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Mixed-bed chromatography is far from being a well-established technology within the panoply of bioseparation tools. Composed of an assembly of distinct sorbents that are mixed in a single bed, they have been mostly developed in the last decade for the reduction of dynamic concentration range where they allowed discovering many low-copy proteins within very complex proteomes. Other interesting preparative applications of mixed-bed chromatography have since been developed. In this chapter the basic concepts first and then detailed application recipes are described for (1) the reduction of protein dynamic concentration range, (2) the removal of impurity traces at the last stage of a biopurification process, and (3) the selection and use of sorbents as mixed bed in protein purification.
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http://dx.doi.org/10.1007/978-1-4939-2447-9_12 | DOI Listing |
Anal Chem
July 2025
Department of Chemistry, University of Wisconsin─Madison, 1101 University Ave., Madison, Wisconsin 53706, United States.
Protein complexes are essential for virtually all biological processes, yet their structural characterization remains a major challenge due to their heterogeneous, dynamic nature and the complexity of the proteome. Native top-down mass spectrometry (nTDMS) has emerged as a powerful tool for comprehensive structural characterization of purified protein complexes, but its application to endogenous protein complexes in the proteome is challenging and typically requires labor-intensive and time-consuming prefractionation. Here, for the first time, we develop a nondenaturing online two-dimensional liquid chromatography (2D-LC) method for native top-down proteomics (nTDP), enabling high-throughput structural analysis of endogenous protein complexes.
View Article and Find Full Text PDFAnal Chem
June 2025
Analytical Chemistry Group, Van't Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam, Science Park 904, Amsterdam 1098 XH, The Netherlands.
Native size-exclusion chromatography (SEC) coupled with native mass spectrometry (nMS) enables the characterization of proteins and protein complexes by combining liquid-phase separation (SEC) and mass measurement (nMS). This approach allows for an increase in the throughput of nMS experiments, reduces the bias that may be present due to the co-ionization of oligomers, and facilitates online sample buffer exchange. Conventional SEC-nMS uses volatile buffers and relatively wide-diameter columns (e.
View Article and Find Full Text PDFProtein complexes are essential for virtually all biological processes, yet their structural characterization remains a major challenge due to their heterogeneous, dynamic nature and the complexity of the proteome. Native top-down mass spectrometry (nTDMS) has emerged as a powerful tool for comprehensive structural characterization of purified protein complexes, but its application to endogenous protein complexes in the proteome is challenging and typically requires labor-intensive and time-consuming prefractionation. Here, for the first time, we develop a nondenaturing online two-dimensional liquid chromatography (2D-LC) method for native top-down proteomics (nTDP), enabling high-throughput structural analysis of endogenous protein complexes.
View Article and Find Full Text PDFJ Proteome Res
July 2024
Department of Chemistry, University of Wisconsin - Madison, 1101 University Ave., Madison, Wisconsin 53706, United States.
Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions.
View Article and Find Full Text PDFJ Chromatogr A
August 2024
CryoBioPhysica, Inc., 4620 N. Park Ave., #1502 w Chevy Chase, MD 20815, USA. Electronic address:
In previous publications we have described the pISep dual simultaneous, independent gradients (DSIGs) liquid chromatography (LC) for uncoupling gradients of non-buffering solute (NaCl, urea or acetonitrile) from externally generated pH gradients. In DSIGs the shape and slope of the [salute] gradient does not depend on the shape and slope of the pH gradient. The technique allows in a single run true simultaneous two dimensional LC separation of complex protein mixtures on various stationary phases including anion, cation exchangers (AEX, CEX), reversed phase (RP), mixed mode and mixed bed.
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