Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry

Area of Science:

• Biochemistry
• Proteomics
• Cell Signaling

Background:

• Proteins with multiple post-translational modifications (PTMs) have functions modulated by their specific modification patterns.
• Precise characterization of these complex proteoforms is analytically challenging.
• MEK1 (MAP2K1) is a key kinase in the mitogen-activating protein kinase (MAPK) pathway, regulating cell signaling.

Purpose of the Study:

• To develop and apply a mass spectrometry-based method for comprehensive characterization of MEK1 phosphoproteoforms.
• To map the landscape of MEK1 phosphorylation states in a relevant biological context.
• To provide a "bird's eye" view of pathway signaling activity through proteoform analysis.

Main Methods:

• Utilized individual ion mass spectrometry, a charge-detection method, for top-down analysis of intact protein ions.
• Applied fragmentation techniques to analyze labile modifications on MEK1 proteoforms.
• Quantified the stoichiometry and distribution of MEK1 phosphorylations in a cellular model.

Main Results:

• Demonstrated reproducible handling of complex phosphoproteoform mixtures and their fragment ions.
• Successfully mapped the proteoform landscape of MEK1, revealing the distribution of 0-4 phosphorylations.
• Determined MEK1 phosphorylation patterns in a cellular model of drug-resistant metastatic melanoma.

Conclusions:

• Individual ion mass spectrometry enables detailed characterization of multiply modified proteoforms.
• This approach provides insights into signaling pathway dynamics by mapping proteoform landscapes.
• The method is generalizable to other complex proteoforms where PTM combinations are critical for function and drug response.