Supplementary Figure 3: The phosphorylation activities of kinases measured with biological peptides derived from phosphorylatable substrate protein regions, are significantly higher than activity levels detected with control peptides.

(a) Activity profiles of recombinant kinases measured in presence of their respective biological peptide subsets. Individual peptide-phosphorylation activities measured with kinases’ biological peptides are significantly greater than activities measured with the 63-reference peptide set (27.2%: p≤0.01; 66.7%: p≤0.05), and >94% are greater than activities measured with the 5-random peptide set. The multi-peptide activity profiles of kinases measured with kinases’ biological peptide subsets are significantly different from profiles measured with the control set of 63-reference peptides (p<7E-11 for AKT1, AKT3, MAPK1/ERK2, FYNA, HCK, LYNA, ABL1, and JAK2; p<4E-2 for AKT2, MAPK14/p38a, BLK, BRK, LCK, LYNB, SRC, HER2, HER4; Supplementary Table 9). (b) Pairwise comparison of differences in levels of kinase activities measured in presence of biological peptides versus modified counterparts (results derived from all experimental readouts). (c-d) Overall comparison of activity profiles of Tyrosine Kinases or Serine/Threonine Kinases measured in presence of any of their predicted Y- or S/T- containing biological peptides, versus any Y- or S/T- free biological or reference peptides (see Supplementary Tables 11-12). Results in (a-d) show that kinases are significantly more capable of phosphorylating a vast majority of their biological peptides than control reference peptides, including mutated or pre-phosphorylated biological peptides (box plot length: 25% and 75% of data, center line: median, whiskers 25% - (or 75% +) 1.5 x IQR). As well, since (i) AUCs of kinase activity profiles calculated in Figs. 1d, 2d and Supplementary Fig. 2f, j show that kinases’ subsets of biological peptides systematically provide excellent specificity/sensitivity to identify kinases; and since (ii) computational analyses in Fig. 1e and Supplementary Fig. 2g, h show that the majority of peptides most capable of predictably discerning kinases’ unique and differential phospho-catalytic activities are biological peptides; and since (iii) our strategy utilizes peptide collections that inherently encompass a wide spectrum of biological peptide sensors that correspond to distinct peptide combinations specific to each individual kinase (owing kinases’ essentially exclusive pools of substrate proteins and phospho-target sites; as defined in PhosphoAtlas ³⁰ (http://cancer.ucsf.edu/phosphoatlas) and represented in Supplementary Fig. 1b); then our findings collectively establish that libraries of biological peptides and control peptides are effective combinatorial sensors to functionally differentiate kinases from each other, and that the biological peptide subsets of kinases can reasonably be used to measure kinases’ enzymatic activity.