skip to main content

Title: Recent advances (2019–2021) of capillary electrophoresis‐mass spectrometry for multilevel proteomics
Multilevel proteomics aims to delineate proteins at the peptide (bottom-up proteomics), proteoform (top-down proteomics), and protein complex (native proteomics) levels. Capillary electrophoresis-mass spectrometry (CE-MS) can achieve highly efficient separation and highly sensitive detection of complex mixtures of peptides, proteoforms, and even protein complexes because of its substantial technical progress. CE-MS has become a valuable alternative to the routinely used liquid chromatography-mass spectrometry for multilevel proteomics. This review summarizes the most recent (2019-2021) advances of CE-MS for multilevel proteomics regarding technological progress and biological applications. We also provide brief perspectives on CE-MS for multilevel proteomics at the end, highlighting some future directions and potential challenges.  more » « less
Award ID(s):
Author(s) / Creator(s):
; ; ; ; ; ; ;
Date Published:
Journal Name:
Mass Spectrometry Reviews
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    Protein O -GlcNAcylation refers to the covalent binding of a single N -acetylglucosamine (GlcNAc) to the serine or threonine residue. This modification primarily occurs on proteins in the nucleus and the cytosol, and plays critical roles in many cellular events, including regulation of gene expression and signal transduction. Aberrant protein O -GlcNAcylation is directly related to human diseases such as cancers, diabetes and neurodegenerative diseases. In the past decades, considerable progress has been made for global and site-specific analysis of O -GlcNAcylation in complex biological samples using mass spectrometry (MS)-based proteomics. In this review, we summarized previous efforts on comprehensive investigation of protein O -GlcNAcylation by MS. Specifically, the review is focused on methods for enriching and site-specifically mapping O -GlcNAcylated peptides, and applications for quantifying protein O -GlcNAcylation in different biological systems. As O -GlcNAcylation is an important protein modification for cell survival, effective methods are essential for advancing our understanding of glycoprotein functions and cellular events. 
    more » « less
  2. null (Ed.)
    Mass spectrometry (MS)-based top-down proteomics (TDP) requires high-resolution separation of proteoforms before electrospray ionization (ESI)-MS and tandem mass spectrometry (MS/MS). Capillary isoelectric focusing (cIEF)-ESI-MS and MS/MS could be an ideal method for TDP because cIEF can enable separation of proteoforms based on their isoelectric points (pIs) with ultra-high resolution. cIEF-ESI-MS has been well-recognized for protein characterization since 1990s. However, the widespread adoption of cIEF-MS for the characterization of proteoforms had been impeded by several technical challenges, including the lack of highly sensitive and robust ESI interface for coupling cIEF to MS, ESI suppression of analytes from ampholytes, and the requirement of manual operations. In this mini review, we summarize the technical improvements of cIEF-ESI-MS for characterizing proteoforms and highlight some recent applications to hydrophobic proteins, urinary albumin variants, charge variants of monoclonal antibodies, and large-scale TDP of complex proteomes. 
    more » « less
  3. Recent advances in mass spectrometry (MS)-based proteomics have enabled tremendous progress in the understanding of cellular mechanisms, disease progression, and the relationship between genotype and phenotype. Though many popular bioinformatics methods in proteomics are derived from other omics studies, novel analysis strategies are required to deal with the unique characteristics of proteomics data. In this review, we discuss the current developments in the bioinformatics methods used in proteomics and how they facilitate the mechanistic understanding of biological processes. We first introduce bioinformatics software and tools designed for mass spectrometry-based protein identification and quantification, and then we review the different statistical and machine learning methods that have been developed to perform comprehensive analysis in proteomics studies. We conclude with a discussion of how quantitative protein data can be used to reconstruct protein interactions and signaling networks. 
    more » « less
  4. null (Ed.)
    Mass spectrometry (MS)-based proteomics has enabled the identification and quantification of thousands of proteins from complex proteomes in a single experiment. However, its performance for mass-limited proteome samples ( e.g. , single cells and tissue samples from laser capture microdissection) is still not satisfying. The development of novel proteomic methodologies with better overall sensitivity is vital. During the last several years, substantial technical progress has been achieved for the preparation and liquid-phase separation-MS characterization of mass-limited proteome samples. In this review, we summarize recent technological progress of sample preparation, liquid chromatography (LC)-MS, capillary zone electrophoresis (CZE)-MS and MS instrumentation for bottom-up proteomics of trace biological samples, highlight some exciting applications of the novel techniques for single-cell proteomics, and provide a very brief perspective about the field at the end. 
    more » « less
  5. Abstract

    Mass spectrometry (MS)‐based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high‐throughput proteomics. Herein, we report a high‐throughput bottom‐up proteomic method enabled by a newly developed MS‐compatible photocleavable surfactant, 4‐hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS‐analysis following UV degradation. Moreover, we developed an Azo‐aided bottom‐up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an “all‐in‐one” MS‐compatible surfactant for both top‐down and bottom‐up proteomics, with streamlined workflows for high‐throughput proteomics amenable to clinical applications.

    more » « less