Abstract The MAPK signaling pathway is a central element of normal development and growth and is a common driver in many cancer types. Although typically described solely by their canonical phosphorylation sites, the downstream components of the MAPK pathway, including MEK1/2 and ERK1/2, also integrate signals from other inputs. This behavior is especially important in elucidating the mechanisms of intrinsic and acquired resistance to drug therapies that target the MAPK pathway. Despite the clear importance of signal integration, almost nothing is known about the combinations of post-translational modifications (PTMs) present on MEK1/2 and ERK1/2 in cancer, particularly in response to drug treatment. These PTM combinations are not effectively detected by standard bottom-up proteomic approaches due to proteolytic digestion and resulting protein inference problem. Here, we propose utilizing an alternative approach comprising immunoprecipitation and subsequent top-down mass spectrometry (IP-TDMS), in which the protein of interest is enriched, ionized, and fragmented to provide precise PTM characterization of intact modified protein forms, or proteoforms. This IP-TDMS approach will be initially executed in cell culture models of resistant metastatic melanoma and colorectal carcinoma. In order to determine if these proteoforms are unique to cancer type and drug responsiveness, IP-TDMS will next be translated to patient derived xenografts. Findings from this study are anticipated to provide considerable insight into how tumors can overcome addiction to MAPK signaling and potential new drug development leads. The proposed work provides an excellent training opportunity in mass spectrometry and proteomics, and the Kelleher laboratory is the ideal environment for this training. The Kelleher group has worked at the forefront of top-down mass spectrometry for the last decade and has extensive knowledge and capability in this area. Having received training in chemical biology and organic chemistry in graduate school, I have constructed a detailed training plan in close collaboration with Prof. Kelleher to rapidly build expertise in this new field. The Kelleher group has a highly structured training program for with the advanced instrumentation in the lab that will establish mass spectrometry as a core expertise. This training program will afford me with the capacity to acquire and analyze targeted proteomics data, a critical skill set for establishing my independent career. Northwestern University features state-of-the-art facilities and many collaborative opportunities with world renowned scientists. We are highly optimistic of the prospect of this combined research proposal and training plan given the intra- laboratory and institutional support for this work.