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.

Deep top-down proteomics revealed significant proteoform-level differences between metastatic and nonmetastatic colorectal cancer cells.

Understanding cancer metastasis at the proteoform level is crucial for discovering previously unknown protein biomarkers for cancer diagnosis and drug development. We present the first top-down proteomics (TDP) study of a pair of isogenic human nonmetastatic and metastatic colorectal cancer (CRC) cell lines (SW480 and SW620). We identified 23,622 proteoforms of 2332 proteins from the two cell lines, representing nearly fivefold improvement in the number of proteoform identifications (IDs) compared to previous TDP datasets of human cancer cells. We revealed substantial differences between the SW480 and SW620 cell lines regarding proteoform and single amino acid variant (SAAV) profiles. Quantitative TDP unveiled differentially expressed proteoforms between the two cell lines, and the corresponding genes had diversified functions and were closely related to cancer. Our study represents a pivotal advance in TDP toward the characterization of human proteome in a proteoform-specific manner, which will transform basic and translational biomedical research.

Top-Down Proteomics by Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Characterization of Proteoforms in Complex Samples.

Capillary zone electrophoresis (CZE) is a fundamentally simple and highly efficient separation technique based on differences in electrophoretic mobilities of analytes. CZE-mass spectrometry (MS) has become an important analytical tool in top-down proteomics which aims to delineate proteoforms in cells comprehensively, because of the improvement of capillary coatings, sample stacking methods, and CE-MS interfaces. Here, we present a CZE-MS/MS-based top-down proteomics procedure for the characterization of a standard protein mixture and an Escherichia coli (E. coli) cell lysate using linear polyacrylamide-coated capillaries, a dynamic pH junction sample stacking method, a commercialized electro-kinetically pumped sheath flow CE-MS interface and an Orbitrap mass spectrometer. CZE-MS/MS can identify hundreds of proteoforms routinely from the E. coli sample with a 1% proteoform-level false discovery rate (FDR).

Evaluation of Machine Learning Models for Proteoform Retention and Migration Time Prediction in Top-Down Mass Spectrometry.

Reversed-phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) are two primary proteoform separation methods in mass spectrometry (MS)-based top-down proteomics. Proteoform retention time (RT) prediction in RPLC and migration time (MT) prediction in CZE provide additional information for accurate proteoform identification and quantification. While existing methods are mainly focused on peptide RT and MT prediction in bottom-up MS, there is still a lack of methods for proteoform RT and MT prediction in top-down MS. We systematically evaluated eight machine learning models and a transfer learning method for proteoform RT prediction and five models and the transfer learning method for proteoform MT prediction. Experimental results showed that a gated recurrent unit (GRU)-based model with transfer learning achieved a high accuracy (R = 0.978) for proteoform RT prediction and that the GRU-based model and a fully connected neural network model obtained a high accuracy of R = 0.982 and 0.981 for proteoform MT prediction, respectively.

Automated Capillary Isoelectric Focusing-Tandem Mass Spectrometry for Qualitative and Quantitative Top-Down Proteomics.

Top-down proteomics (TDP) aims to delineate proteomes in a proteoform-specific manner, which is vital for accurately understanding protein function in cellular processes. It requires high-capacity separation of proteoforms before mass spectrometry (MS) and tandem MS (MS/MS). Capillary isoelectric focusing (cIEF)-MS has been recognized as a useful tool for TDP in the 1990s because cIEF is capable of high-resolution separation of proteoforms. Previous cIEF-MS studies concentrated on measuring the protein's mass without MS/MS, impeding the confident proteoform identification in complex samples and the accurate localization of post-translational modifications on proteoforms. Herein, for the first time, we present automated cIEF-MS/MS-based TDP for large-scale delineation of proteoforms in complex proteomes. Single-shot cIEF-MS/MS identified 711 proteoforms from an Escherichia coli (E. coli) proteome consuming only nanograms of proteins. Coupling two-dimensional size-exclusion chromatography (SEC)-cIEF to ESI-MS/MS enabled the identification of nearly 2000 proteoforms from the E. coli proteome. Label-free quantitative TDP of zebrafish male and female brains using SEC-cIEF-MS/MS quantified thousands of proteoforms and revealed sex-dependent proteoform profiles in brains. Particularly, we discovered several proteolytic proteoforms of pro-opiomelanocortin and prodynorphin with significantly higher abundance in male zebrafish brains as potential endogenous hormone proteoforms. Multilevel quantitative proteomics (TDP and bottom-up proteomics) of the brains revealed that the majority of proteoforms having statistically significant difference in abundance between genders showed no abundance difference at the protein group level. This work represents the first multilevel quantitative proteomics study of sexual dimorphism of the brain.

