Array assembly, printing method, topographic normalization, and example slide. (A) Each letter represents a group of specimens. Replicates of the same patient sample were printed in area “A” and in a reversed orientation in area “B.” As controls, 18 cell lines (MDA-MD-231, MDA-MD-231 stimulated with IGF, MDA-MD-468, MDA-MD-468 stimulated with EGF, Hela HL-60, Jurkat [× 2 from separate sources], Jurkat incubated with anti-FAS antibody, K562, Kasumi-1, MV4-11, NB4, OCIAML3, Raji, THP-1, U937, Y79) were printed in area “C” and 18 normal peripheral blood samples in area “N.” Purified peptides (n = 138) were printed encircling the patient samples, as shown by the purple band. (B) Schematic showing the dilution series of patient samples. At the end of each row of patient samples, a positive control made of a mixture of 11 cell lines (Hela, HL60, Jurkat, Kasumi1, K562, KG1, MV4-11, OCI-AML3 NB4, U937, Y79) (pooled control, shown as a red “+”) or negative control (protein lysis buffer, shown as a “−”) is printed, creating a grid across the whole slide for negative and positive controls to permit topographic normalization. (C) Representative slide of phospho-AKT (Thr308). The overlays in purple, red, and green covers the areas where the purified peptides, cell lines, and normal peripheral blood samples and positive and negative control are located as depicted in Figure 1A. The insert demonstrates phosphorylated and unphosphorylated AKT peptide specifically detected by the antiphospho-AKT (Thr308) antibody. (D,E) The 3-dimensional (D) topographic map of the negative control generates a 3-dimensional topographic map that can be used to correct for background, whereas the 3-dimensional grid of the positive controls sets the scale for quantification. A topographic map from a slide (probed for BAK) with low background that is even across the slide (D) and another (E) from the slide (probed for MCL1) with the most background variation across the slide are shown.