/antibody assays saves assay samples, reagents, labor and time in generating binding affinity or enzymatic reaction curves with a single experiment, allowing cross comparison of the systems assayed. The use of comparisons provides a more comprehensive picture and more reliable results.

Close up view of a µParaFlo® biochip

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Peptide Concentration Variation  Across the Microarray Titer Plate

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Pico-liter Scale Microarray Titer Plate

Peptide sequences are synthesized on the array at different molecular densities and thus are presented at different “concentrations”.

A 4000 feature microarray titer plate contains the molecular content equivalent to 40 microtiter plates.

Titration curves are obtained by concentration variations of the peptide substrates or the binding proteins.  Similarly, time-curves and inhibition curves can be measured simultaneously for a large number of peptides.

Quantitative Peptide Microarray Applications

Multiplex Protein Kinase Assays

LC Sciences provides a comprehensive kinase analysis service utilizing high density protein kinase substrate (PKS) peptide microarrays synthesized for proteomic scale kinase profiling, quantitative measurement of kinase kinetic activities, and drug discovery research.

Known Kinases

  • Identify specific phosphorylation peptide substrates.
  • Characterize kinase kinetics and determine relative reaction rates.
  • Assay kinase inhibitors on an array of hundreds of peptide substrates for development of kinase regulatory agents.
  • Profile large sets of kinases with an array of signature “known” peptide substrates.
  • Make quantitative measurements for comparison of normal and mutant kinases reactivities.
  • Study signaling pathway proteins and mechanisms of disease.

Unknown Kinases

  • Identify substrate phosphorylation patterns and provide PK family assignments.
Large Scale Epitope Mapping

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LC Sciences provides a comprehensive epitope peptide microarray assay service to make both qualitative and quantitative measurements of antibody/epitope binding.  Screening on a peptide microarray offers the opportunity to study thousands (up to thousands of peptides/chip) of potential epitopes in a single experiment utilizing only sub-µg quantity of protein.  Using a tiling method, we can systematically map the binding sites on a pathogen/protein at single amino acid resolution.

Immunological Studies

  • Quantitate and optimize antibody binding affinity.
  • Optimize of phage-display lead peptides.
  • Identify immunodominant regions in antigens.
  • Screen antibody-targeting therapeutic peptides.

Vaccine Development

  • Screen vaccine peptides.
  • Develop specific antibodies by prescreening cross-reactivity.

Biomarker Screening

  • Detect autoimmune-response antibodies.
  • Develop biosensor arrays
Phosphopeptide-Binding Protein Studies

LC Sciences provides a comprehensive phosphopeptide-binding protein assay service for profiling SH2-containing proteins in complex biological samples by an array of phosphopeptides which are carefully curated from literature.

  • Screen effective SH2 binding inhibitors and measure inhibitory activities.
  • Screen signature phosphopeptides for protein capture as a tool for signaling pathway characterization.
  • Optimize phosphopeptide domain binding.

Multiplex Protein Kinase Assays

LC Sciences Standard Kinase Array Content
  • Covers the entire human kinome with substrates representative of all PK families.
  • Contains over a thousand unique peptide substrates.
  • Also contains corresponding negative controls (Ser/Thr/Tyr substituted by Ala) and
  • Replicates of each sequence.
Custom Content Kinase Arrays
  • Microfluidic platform and in situ synthesis permit total customization of sequence content.
  • Thousands of customer defined unique peptide substrates are synthesized per array. No minimum limit on the number of unique arrays synthesized.
  • LC Sciences can assist with sequence design.

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Characterization of a Novel Protein Kinase

The preferred binding pattern of an unknown kinase revealed it is a serine/threonine kinase and belongs to PKA family.

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Kinetic Studies of PKA Reaction

Parallel measurements of reaction curve as a function of time and substrate concentration.  These measurements lead to derivation of VMAX and KM. Pico-liter titer plate arrays enable simultaneous measurement of hundreds of such curves increasing workflow efficiency 40x.

