Top-down mass spectrometry (MS), pioneered by Fred McLaffery, is a growing technology for protein structure elucidation and proteoform assessment. Fragmentation of the gas-phase protein (i.e., MS/MS) yields not only primary structure (sequence) information, but also reveals aspects of tertiary and quaternary structure. Dissociation of native protein complexes can uncover solvent accessible regions, sites of post-translational modifications, and ligand binding. Structural information from native top-down MS can be correlated to solution-phase structures. Here we show that protein top-down MS is complementary to other biophysical techniques employed by structural biologists. Moreover, collision-based fragmentation (e.g., collisionally activated dissociation, CAD, and high-energy C-trap dissociation, HCD) coupled with high resolution mass spectrometers (e.g., Fourier-transform ion cyclotron resonance and Orbitrap systems) are complementary to each other in which the sum of the information they return can yield more complete sequencing than each individual method. Internal fragments (i.e., fragments containing neither terminus) increase sequence coverage of proteoforms and gives information on higher order structure. Raw MS/MS spectra can be deconvoluted and uploaded into the ClipsMS computer program for terminal and internal product ion assignment. These aspects will be shown with examples from top-down MS of membrane proteins, metal-binding and cofactor-ligated proteins, and disulfide-bonded proteins, including monoclonal antibodies.

Top-down mass spectrometry: Why shouldn’t everyone be doing it?

Where thousands of chemistry professionals meet to share ideas and advance scientific and technical knowledge. Spring 2023 abstracts, posters, and presentations will launch at the end of March.

ACS Spring 2023

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.