posted on 2024-03-09, 04:03authored byBryon
S. Drown, Raveena Gupta, John P. McGee, Michael A. R. Hollas, Paul J. Hergenrother, Jared O. Kafader, Neil L. Kelleher
The
functions of proteins bearing multiple post-translational modifications
(PTMs) are modulated by their modification patterns, yet precise characterization
of them is difficult. MEK1 (also known as MAP2K1) is one such example
that acts as a gatekeeper of the mitogen-activating protein kinase
(MAPK) pathway and propagates signals via phosphorylation by upstream
kinases. In principle, top-down mass spectrometry can precisely characterize
whole MEK1 proteoforms, but fragmentation methods that would enable
the site-specific characterization of labile modifications on 43 kDa
protein ions result in overly dense tandem mass spectra. By using
the charge-detection method called individual ion mass spectrometry,
we demonstrate how complex mixtures of phosphoproteoforms and their
fragment ions can be reproducibly handled to provide a “bird’s
eye” view of signaling activity through mapping proteoform
landscapes in a pathway. Using this approach, the overall stoichiometry
and distribution of 0–4 phosphorylations on MEK1 was determined
in a cellular model of drug-resistant metastatic melanoma. This approach
can be generalized to other multiply modified proteoforms, for which
PTM combinations are key to their function and drug action.