Standard procedures for native CZE-MS of proteins and protein complexes up to 800 kDa

Kevin Jooß, John P. McGee, Rafael D. Melani, Neil L. Kelleher

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Native mass spectrometry (nMS) is a rapidly growing method for the characterization of large proteins and protein complexes, preserving “native” non-covalent inter- and intramolecular interactions. Direct infusion of purified analytes into a mass spectrometer represents the standard approach for conducting nMS experiments. Alternatively, CZE can be performed under native conditions, providing high separation performance while consuming trace amounts of sample material. Here, we provide standard operating procedures for acquiring high-quality data using CZE in native mode coupled online to various Orbitrap mass spectrometers via a commercial sheathless interface, covering a wide range of analytes from 30–800 kDa. Using a standard protein mix, the influence of various CZE method parameters were evaluated, such as BGE/conductive liquid composition and separation voltage. Additionally, a universal approach for the optimization of fragmentation settings in the context of protein subunit and metalloenzyme characterization is discussed in detail for model analytes. A short section is dedicated to troubleshooting of the nCZE-MS setup. This study is aimed to help normalize nCZE-MS practices to enhance the CE community and provide a resource for the production of reproducible and high-quality data.
Original languageEnglish
Pages (from-to)1050-1059
JournalElectrophoresis
Volume42
Issue number9-10
DOIs
Publication statusPublished - 1 May 2021
Externally publishedYes

Funding

This work was supported by the National Institute of General Medical Sciences P41 GM108569 for the National Resource for Translational and Developmental Proteomics at Northwestern University, and the instrumentation award (S10OD025194) from NIH Office of Director. We thank SCIEX for their support including Dr. Fang Wang for the valuable discussions and insightful suggestions throughout this research.

FundersFunder number
National Institutes of Health
NIH Office of the DirectorS10OD025194
National Institute of General Medical SciencesP41 GM108569
Northwestern University

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