TY - JOUR
T1 - Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry
AU - Drown, Bryon S.
AU - Gupta, Raveena
AU - McGee, John P.
AU - Hollas, Michael A.R.
AU - Hergenrother, Paul J.
AU - Kafader, Jared O.
AU - Kelleher, Neil L.
N1 - Publisher Copyright:
© 2024 American Chemical Society.
PY - 2024/3/19
Y1 - 2024/3/19
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85187521575&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85187521575&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.3c04758
DO - 10.1021/acs.analchem.3c04758
M3 - Article
C2 - 38458998
AN - SCOPUS:85187521575
SN - 0003-2700
VL - 96
SP - 4455
EP - 4462
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 11
ER -