Temporal Phosphoproteomic Network Reconstruction of Condition-Dependent EGFR Signaling Routes Through Unmeasured Proteins

Time-resolved phosphoproteomics captures fast signaling dynamics, but proteins that show no phosphorylation change, or escape detection, leave gaps the data cannot bridge. TS-BFS-BS uses the temporal order of a phosphoproteomic time course to guide a beam-pruned breadth-first search over STRING v12.5 functional, physical, and regulatory priors, returning temporally continuous routes that can pass through unmeasured bridge proteins. In EGF time courses from MDA-MB-468, EGFR Flp-In HEK293T and HeLa cells, endpoints and short routes were stable across search settings, and endpoints were largely shared across STRING priors, whereas long routes depended on settings and prior. When measured hubs CBL and SRC were removed, both were recovered as bridges and 94-99% of endpoints were retained. Incorrect temporal orders gave endpoint Jaccard indices of only 0.23-0.33 with the correct order, comparable to SHP2 inhibition (0.25-0.35), under which PTPN11 was excluded from all routes by its measured pretreatment response and returned after washout. TS-BFS-BS generates time-resolved, testable signaling hypotheses for iterative refinement.