Details of recombinant protein binding assay on peptide microarray.
2(a) Flow diagram showing the steps of recombinant protein binding assay on the pY peptide microarray synthesized on the chip surface. The recombinant protein containing an affinity tag (such as HIS, or GST) was applied to the microfluidic chip to be in contact with the pY peptide probes for about 1 hour. The specific interaction (binding) occurs between the protein and the pY peptide probe peptides based on the affinity of SH2 domain with phosphotyrosine residues on specific protein motifs. The binding was recognized by primary antibody (such as anti-HIS or anti-GST). Detection of the binding is through fluorescence dye (Hylite)-conjugated primary antibody. 2(b) Recombinant protein binding on peptide array: Quality of binding image, data processing and analysis: The chip contained 3986 peptides synthesized in situ on a microfluidic chip surface containing 3,968 picoliter reaction wells with 1,227 peptides were tyrosine phosphorylated (pY-peptides) and 1227 corresponding control peptides (A-peptides) with technical controls for monitoring synthesis quality and protein binding assays. A. Binding image of GST-Grb2 SH2 domain on phosphopeptide chip: The array was probed with GST-tagged Grb2-SH2 domain and detected by antiGST-Hylite 647 conjugated antibody. Signals from proteins bound to phosphopeptides compared and respective control peptides wells were scored. B. Representative image of a small region from panel A. Peptide sequences (pY-peptides and A-peptides) are provided on either side of the image panel with the pY sequences marked in red and A-peptide sequences in black. The pY-peptide and corresponding A-peptide were synthesized at the two adjacent positions in the same column for conveniently visualizing pY specific binding. C. Histogram of the Grb2 binding (log10 scale) showing the distribution of binding intensities ranging from 500 to 60000 with a mean intensity around 1500. D. Histogram plot of the spot to spot CV (covariance of three replicate peptide-probes on chip) distribution of Grb2 binding: 3,695 (93%) spots have CVs less than 0.25, showing both peptide synthesis and Grb2 binding are uniform. E. Bar graph plot of the detected intensities of Grb2 SH2 domain binding shown as blue (pY-peptide probe signals) or brown (A-peptide probe signals). Grb2 SH2 domain binding affinity varied from 5 to 30 folds compared to control peptides. 2(c) Comparison of peptide microarray SH2 domain binding motif consensus of this study (BTK, Grb2, Src and ZAP70) with those of previously published in vitro peptide binding results: Binding patterns and consensus sequences of four SH2 domain containing proteins (BTK, Grb2, Src and ZAP70). A. Binding images of four SH2 domains on the same region of four independent phosphopeptide chips. B. Heat map showing differential binding intensities (on Z value scale) of 4 SH2 containing proteins to related PPEPs on the chips. The pY probes with Z values ranked from 1.0–3.0 were plotted. C. Consensus sequences of binding sequences of 4 SH2 containing proteins. The consensus sequences were aligned by using WebLogo (version 2.8.2 http://weblogo.berkeley.edu/logo.cgi/). For each SH2 domain, the binding PPEPs with Z values greater than 1 were selected. The number of high confidence peptide probes selected to BTK, Grb2, Src and ZAP70 are 35, 133, 86 and 45, respectively.