Large-Scale Qualitative and Quantitative Top-Down Proteomics Using Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry with Nanograms of Proteome Samples

Abstract

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has attracted attention recently for top-down proteomics because it can achieve highly efficient separation and very sensitive detection of proteins. However, separation window and sample loading volume of CZE need to be boosted for a better proteome coverage using CZE-MS/MS. Here, we present an improved CZE-MS/MS system that achieved a 180-min separation window and a 2-μL sample loading volume for top-down characterization of protein mixtures. The system obtained highly efficient separation of proteins with nearly one million theoretical plates for myoglobin and enabled baseline separation of three different proteoforms of myoglobin. The CZE-MS/MS system identified 797±21 proteoforms and 258±7 proteins (n=2) from an Escherichia coli (E. coli) proteome sample in a single run with only 250 ng of proteins injected. The system still identified 449±40 proteoforms and 173±6 proteins (n=2) from the E. coli sample when only 25 ng of proteins were injected per run. Single-shot CZE-MS/MS analyses of zebrafish brain cerebellum (Cb) and optic tectum (Teo) regions identified 1 730±196 proteoforms (n=3) and 2 024±255 proteoforms (n=3), respectively, with only 500-ng proteins loaded per run. Label-free quantitative top-down proteomics of zebrafish brain Cb and Teo regions revealed significant differences between Cb and Teo regarding the proteoform abundance. Over 700 proteoforms from 131 proteins had significantly higher abundance in Cb compared to Teo, and these proteins were highly enriched in several biological processes, including muscle contraction, glycolytic process, and mesenchyme migration.

Recent (2018–2020) development in capillary electrophoresis

“…Such focusing is generally utilized for analysis of peptides and intact proteins by CE-MS, and achieved by generating a pH-induced electrophoretic mobility variation at the sample/pH barrage boundary. It has been successfully applied for the study of the MCF7 breast cancer cell proteome using the bottom-up approach [10], and for top-down proteomic analysis of the zebrafish brain cerebellum (Cb) and optic tectum (Teo) regions [11]. Also, quantitative analysis of bisphosphonate compounds [12] and digested monoclonal antibodies [13] was achieved by this technique.…”

Qualitative and quantitative top-down proteomics of human colorectal cancer cell lines identified 23000 proteoforms and revealed drastic proteoform-level differences between metastatic and non-metastatic cancer cells

“…[18][19][20][21][22] The data clearly demonstrate the power of our CZE-MS/MS-based TDP workflow for comprehensive characterization of proteoforms in complex proteome samples. We attribute the drastic improvement of proteoform IDs to the high separation efficiency of CZE for proteoforms, [23] high sensitivity of CZE-MS for proteoform detection, [23][24][25] and high orthogonality of LC and CZE for biomolecule separations. [21,26] The features of CZE-MS/MS for TDP have been systematically reviewed recently.…”

Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research

“…Quantitative information was based on the relative intensities of the detected glycoforms where glycoform concentrations were estimated to range between 0.35 and 630 nM. In a TDMS study, Lubeckyj et al () demonstrated a label‐free strategy for the quantification of thousands of proteoforms from zebrafish brain using CZE‐MS. This quantitative approach revealed that the abundance of proteoforms differs significantly in different regions of the zebrafish brain.…”