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Heart-cut nano-LC-CZE-MS for the characterization of proteins on the intact level

  • Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed-phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well-suited geometries/dimensions. Here, a heart-cut nano-LC-CZE-MS setup was developed utilizing for the first time a mechanical 4-port valve as LC-CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart-cut transfer of individual LC peaks and subsequent CZE-MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower μg/mL range were determined, which are considerably lower compared to traditional CZE-MS. In addition, this study represents the first application of an LC-CE-MS system for intact protein analysis. The nano-LC-CZE-MS system is expected to be applicable to various other analytical challenges.

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Author:Kevin Jooß, Nico Scholz, Jens Meixner, Christian Neusüß
Institutional Author:Kevin Jooß
Institutional Author:Nico Scholz
Institutional Author:Jens Meixner
Institutional Author:Christian Neusüß,
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/30575976
Source Title (English):Electrophoresis
Document Type:Article
Year of Completion:2019
Release Date:2021/05/11
Tag:Chromatography, Reverse-Phase/instrumentation/methods; Electrophoresis, Capillary/instrumentation/methods; Glycosylation; Hydrogen-Ion Concentration; Limit of Detection; Ribonucleases/analysis; Tandem Mass Spectrometry/instrumentation/methods
First Page:1061
Last Page:1065
Relevance:peer reviewed