Three observations were made from the analysis of different window sizes. First, as the window size increases, the computational time used for the normalization also increases. Second, no obvious spatial artifacts were left after the normalization with any of the window sizes tested ( Figure  5b ). Third, a small window size diminishes any signal inequality that exists between positive signals and background noise. Indeed, a small scaling window tends to introduce extreme changes to the original signals and, therefore, increases the discrepancy between the duplicate spots of the same protein. The variance of the signals for the same protein after normalization with different window size was calculated. In five out of the six cases (three dilutions of two proteins) the scaling window 9 can successfully reduce the signal variance in a range from 31% to 90% (Figure 5c ). Decrease of signal variation suggests that a large scaling window will help to reduce spatial artifacts. Although larger window sizes are possible, 9 was used as the default number for ProCAT because the analysis can be done in a reasonable time and minimal improvement has been achieved after window size 7 (Additional data file 1).