In the analysis of the phosphorylome dataset [6] , we applied the neighborhood background correction and observed a high sensitivity in identifying positive targets. To further characterize the effects of neighborhood background correction, we performed a test kinase assay with 100 nM protein kinase A (PKA) spotted at 96 locations on one slide (Figure 2a) . Each of the 48 blocks on the slide contains two PKA pairs with random yeast proteins spotted elsewhere (approximately 12,000 spots). After incubating the slide with 33 P-γ-ATP, all of the PKA spots autophosphorylated and showed strong signals, and in many cases the signal went beyond the grid circle boundaries (Figure 2b ). We then applied the neighborhood background correction to the PKA spots. As expected, the median for PKA signal intensities was enhanced by 53%. Furthermore, the PKA signals from different positions are more similar to each other; the variance within them is decreased by 41% (p value = 0.006; Fig 2d) . Therefore, the neighborhood method for accessing background provides more robust measurements than that of the adjacent background method.