Predicting Electrophoretic Mobility of Proteoforms for Large-Scale Top-Down Proteomics.

Large-scale top-down proteomics characterizes proteoforms in cells globally with high confidence and high throughput using reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) or capillary zone electrophoresis (CZE)-MS/MS. The false discovery rate (FDR) from the target-decoy database search is typically deployed to filter identified proteoforms to ensure high-confidence identifications (IDs). It has been demonstrated that the FDRs in top-down proteomics can be drastically underestimated. An alternative approach to the FDR can be useful for further evaluating the confidence of proteoform IDs after the database search. We argue that predicting retention/migration time of proteoforms from the RPLC/CZE separation accurately and comparing their predicted and experimental separation time could be a useful and practical approach. Based on our knowledge, there is still no report in the literature about predicting separation time of proteoforms using large top-down proteomics data sets. In this pilot study, for the first time, we evaluated various semiempirical models for predicting proteoforms' electrophoretic mobility (µef) using large-scale top-down proteomics data sets from CZE-MS/MS. We achieved a linear correlation between experimental and predicted µef of E. coli proteoforms (R2 = 0.98) with a simple semiempirical model, which utilizes the number of charges and molecular mass of each proteoform as the parameters. Our modeling data suggest that the complete unfolding of proteoforms during CZE separation benefits the prediction of their µef. Our results also indicate that N-terminal acetylation and phosphorylation both decrease the proteoforms' charge by roughly one charge unit.

Capillary zone electrophoresis-tandem mass spectrometry using ultraviolet photodissociation (213 nm) for large-scale top-down proteomics.

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has attracted attention recently for large-scale top-down proteomics that aims to characterize proteoforms in cells at a global scale and with high throughput. In this work, CZE-MS/MS with ultraviolet photodissociation (UVPD) was evaluated for large-scale top-down proteomics for the first time. Roughly, 600 proteoforms and 369 proteins were identified from a zebrafish brain sample via coupling size exclusion chromatography (SEC) fractionation to CZE-UVPD. The dataset represents one of the largest top-down proteomics datasets using UVPD. Single-shot CZE-UVPD identified 227 proteoforms of 139 proteins from one SEC fraction of the zebrafish brain sample. The SEC-CZE-UVPD system identified zebrafish brain proteoforms in a mass range of 3-21 kDa. The UVPD with 213-nm photons produced reasonably good gas-phase fragmentation of proteoforms. For instance, 75% backbone cleavages were observed for Parvalbumin-7 with about 12-kDa molecular weight. The system detected various post-translational modifications (PTMs) from the zebrafish brain sample, including N-terminal acetylation, trimethylation and myristoylation of N-terminal glycine. Two different proteoforms of calmodulin, with either only N-terminal acetylation or both N-terminal acetylation and K115 trimethylation, were identified in the zebrafish brain sample. To our best knowledge, there is no experimental evidence reported in the literature on the two proteoforms of calmodulin in the zebrafish brain.

[Comparing nanoflow reversed-phase liquid chromatography-tandem mass spectrometry and capillary zone electrophoresis-tandem mass spectrometry for top-down proteomics].

One of the major shortcomings in top-down proteomics is the lack of efficient separations for intact proteins that can be effectively coupled to mass spectrometry. Capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (nanoRPLC) are two methods that can be coupled to mass spectrometry directly and have been recently advanced in terms of their ability to separate intact proteins in complex biological mixtures. In this work, for the first time, we compared the state-of-the-art nanoRPLC-MS/MS and CZE-MS/MS platforms for top-down characterization of a standard protein mixture and an Escherichia coli (E. coli) proteome sample. CZE-MS produced comparable signals of standard proteins to RPLC-MS with 10-times less sample consumption. Interestingly, the proteins in RPLC-MS tended to have higher charge states than in CZE-MS, most likely due to the high acetonitrile concentration in RPLC mobile phase, leading to the more extensive unfolding of proteins in RPLC compared to in CZE. CZE-MS/MS identified 159 proteins and 513 proteoforms using 1-µg E. coli proteins in a single run and outperformed RPLC-MS/MS using 1-µg E. coli proteins in terms of protein and proteoform identifications (159 vs. 105 proteins and 513 vs. 277 proteoforms). The RPLC-MS/MS using 8-µg E. coli proteins identified 245 proteins and 1004 proteoforms in a single run, and the data was much better than that from CZE-MS/MS (1-µg E. coli proteins) regarding the number of identifications because of the 8-times higher sample loading amount and significantly wider separation window of RPLC-MS/MS compared to CZE-MS/MS.

