Peptide Microarray Applications

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Kinase Substrate Profiling 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. Kinetic Studies of PKA Reaction Parallel measurements of reaction curve as a function of time and substrate concentration. These measurements lead to derivation of V MAX and K M . Pico-liter titer plate arrays enable simultaneous measurement of hundreds of such curves increasing workflow efficiency 40x. [Substrate] ( µ M ) 0 20 40 60 v( µ mol/min/mg) 0 1 2 3 4 5 6 LRRGSLG LRRMSLG LRRYSLG LRRSSLG LRRASLG 1/[S] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 1/v (min*mg/ m mol) 0 1 2 3 4 5 LRRASLG

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Large Scale Epitope Mapping 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 ( D 4 , D 6 and Y 7 ) are essential. Anti-HA Epitope Binding Array Cy5-IgG Staining 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) Ala-scanning YPY D VP DY A red: important residues

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Phosphoprotein Binding Studies 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. Web-based tools are available to assist with experimental design. PepCyber - A searchable database of human protein–protein interactions mediated by phosphoprotein-binding domains ( PPBD s). http://pepcyber.umn.edu/PPEP/ IL6R4 (Negative) CagA-A CagA-B CTLA-4 IL6R3 EGFR-1173 IL4Ra1 IL6R4 EGFR-954 VEGFR-1 S01 - Treated Serum S02 - Normal Serum -3.0 0.0 3.0 CagA-E Gab1-659 IRS-1 AFAP110-3 Gab-3 FRS2-392 PDGFRB_Y1009 STAT3-1642 CagA-A Met2 SRC-1589 IL-6R-1 S01-CSH2 S02-NSH2 S03-2SH2 -3.0 0.0 3.0 = Phosphorylated Tyrosine P A=Ala Y=Tyr A Y P