Top-Down Proteomics
Our research focuses on the development of novel analytical methodologies based on CZE-MS/MS for high-resolution and ultra-sensitive proteomics and applications of the new methodologies for answering important questions in biology.
Figure 1. Schematic strategy for creating the Human Proteoform Atlas. Adapted from reference [1].
Top-down proteomics (TDP), aims to define the human proteome in a proteoform-specific manner (human proteoform project) and create an integrated Human Proteoform Atlas, Figure 1. [1,2] It is well known that one human gene can produce many different forms of protein molecules (proteoforms) due to genetic variations, RNA alternative splicing, and protein post-translational modifications (PTMs). Proteoforms from the same gene can have drastically different biological functions. [2] Delineation of proteins in a proteoform-specific manner is vital for revolutionizing our understanding of molecular mechanisms of diseases, developing more reliable approaches for disease diagnosis, and making drugs that are more effective. [3-5] Unfortunately, the mature proteomics approach is blind to proteoforms. The TDP-based human proteoform project requires high-capacity separation and ultrasensitive MS-based detection of complex proteoform mixtures extracted from human cells and tissues. We recently performed deep TDP of colorectal cancer cell lines using CE-MS and identified over 20,000 proteoforms, Figure 2. It is a world record regarding the number of proteoforms identified from human cells and our technique pushed the TDP to a new level.
Figure 2. The abstract overview of our recent work which was accepted in Science Advance.
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(I) Couple multi-dimensional LC to CZE-MS/MS for high-resolution and ultrasensitive proteomics. We employ orthogonal separation techniques to improve the separation of peptides and intact proteins in complex proteomes, boosting the proteome coverage from proteomics. We integrate microscale RPLC (µRPLC) with CZE-MS/MS to improve the sensitivity of proteomics, enabling deep proteomics of mass-limited samples. We collaborate with developmental biologists to apply our techniques for understanding important questions in vertebrate early embryogenesis using zebrafish as a model system. We are particularly interested in two important questions. First, how do proteins and/or their PTMs accurately control the zygotic genome activation at mid-blastula transition? Second, when and how do interblastomere differences arise during early cellular differentiation? We believe quantitative proteomics of zebrafish embryos and blastomeres across multiple developmental stages will provide valuable insight into those questions.
(II) Develop analytical methods for native proteomics. We couple size exclusion chromatography (SEC) to CZE-MS/MS for high-resolution separation of complex proteomes under native conditions. The SEC-CZE-MS/MS will enable large-scale identification and relative quantification of protein complexes directly from complex proteome samples and in discovery mode. We are particularly interested in characterization of protein-metal complexes in cells.
Reference:
[1] Smith L, et al. Preprints. 2020. doi: 10.20944/preprints202010.0368.v1
[2] Smith L, et al. Science. 2018. PMID: 29590032
[3] Ntai I, et al. Proc Natl Acad Sci U S A. 2018. PMID: 29610327
[4] Tucholski T, et al. Proc Natl Acad Sci U S A. 2020. PMID: 32968017
[5] Tran JC, et al. Nature. 2011. PMID: 22037311
[6] Chen D, et al. Mass Spectrom Rev. 2021. PMID: 34128246
[7] Shen X, et al. Trends Analyt Chem. 2019. PMID: 31537953
