Recent advances (2019-2021) of capillary electrophoresis-mass spectrometry for multilevel proteomics

Abstract

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.

Keywords: capillary isoelectric focusing-mass spectrometry; capillary zone electrophoresis-mass spectrometry; multilevel proteomics; protein complex; proteoform.

Figure 1.

Schematic about the differences of BUP, TDP, and native proteomics regarding proteoform and protein complex characterizations.

Figure 2.

Summary of the history of CZE-MS (A) and cIEF-MS (B) for multi-level proteomics.

Figure 3.

Schematic designs of the electrokinetically pumped sheath flow CE-MS interface (A) and its three different generations (B). reproduced from Sun et al. (2015) with permission from American Chemical Society, copyright (2015).

Figure 4.

Schematic designs of the CZE-MS system with the dynamic pH barrage junction stacking and the flow through microvial CE-MS interface (A) and the basic principle of the dynamic pH barrage junction stacking using a PEI-coated separation capillary (B). Reproduced from Wang et al. (2020) with permission from John Wiley and Sons, copyright (2020).

Figure 5.

(A) Schematic of the automated cIEF-MS system based on the “sandwich” injection approach. (B) Base peak electropherograms of an E. coli lysate after triplicate analyses by the high-throughput cIEF-MS/MS. (C) Base peak electropherograms of an E. coli lysate after analyses by automated cIEF-MS/MS using an 80-cm LPA-coated capillary and 0.1% FA as the anolyte (high-throughput, red), a 150-cm LPA-coated capillary and 0.1% FA as the anolyte (blue), and a 150-cm LPA-coated capillary and 5% AA as the anolyte (high-capacity, dark cyan). Reproduced from Xu et al. (2020) with permission from American Chemical Society, copyright (2020).

Figure 6.

(A) Hierarchical-cluster-analysis heat map of quantified proteins across blastomeres from 16-cell, 32-cell, 64-cell, and 128-cell Xenopus embryos. Blastomeres from the same embryonic stage are grouped together and blastomeres from the 128-cell stage show significantly higher protein abundance heterogeneity compared to that from the 16-cell stage. Protein examples with quantifiable cell heterogeneity in the 16-cell and 128-cell blastomeres are marked with asterisks (*). Reproduced from Lombard-Banek et al. (2019) with permission from American Chemical Society, copyright (2019). (B) Illustration of nanoRPLC-CZE-MS/MS for orthogonal and high-capacity separations of peptides. Eluates from nanoRPLC are collected every several minutes, followed by dynamic pH junction-based CZE-MS/MS analysis.

Figure 7.

Base peak electropherogram of an enriched phosphopeptide sample from a mouse brain digest analyzed by CZE-MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer. A 1-meter-long LPA-coated capillary (50 μm i.d.) and the electrokinetically pumped sheath flow CE-MS interface were used. About 220 ng of enriched phosphopeptides were loaded for the analysis. Reproduced from Zhang et al. (2019) with permission from American Chemical Society, copyright (2019).

Figure 8.

Linear correlation between predicted electrophoretic mobility (μ_ef) and experimental μ_ef of proteoforms without PTMs from E. coli cells identified by CZE-MS/MS. The μ_ef prediction is based on the equation labeled in the figure and the predicted μ_ef relates to the charge (Q) and mass (M) of each proteoform. Reproduced from Chen et al. (2020) with permission from American Chemical Society, copyright (2020).

Figure 9.

CE-MS electropherograms of a fusion protein analyzed by the ZipChip technique with commercially available separation buffer (A), and the optimized separation buffer containing 10% 2-propanol and 0.2% acetic acid (pH 3.2) (B). Reproduced from Deyanova et al. (2021) with permission from John Wiley and Sons, copyright (2021).

Figure 10.

Schematic of the native CZE-MS and MS/MS using UVPD for delineation of E. coli 70S ribosomes. The commercialized electrokinetically pumped sheath flow CE-MS interface (EMASS-II) was used for coupling native CZE to MS. Reproduced from Mehaffey et al. (2020) with permission from American Chemical Society, copyright (2020).