Emerging proteomic technologies for elucidating context‐dependent cellular signaling events: A big challenge of tiny proportions

Aberrant cell signaling events either drive or compensate for nearly all pathologies. A thorough description and quantification of maladaptive signaling flux in disease is a critical step in drug development, and complex proteomic approaches can provide valuable mechanistic insights. Traditional proteomics‐based signaling analyses rely heavily on in vitro cellular monoculture. The characterization of these simplified systems generates a rich understanding of the basic components and complex interactions of many signaling networks, but they cannot capture the full complexity of the microenvironments in which pathologies are ultimately made manifest. Unfortunately, techniques that can directly interrogate signaling in situ often yield mass‐limited starting materials that are incompatible with traditional proteomics workflows. This review provides an overview of established and emerging techniques that are applicable to context‐dependent proteomics. Analytical approaches are illustrated through recent proteomics‐based studies in which selective sample acquisition strategies preserve context‐dependent information, and where the challenge of minimal starting material is met by optimized sensitivity and coverage. This review is organized into three major technological themes: (i) LC methods in line with MS; (ii) antibody‐based approaches; (iii) MS imaging with a discussion of data integration and systems modeling. Finally, we conclude with future perspectives and implications of context‐dependent proteomics.