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Development and Implementation of Online Separation Techniques Coupled to Native Mass Spectrometry for Characterization of Endogenous Proteins Extracted from Biological Matrices

EMSL Project ID


Recent developments of state-of-the-art mass spectrometry (MS) instrumentation at EMSL, including the 21T Fourier Transform Ion Cyclotron Resonance (FTICR) MS, have enabled high resolution and high mass accuracy detection of intact proteins and native protein complexes for deep characterization of protein assemblies at the molecular level. However, intact proteins extracted from biological matrices, often have various post-translational modifications (PTMs), and are challenging to study even by mass spectrometers with the highest performance, due to heterogeneity and heavy interference from the chemical background. Yet these PTMs, many of them uncharacterized and not directly encoded in the genome, can play important roles in regulating protein functions. For example, cellulase enzymes produced by microbes are heavily glycosylated in a highly heterogeneous manner. The glycosylation patterns are directly linked to the activity of cellulose degradation. While these proteins can be characterized by EMSL MS instrumentation in their highly purified forms, it is still difficult to detect them effectively from biological matrices with the existing methods. Over the years, various methods for separation of intact proteins and native protein complexes have been developed in the scientific community. However, the existing EMSL proteomics pipelines, despite their robustness, largely rely on a single type of the reversed-phase liquid chromatography to analyze proteins under their denatured states. Therefore, we aim to couple the latest solution-phase separation techniques (liquid chromatography, LC, and capillary electrophoresis, CE) with the cutting-edge MS instrumentation to better serve functional proteomics applications by offering comprehensive characterization of PTMs, ligand binding, and noncovalent interactions on proteins directly extracted from biological matrices. We will work with external collaborators who are experts in this area to implement the new methods and explore for further improvements by testing them on several EMSL user samples (plant and fungal enzymes). The proposed development and implementation of separation methods will create new science capabilities at EMSL to understand biology at the subcellular level in detail.

Project Details

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Principal Investigator

Mowei Zhou
Environmental Molecular Sciences Laboratory

Team Members

Liangliang Sun
Michigan State University

Jesse Wilson
Environmental Molecular Sciences Laboratory

Andrea Gargano
Vrije Universiteit Amsterdam

Roza Wojcik
Pacific Northwest National Laboratory

Jared Shaw
Environmental Molecular Sciences Laboratory

Rui Zhao
Environmental Molecular Sciences Laboratory