Capillary zone electrophoresis-mass spectrometry for top-down proteomics.

Mass spectrometry (MS)-based top-down proteomics characterizes complex proteomes at the intact proteoform level and provides an accurate picture of protein isoforms and protein post-translational modifications in the cell. The progress of top-down proteomics requires novel analytical tools with high peak capacity for proteoform separation and high sensitivity for proteoform detection. The requirements have made capillary zone electrophoresis (CZE)-MS an attractive approach for advancing large-scale top-down proteomics. CZE has achieved a peak capacity of 300 for separation of complex proteoform mixtures. CZE-MS has shown drastically better sensitivity than commonly used reversed-phase liquid chromatography (RPLC)-MS for proteoform detection. The advanced CZE-MS identified 6,000 proteoforms of nearly 1,000 proteoform families from a complex proteome sample, which represents one of the largest top-down proteomic datasets so far. In this review, we focus on the recent progress in CZE-MS-based top-down proteomics and provide our perspectives about its future directions.

Capillary zone electrophoresis-tandem mass spectrometry for top-down proteomics using attapulgite nanoparticles functionalized separation capillaries.

Attapulgite nanoparticles have good chemical properties and can be modified easily for broad applications. In this work, for the first time, attapulgite nanoparticles were employed to modify the inner wall of separation capillaries for capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS)-based top-down proteomics. The attapulgite nanoparticles and the inner wall of a fused silica capillary were first functionalized with γ-methacryloxypropyl trimethoxysilane. Then the modified nanoparticles and acrylamide were copolymerized in the fused silica capillary with the assistance of azobisisobutyronitrile and heat. The incorporation of high-surface-area nanoparticles in the linear polyacrylamide (LPA) coating resulted in significantly lower electroosmotic mobility compared with the typical LPA coating (3.48 × 10-5 vs. 9.03 × 10-5 cm2 V-1 S-1), most likely because more LPA molecules were immobilized on the inner wall of the separation capillary. The attapulgite nanoparticles functionalized separation capillaries have shown great stability and reproducibility across 43 discontinuous CZE-MS runs of a standard protein mixture. We applied the CZE-MS/MS system for top-down proteomics of Escherichia coli cells. In a proof-of-principle experiment, the CZE-MS/MS system achieved a 90-min separation window and a 1-µL sample loading volume, leading to nearly 300 proteoform and 135 protein identifications in a single run. Many post-translational modifications (PTMs) were identified, including methylation, acetylation, phosphorylation, biotinylation, succinylation, and disulfide bond.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics.

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

Deep Top-Down Proteomics Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry: Identification of 5700 Proteoforms from the Escherichia coli Proteome.

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.

Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics using CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction-based CZE-MS/MS approached a 1 µL loading capacity, 90 min separation window, and high peak capacity (∼280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified proteoforms in this work is over three times higher than that in previous single-shot CZE-MS/MS studies. Truncations, N-terminal methionine excision, signal peptide removal, and some post-translational modifications including oxidation and acetylation were detected.

The "PepSAVI-MS" Pipeline for Natural Product Bioactive Peptide Discovery.

The recent increase in extensively drug-resistant bacterial pathogens and the associated increase of morbidity and mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. Here, we report the development of the PepSAVI-MS pipeline for bioactive peptide discovery. This highly versatile platform employs mass spectrometry and statistics to identify bioactive peptide targets from complex biological samples. We validate the use of this platform through the successful identification of known bioactive peptides from a botanical species, Viola odorata. Using this pipeline, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cycloviolacin O2 against A. baumannii. We further demonstrate the broad applicability of the platform through the identification of novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast, and prostate cancer cell lines.