Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as a useful tool for top-down proteomics. However, its performance for deep top-down proteomics is still dramatically lower than widely used reversed-phase liquid chromatography (RPLC)-MS/MS. We present an orthogonal multidimensional separation platform that couples size exclusion chromatography (SEC) and RPLC based protein prefractionation to CZE-MS/MS for deep top-down proteomics of Escherichia coli. The platform generated high peak capacity (∼4000) for separation of intact proteins, leading to the identification of 5700 proteoforms from the Escherichia coli proteome. The data represents a 10-fold improvement in the number of proteoform identifications compared with previous CZE-MS/MS studies and represents the largest bacterial top-down proteomics data set reported to date. The performance of the CZE-MS/MS based platform is comparable to the state-of-the-art RPLC-MS/MS based systems in terms of the number of proteoform identifications and the instrument time.
Native proteomics aims to characterize complex proteomes under native conditions and ultimately produces a full picture of endogenous protein complexes in cells. It requires novel analytical platforms for high-resolution and liquid-phase separation of protein complexes prior to native mass spectrometry (MS) and MS/MS. In this work, size-exclusion chromatography (SEC)-capillary zone electrophoresis (CZE)-MS/MS was developed for native proteomics in discovery mode, resulting in the identification of 144 proteins, 672 proteoforms, and 23 protein complexes from the Escherichia coli proteome. The protein complexes include four protein homodimers, 16 protein-metal complexes, two protein-[2Fe-2S] complexes, and one protein-glutamine complex. Half of them have not been reported in the literature. This work represents the first example of online liquid-phase separation-MS/MS for the characterization of a complex proteome under the native condition, offering the proteomics community an efficient and simple platform for native proteomics.
Better peptide separation is required for bottom-up proteomics for further improving the proteome coverage. The two-dimensional liquid chromatography (2D-LC) systems only explore differences among peptides in their hydrophobicity (reversed-phase, RP) and charge (strong cation/anion exchange, SCX/SAX). Alternative separation techniques with different separation mechanisms are required to further improve the separation. Capillary zone electrophoresis (CZE) is an attractive alternative because it has high efficiency for separation of biomolecules and it separates analytes based on their size-to-charge ratios, complementary with LC. However, the low loading capacity and narrow separation window of CZE limit its wide application for large-scale proteomics. In this manuscript, we present an automated CZE-mass spectrometry (MS) system for solving those issues. The CZE-MS system can approach at least half-a-microliter loading capacity with good robustness and reproducibility, can routinely use over 12% of the available sample in the sample vial for analysis, and can generate a 140 min separation window and high peak capacity (∼380) for complex proteome analysis. The results represent the highest peak capacity and the widest separation window of CZE for peptide separation with a microliter-scale loading capacity. It is the first time that CZE-MS approaches both the microliter-scale loading capacity and over 2-hour separation window for analysis of complex samples. The automated CZE-MS system dramatically reduces the gap between CZE-MS and RPLC-MS in terms of loading capacity, separation window and peak capacity. It truly opens the door for large-scale bottom-up proteomics using CZE-MS.
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.capillary isoelectric focusing-mass spectrometry, capillary zone electrophoresis-mass spectrometry, multilevel proteomics, protein complex, proteoform
| INTRODUCTIONProteomics aims to characterize proteins in cells comprehensively as a function of biological conditions, including but not limited to their expression, posttranslational modifications (PTMs), interactions, locations, and turnover (Aebersold & Mann, 2016;Zhang et al., 2013). Mass spectrometry (MS)-based proteomics has become crucial for pursuing better understandings of molecular mechanisms of fundamental cellular processes and disease development (Aebersold & Mann, 2016). There are three main strategies: bottom-up proteomics (BUP), top-down proteomics (TDP), and native proteomics.BUP is the most mature and widely used strategy. It identifies proteins through the MS and tandem MS (MS/MS) measurements of peptides derived from the enzymatic
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