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Protein phosphorylation is catalyzed by kinases to regulate a large variety of cellular activities, including growth and signal transduction. Methods to identify kinase substrates are crucial to fully understand phosphorylation‐mediated cellular events and disease states. Here, we report a set of protocols to identify substrates of a target kinase using Kinase‐catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K‐BILDS). As described in these protocols, K‐BILDS involves inactivation of endogenous kinases in lysates, followed by addition of an active exogenous kinase and the γ‐phosphate‐modified ATP analog ATP‐biotin for kinase‐catalyzed biotinylation of cellular substrates. Avidin enrichment isolates biotinylated substrates of the active kinase, which can be monitored by western blot. Substrates of the target kinase can also be discovered using mass spectrometry analysis. Key advantages of K‐BILDS include compatibility with any lysate, tissue homogenate, or complex mixture of biological relevance and any active kinase of interest. K‐BILDS is a versatile method for studying or discovering substrates of a kinase of interest to characterize biological pathways thoroughly. 

Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase‐catalyzed crosslinking and immunoprecipitation (K‐CLIP). K‐CLIP uses a photocrosslinking γ‐phosphoryl‐modified ATP analog, such as ATP‐arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase‐substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K‐CLIP is an adaptable chemical tool to investigate and discover kinase‐substrate pairs, which will promote characterization of complex phosphorylation‐mediated cell biology. 

This Perspective covers discovery and mechanistic aspects as well as initial applications of novel ioni-zation processes for use in mass spectrometry that guided us in a series of subsequent discoveries, in-strument developments, and commercialization. With all likelihood, vacuum matrix-assisted ionization on an intermediate pressure matrix-assisted laser desorption/ionization source without the use of a laser, high voltages, or any other added energy was the defining turning point from which key developments grew that were at the time unimaginable, and continue to surprise us in its simplistic preeminence, and is therefore a special focus here. We, and others, have demonstrated exceptional analytical utility with-out a complete understanding of the underlying mechanism. Our current research is focused on how best to understand, improve, and use these novel ionization processes through dedicated platform and source developments which convert volatile and nonvolatile compounds from solid or liquid matrices into gas-phase ions for analysis by mass spectrometry using e.g., mass-selected fragmentation and ion mobility spectrometry to provide reproducible, accurate, and sometimes improved mass and drift time resolution. The combination of research and discoveries demonstrated multiple advantages of the new ionization processes and established the basis of the successes that lead to the Biemann Medal and this Perspective. How the new ionization processes relate to traditional ionization is also presented, as well as how these technologies can be utilized in tandem through instrument modification and implementa-tion to increase coverage of complex materials through complementary strengths.