Top-down proteomic characterization of MEK/ERK in MAPK-driven cancers - 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.