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Measurement of Kinase Inhibition Curves

A powerful tool for protein kinase inhibitor screen assays – A Single array assay measures multiple inhibitory constants on various PK substrate peptides, allowing comparison of specificity of PK inhibition and identification of potential major and off-site phosphorylation sites.

(D1, D2, D3 – peptide surface density)

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Large Scale Epitope Mapping

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Epitope Discovery

Measure relative binding affinity to epitope sequence variants (N-truncation, C-truncation, Ala scan, tiling).  Achieve single amino acid resolution for the binding core.

Antibody: anti-HA

Epitope (known): YPYDVPDYA

Chip #1: HA and flag epitope and variant sequences

Chip #2: HA, c-myc, VSVG, and flag epitope and other peptide sequences

Assay solution: 200 ml TBS (pH 6.8)

Temperature: 4 ˚C

Experimental Time: 1 hour

Detection: Cy5-IgG

Results: HA epitope is a hexapeptide DVPDYA. Three amino acids (D4, D6 and Y7) are essential.

 

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Results of epitope high resolution mapping are consistent with published data by protein crystallography method.

(Science 1992, 255:959; Curr Opin Immunol 1999, 11:193-202)

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Measurement of Association Constants

Make high-throughput quantitative measurements of antibody binding curves and calculate association constants.

Association curves measured with protein concentration titration on an epitope peptide microarray

  • Microfluidic design enables multiple concentration bindings on the same chip to generate not only a single titration curve for the known epitope but also titration curves for all the epitope variants.
  • Anti-HA varied from 0.1 to 60,000 ng/ml, signal intensities acquired at different scanning gains were scaled according to a calibration curve, curve fitting used Origin (Origin Lab).
  • Kd values measured are in the range of 2-4 ug/ml.

Association curves measured with peptide concentration (density) variation on an epitope peptide microarray

  • Unique synthesis chemistry makes it possible to vary the substrate density across the array. “Pico-titer” plate.
  • Thousands of binding curves can be measured simultaneously by one assay incubation. Equivalent to 40 microtiter plates.
  • In situ synthesis and innovative chemistry can provide more than 25-fold change in peptide density on the surface.
  • Measured a 31-fold change in anti-HA binding for highest density vs regular peptide density.

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Phosphopeptide-Binding Protein Studies

LC Sciences Standard Profiling Array Content

Phosphoprotein binding proteins (PPBP) contain domains which binds to phosphorylation sites in phosphoprotein or phosphopeptide (PPEP).

Through the use of high density arrays with addressable “signature” peptides for specific kinds of protein binding domains we can detect the presence or absence of these kinds of proteins in complex biological samples (cell lysate, human serum).

We have standard content phosphopeptide binding arrays with information from literature incorporated into the standard array design.  Important signaling protein binding domains are represented on the array.

Drug development studies can be performed by measuring differential binding affinity to compare various phosphoprotein binding proteins or to quantitatively measure inhibitor effect.

Standard SH2 Profiling Array

SH2 is an example of PPBD which recognizes phosphorylated tyrosine (pY).

Total 1122 unique signature phosphopeptides from 87 phosphopeptide binding domains with 2 replicates and one set of negative controls (with phosphotyrosine substituted by alanine)

Measuring the relative binding of built-in controls on the array including natural Tyrosine, Phosphorylated Tyrosine, and Alanine (negative) enables us to make accurate quantitative measurements of protein binding and to minimize false positive signals due to non-specific phosphate binding.

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Quantitative Analysis of Phosphopeptide-Binding on a Profiling Array

Binding specificity of SHP2-N-terminal SH2 versus an alternate domain (XSH2) to PPEP

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Differential Binding Affinity Analysis

Measure relative binding affinity to various recombinant phosphoprotein binding proteins.

Samples: GST-tagged recombinant fusion proteins: S01-SHP2 CSH2, S02-SHP2 NSH2, S03-SHP2 2SH2

Chip: LC Sciences standard SH2 profiling array.

Detection: Anti-GST Ab Cy5 conjugate

Data Processing: Global background subtraction and Z transformation, hierarchical clustering analysis.

