Handling large data sets exemplified by protease recognition of high-density peptide microarrays A peptide microarray containing a total of .100,000 peptides (49,838 of which were unique) was digested with the protease trypsin. The peptide sequences had been synthesized using the theme Ac-GAGAXXXXXGAGA, where Ac-is acetyl blocking the peptide alpha-amino group prior to digestion, and X represents amino acids chosen randomly from the 20 natural amino acids (except lysine, as this residue contains an epsilon-amino group, which even without digestion would be detectable (see Materials and Methods for details)). As a result, free amino groups can only be expressed by trypsin cleaved peptides, which then can then be labeled with Dylight549 and quantitated by fluorescence microscopy. A fluorescence microscopy picture of such a digested and stained peptide microarray (Figure 5a ) demonstrates both the resolution of the photolithographic peptide synthesis strategy and the dynamic range of the free amino group detection strategy. The resulting data was log-transformed and rescaled to obtain a data distribution covering the spectrum between 0 and 1, which -along with the corresponding peptide sequences encoded as Blosum scores without flanking regionswere used to train an NNAlign method. Training was done with a motif length of 5, a fixed number of 3 hidden neurons, 5-fold exhaustive validation, and stopping at the best test set performance. The prediction method yielded a Pearson correlation between measured values and predictions of r = 0.971, a Spearman correlation of r = 0.910, and receiver operating characteristics (ROC) area under the curve (AUC) of 0.997 (using The fundamental pattern appears in all these networks, but they place the anchors at different position of the core. e) shows the core of the 20 networks ensemble without offset correction; in f) offset correction was used to realign the logos to a common register. doi:10.1371/journal.pone.0026781.g003 a target threshold of t = 0.5). The very high performance measures of the resulting NNAlign method demonstrate both that the recorded peptide digestion data contains a consistent and intelligible signal, and that the NNAlign method is capable of deciphering and predicting this extraordinary large number of sequence-dependent peptide signals. The correlation scatterplot feature of the NNAlign web-server output, which compares predicted vs. observed values, further supports the validity of both the peptide microarray and of the NNAlign method. The correlation scatterplot for the trypsin digestion data reveals two major populations of peptides, one composed of non-degradable, non-predicted peptides and one containing weakly to strongly degradable, predicted peptides ( Figure 5b) . Few (0.7%) of the former peptides contained Arginine, whereas most (97.1%) of latter peptides contained Arginine. This is exactly what one would have expected from a peptide digestion with trypsin, which is known to cleave at the C-terminal side of amino acids Arginine (and Lysine, which has been excluded here, see above) [34] . For illustration purposes, Figure 5b includes a color-enhanced visualization of certain dipeptide sequences (note, this is not a standard feature of the NNAlign server) showing that RP sequences are resistant, RA sequences are quite susceptible, and RR sequences appear extremely susceptible to trypsin digestion. Thus, the known trypsin resistance of RP sequences is both demonstrated by the peptide microarray and subsequently captured by the NNAlign method. Note, that both the peptide microarray and the NNAlign generate a continuous set of measurements and predictions showing that trypsin cleavage involves a more complex interaction than a simple recognition solely of an Arginine residue (and by inference a Lysine residue), which would have resulted in a cleaved/non-cleaved classification [35] . It is also important to note that the detection strategy employed here does not reveal where the protease cleavage has occurred, but merely that the protease has recognized the peptide as a substrate and cleaved it somewhere.