Fig 1.
A schematic of the toolbox organization and analytic tools are shown. The three major components are the Library, Server, and Client.
Fig 2.
Data grouping and outlier detection.
A) shows the sample list screen under the Data tab. Drop down menu allows for selecting samples to be analyzed as particular groups. The data levels can be selected using the links at the bottom with corresponding descriptions. B) shows the Data Visualization tool under the Analyses Available tab. The peptide ENOG_37_49 has been selected and peptide information, cycle number, exposure, data measurements and curve fits are shown. Red dots on the fitted curve indicate spots that were auto-flagged as outliers using the stringent cutoffs described in the Results section.
Fig 3.
Background correction of a single array.
One array image was chosen for visualization. Panel A and C represent the background before normalization. Panel A shows the background values plotted against position. Panel C shows the spot intensity versus background intensity and has a correlation of 0.9642 (p-val: <0.0001). Panels B and D represent the background after normalization. Panel B is background plotted against position. Panel D is spot intensity versus background intensity and has a correlation of 0.2272 (p-val: 0.0062). These figures can be generated for every image presented here at http://bit.kinomecore.com/?fig3.
Fig 4.
Measurement reproducibility for kinetic parameterization.
In panels A-C, kinetic curves created by images series of varying exposure times (vi,e) are compared to the more robust vi,·. The data presented is 144 peptides for all 12 samples discussed in Methods. Panel A is a 10 ms exposure time and has a Pearson's r = 0.9981 and a Spearman's ρ = 0.9097. Panel B is a 50 ms exposure time and has a Pearson's r = 0.9989 and a Spearman's ρ = 0.9920. Panel C is a 200 ms exposure time and has a Pearson's r = 0.9807 and a Spearman's ρ = 0.9995. Panels D compares ls(vi,50 (s), vi,50,b(b)) to ls(vi,· (s), vi,·,b(b)) and has a Pearson's r = 0.9887 and a Spearman's ρ = 0.9279. Panels E compares to
and has a Pearson's r = 0.9941; Spearman's ρ = 0.9681. These figures and all similar variations can be further investigated and explored at http://bit.kinomecore.com/?fig4a for b and at http://bit.kinomecore.com/?fig4b for
.
Table 1.
Reproducibility of background corrected technical replicates.
The correlation of the parameters for 144 peptides across 4 sets of 3 technical replicates (3,456 points). These and similar comparisons can be visualized for background normalized data: http://bit.kinomecore.com/?tab1a and for non-normalized: http://bit.kinomecore.com/?tab1b.
Table 2.
List of the top 5 differentially phosphorylated peptides as determined by one-way ANOVA f-statistic.
Ranks are indicated in parenthesis. These values and many more can be visually inspected at http://bit.kinomecore.com/?tab2.
Fig 5.
2-way hierarchal clustering of technical replicates as visualized by heatmap.
Panel A is based on and panel B is based on
. The cluster diagram does not represent the distance between branches, only the branch points of a hierarchal clustering model based on Euclidean distance with average-linkage clustering. Yellow is the highest signal peptides, blue is the lowest. The peptide order varies between the two panels and are based on an identical hierarchal clustering. These figures and several more can be inspected at http://bit.kinomecore.com/?fig5.