One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Many laboratories, however, forgo the challenging mass analysis of intact proteins. Instead, these researchers resort to well-established methods for proteolytic digests. In this work, the Consortium for Top-Down Proteomics (CTDP) addresses the unmet need for robust protocols for obtaining intact protein mass spectra. We quantify and rationalize the signal suppression associated with common interfering substances. A decision tree, based upon sample composition and experimental goals, guides researchers to a given protocol. Cross-platform analytical benchmarks are provided to allow users to gauge their proficiency and to promote method standardization. This work should permit users at any experience level to obtain high-quality intact mass spectra of native and denatured proteins from mixtures of varying complexity, including intact antibodies and membrane proteins.
Revised: September 30, 2019 |
Published: July 1, 2019
Citation
Donnelly D.P., C.M. Rawlins, C.J. Dehart, L. Fornelli, L.F. Schachner, Z. Lin, and J. Lippens, et al. 2019.Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.Nature Methods 16, no. 7:587-594.PNNL-SA-141748.doi:10.1038/s41592-019-0457-0