Plot Information: TMEV (9) image of the Z-values for relative binding affinity ranked for each SH2 protein.  From red to green is stronger to weaker binding.

Quantitative Measurement of Inhibitor Effect

Differential binding measurement in complex biological samples.

Sample 1: Normal Human Serum

Sample 2: Treated Human Serum

Treatment:  Removal of Albumin and IgG

Sample Size: 5ng/µl in 100 µl total volume.

Chip: LC Sciences standard SH2 profiling array.

Detection: Anti-human IgG-Cy5 conjugate

 

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Web-based tools are available to assist with experimental design.

PepCyber – A searchable database of human protein–protein interactions mediated by phosphoprotein-binding domains (PPBDs).

http://pepcyber.umn.edu/PPEP/

 

Comprehensive Sample to Data Service

Sample QC

Appropriate sample requirements are established for each particular array application.  Integrity of the received customer sample is determined via a thorough analysis process.  Samples that do not meet requirements are flagged and notification is sent with a recommendation not to proceed with the microarray assays.

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Chip Synthesis and Control Experiments

A standard or custom peptide microarray is synthesized on a high density peptide µParaflo® microfluidic biochip.  Each chip contains multiple synthesis control sites optimized for rigorous quality analysis.

On-Chip Binding or Enzymatic Assays

Multiplex assays are performed on a µParaFlo® microfluidics chip which ensures uniform flow of the protein samples through closed pico-liter chambers.  The assay is controlled by optimized binding protocols and performed under temperature controlled conditions.  Binding/Enzymatic experiments are monitored to achieve high quality and stringency.

Titer-Plate Binding or Enzymatic Assays

Peptides on reaction sites are synthesized according to pre-determined surface densities as “concen

tration” variation sites.  Parallel protein assays on these titer-plate reaction sites provide hundreds of curves for binding or enzymatic/inhibitory activity measurements with minimal systemic variations.

Time and Concentration Dependent Measurements

Multiple measurements are performed to obtain variable plots using time or concentrations of protein, enzyme, inhibitor, or co-factor.

Microarray Scan and Data Extraction

Microarray images are carefully scanned for a balanced view.  Numerical intensities are extracted for control, background, reference and test peptides.

Data Analysis

Basic data analysis  includes background subtraction, control and reference signal guided data processing, list of detected signals and data averaging results.  Optional in-depth analysis and comprehensive data processing services such as: consensus sequence analysis, statistical and pattern analysis, plot generation and kinetic/thermodynamic parameter calculation is available as requested based on your specific application . A summary of the analysis report is emailed.  The complete data set is saved to a CD, which is shipped via overnight carrier.

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Microfluidic Array Platform

The microfluidic technology produces a uniform distribution of the sample solutions on the array, ensures efficient sample-peptide contact and enhances binding reactions and stringency wash processes.  The microarray chip consists of thousands of three-dimensional chambers and is a closed system.  Under these conditions multiplex protein assays are carried out in a way much like in thousands of pico-liter tubes, enclosure keeps the proteins in a stable environment, in solution and protected from air– oxidation/contamination. The miniaturized system provides automation, sample/reagent-savings and simplicity in operation.

Multiple-Time Repetitive Assays on the Same Chip

Peptide microarrays can be used for time-, protein/antibody/enzyme concentration-, or co-factor/inhibitor-dependent measurements.  The array can be used multiple times, each time assay is carried out and assay signals are collected by fluorescence scan. These assays generate a very large set of data for proteomic assays and valuable information which would take weeks and months to produce using a conventional single protein/antibody/enzyme assay kit.

