Fig. 1.
Addressable peptide arrays provide a reproducible and semiquantitative assay for interactions between SH2 domains and peptides. A, a schematic representation of a high density SPOT peptide array composed of 192 peptides including control peptides (black circles) and “physiological” peptides corresponding to tyrosine-containing sequences from the cytoplasmic regions of 13 proteins involved in phosphotyrosine-mediated signaling. Different proteins are indicated by colored circles with peptides of redundant sequence corresponding to multiple proteins indicated with multiple colors. B, peptide arrays probed with anti-phosphotyrosine antibodies (4G10 and PY20) provide confirmation of phosphotyrosine incorporation in cases where no SH2 domains bound. Staining of the peptides with BPB provides a control for completed peptide synthesis with BPB stain intensity varying according to the acidity of the peptide. Arrays were probed with GST alone to reveal non-selective binding, and peptides binding GST above mean intensity were discarded. C, two independently synthesized arrays probed with independently purified GST-Sh2d1b SH2 reveal reproducible patterns of binding. A direct comparison of measured intensities over all peptides indicates highly similar relative values with r2 = 0.866. Peptides greater than 3× mean are enclosed in the gray box. D, internal reproducibility analysis of our SH2 domain panel is analyzed by binding to Tyr-409 of p62Dok1 (ATDDpYAVPPPR), corresponding to peptide (Pep) 2 (position A2) and peptide 191 (position H23), reveals a correlation coefficient of 0.973. E, the equilibrium dissociation constants (Kd) of 21 array-positive and 20 array-negative interactions were measured by fluorescence polarization in solution. The mean Kd (black line) for array-positive interactions (>3× mean, n = 21) was 2.39 μm. Non-binders (less than 1× mean, n = 20) had an average affinity greater than 30 μm.