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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


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 (US)
Pages (from-to)1050-1059
Number of pages10
Issue number9-10
StatePublished - May 2021


  • CESI
  • Native mass spectrometry
  • Native top-down
  • Sheathless ionization
  • Standard operating procedure

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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