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Kinetic Curve of PKA Reaction
  • LRRXSLG series of peptides. Parallel measurements of hundreds of reaction curves.
  • Kinetic curves on time courses at different concentration of substrate.
  • PKA kinase reaction was performed at 30oC, 0-480 min w/0.2 mM ATP, 0.5 µg/mL PKA.
  • Longer reaction time reaches higher product. At 120 min, reaction at all concentrations reaches the highest product yield (signal plateau).
Kinase Profiling Customer Publications
  1. Wang W, Woodbury NW. (2014) Unstructured interactions between peptides and proteins: Exploring the role of sequence motifs in affinity and specificity. Acta Biomaterialia 11(88-95) [abstract].
  2. González Burón H. (2014) The role of the Tousled Like Kinases in genome stability and mammalian development. 24(11):1872-1885. [abstract].
  3. Jang J, Stella A, Boudou Fd, Levillain F, Darthuy E, Vaubourgeix J, Wang C, Bardou F, Puzo G, Gilleron M. (2010) Functional characterization of the Mycobacterium tuberculosis serine/threonine kinase PknJ. Microbiology 156(6), 1619-1631 [abstract]
Epitope Mapping Customer Publications
  1. Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, Shively J, Cannon GC, Kerfeld CA. (2015) Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component. Life 5(2), 1141-1171 [abstract].
  2. Aloisio GM, Nakada Y, Saatcioglu HD, Peña CG, Baker MD, Tarnawa ED, Mukherjee J, Manjunath H, Bugde A, Sengupta AL. (2014) PAX7 expression defines germline stem cells in the adult testis. Journal of Clinical Investigation 124(9), 3929-3944 [abstract].
  3. Assis DN, Leng L, Du X, Zhang CK, Grieb G, Merk M, Garcia AB, McCrann C, Chapiro J, Meinhardt A. (2013) The role of macrophage migration inhibitory factor (MIF) in autoimmune liver disease. Hepatology 59(2):580-91 [abstract].
  4. Reichmann D, Xu Y, Cremers CM, Ilbert M, Mittelman R, Fitzgerald MC, Jakob U. (2012) Order out of Disorder: Working Cycle of an Intrinsically Unfolded Chaperone. Cell 148(5), 947-57 [abstract].
  5. Butterfield K, Caplan M, Panitch A. (2010) Identification and Sequence Composition Characterization of Chondroitin Sulfate-Binding Peptides through Peptide Array Screening. Biochemistry 49(7), 1549-55 [abstract].
  6. Williams BA, Diehnelt CW, Belcher P, Greving M, Woodbury NW, Johnston SA, Chaput JC. (2009) Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds. J Am Chem Soc 131(47), 17233-41 [abstract].
µParaflo® Peptide Microarray  Synthesis Technology Articles
  1. Zhu Q, Hong A, Sheng N, Zhang X, Matejko A, Jun K-Y, Srivannavit O, Gulari E, Gao X and Zhou X. (2007) µParaflo Biochip for Nucleic Acid and Protein Analysis. Methods in Molecular Biology, 382, 287-312 [abstract].
  2. Gao X, Pellois J P, Kim K, Na Y, Gulari E, and Zhou X. (2004) High density peptide microarrays. In situ synthesis and applications. Molecular Diversity. 8, 177-187 [abstract].
  3. Gao X.(2004) In situ parallel synthesis of addressable peptide microarrays – in Proceedings of the 7th China Peptide Symposium. Peptides. Biology and Chemistry. Eds. Du, Y-C., Zhang, Y. S., and Tam, J. P. Shanghai Scientific & Technology Publishers. pp. 29-33 [abstract].
  4. Gulari E, Gao X, and Zhou X. (2003) Light directed massively parallel on-chip synthesis of peptide arrays with t-Boc chemistry. Proteomics 3, 2135–2141 [abstract].
  5. Pellois J P, Zhou X, Srivannavit O, Zhou T, Gulari E, and Gao X. (2002) Individually addressable parallel peptide synthesis on microchips. Nat. Biotechnol. 20, 922-926 [abstract].
  6. Pellois J P, Wang W and Gao X. (2000) Peptide synthesis based on t-Boc chemistry and solution photogenerated acids. J. Comb. Chem. 2, 355-360 [abstract].

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Peptide Microarray Data Analysis Using SVM-PEPARRAY Peptide Microarray Design Using µPepArray Pro