Top-down mass spectrometry of native proteoforms and their complexes: a community study

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Native electrospray ionization (ESI) coupled with top-down fragmentation in mass spectrometry (MS) enables simultaneous analysis of noncovalent complex stoichiometry and identification of proteoforms.
• Current native MS and top-down MS methods lack standardization, limiting their widespread adoption and reproducibility in research laboratories.
• A limited number of research groups regularly perform these complex analyses, hindering broader scientific community engagement.

Purpose of the Study:

• To develop and rigorously test standardized protocols for native MS combined with top-down fragmentation.
• To establish robust benchmarks for analyzing intact proteins and protein complexes using this integrated MS approach.
• To provide a valuable and accessible entry point for researchers new to native MS and top-down proteomics.

Main Methods:

• A multi-laboratory study involving 11 instruments across nine institutions was conducted.
• Protocols for native MS and subsequent top-down fragmentation of proteins and protein complexes were systematically developed and evaluated.
• Data generated from diverse instruments were compared to identify commonalities and variations in protocol performance.

Main Results:

• The study successfully generated a summary of outcomes from the protocol testing across multiple sites and instruments.
• Key performance metrics and best practices for the combined native MS and top-down fragmentation workflow were identified.
• The results provide a foundation for reproducible analysis of intact protein complexes and their constituents.

Conclusions:

• Standardized protocols for native MS coupled with top-down fragmentation are feasible and beneficial for the proteomics community.
• The established benchmarks will facilitate the adoption and reliable application of these powerful analytical techniques.
• This work represents a significant step towards democratizing advanced top-down proteomics for studying complex biological systems.