Native Proteomics in Discovery Mode Using Size-Exclusion Chromatography–Capillary Zone Electrophoresis–Tandem Mass Spectrometry

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 characterization of a complex proteome under the native condition, offering the proteomics community an efficient and simple platform for native proteomics

Microscale Reversed-Phase Liquid Chromatography/Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Deep and Highly Sensitive Bottom–Up Proteomics: Identification of 7500 Proteins with Five Micrograms of an MCF7 Proteome Digest

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has been well recognized for bottom–up proteomics. It has approached 4000–8000 protein identifications (IDs) from a human cell line, mouse brains, or Xenopus embryos via coupling with liquid chromatography (LC) prefractionation. However, at least 500 μg of complex proteome digests were required for the LC/CZE-MS/MS studies. This requirement of a large amount of initial peptide material impedes the application of CZE-MS/MS for deep bottom–up proteomics of mass-limited samples. In this work, we coupled microscale reversed-phase LC (μRPLC)-based peptide prefractionation to dynamic pH-junction-based CZE-MS/MS for deep bottom–up proteomics of the MCF7 breast cancer cell proteome starting with only 5 μg of peptides. The dynamic pH-junction-based CZE enabled a 500 nL sample injection from as low as a 1.5 μL peptide sample, using up to 33% of the available peptide material for an analysis. Two kinds of μRPLC prefractionation were investigated, C18 ZipTip and nanoflow RPLC. C18 ZipTip/CZE-MS/MS identified 4453 proteins from 5 μg of the MCF7 proteome digest and showed good qualitative and quantitative reproducibility. Nanoflow RPLC/CZE-MS/MS produced over 7500 protein IDs and nearly 60 000 peptide IDs from the 5 μg of MCF7 proteome digest. The nanoflow RPLC/CZE-MS/MS platform reduced the required amount of complex proteome digests for LC/CZE-MS/MS-based deep bottom–up proteomics by 2 orders of magnitude. Our work provides the proteomics community with a powerful tool for deep and highly sensitive proteomics

Improved Nanoflow RPLC-CZE-MS/MS System with High Peak Capacity and Sensitivity for Nanogram Bottom-up Proteomics

Novel mass spectrometry (MS)-based proteomic tools with extremely high sensitivity and high peak capacity are required for comprehensive characterization of protein molecules in mass-limited samples. We reported a nanoRPLC-CZE-MS/MS system for deep bottom-up proteomics of low micrograms of human cell samples in previous work. In this work, we improved the sensitivity of the nanoRPLC-CZE-MS/MS system drastically via employing bovine serum albumin (BSA)-treated sample vials, improving the nanoRPLC fraction collection procedure, and using a short capillary for fast CZE separation. The improved nanoRPLC-CZE produced a peak capacity of 8500 for peptide separation. The improved system identified 6500 proteins from a MCF7 proteome digest starting with only 500 ng of peptides using a Q-Exactive HF mass spectrometer. The system produced a comparable number of protein identifications (IDs) to our previous system and the two-dimensional (2D) nanoRPLC-MS/MS system developed by Mann’s group with 10-fold and 4-fold less sample consumption, respectively. We coupled the single-spot solid phase sample preparation (SP3) method to the improved nanoRPLC-CZE-MS/MS for bottom-up proteomics of 5000 HEK293T cells, resulting in 3689 protein IDs with the consumption of a peptide amount that corresponded to only roughly 1000 cells

Improved Nanoflow RPLC-CZE-MS/MS System with High Peak Capacity and Sensitivity for Nanogram Bottom-up Proteomics

Novel mass spectrometry (MS)-based proteomic tools with extremely high sensitivity and high peak capacity are required for comprehensive characterization of protein molecules in mass-limited samples. We reported a nanoRPLC-CZE-MS/MS system for deep bottom-up proteomics of low micrograms of human cell samples in previous work. In this work, we improved the sensitivity of the nanoRPLC-CZE-MS/MS system drastically via employing bovine serum albumin (BSA)-treated sample vials, improving the nanoRPLC fraction collection procedure, and using a short capillary for fast CZE separation. The improved nanoRPLC-CZE produced a peak capacity of 8500 for peptide separation. The improved system identified 6500 proteins from a MCF7 proteome digest starting with only 500 ng of peptides using a Q-Exactive HF mass spectrometer. The system produced a comparable number of protein identifications (IDs) to our previous system and the two-dimensional (2D) nanoRPLC-MS/MS system developed by Mann’s group with 10-fold and 4-fold less sample consumption, respectively. We coupled the single-spot solid phase sample preparation (SP3) method to the improved nanoRPLC-CZE-MS/MS for bottom-up proteomics of 5000 HEK293T cells, resulting in 3689 protein IDs with the consumption of a peptide amount that corresponded to only roughly 1000 cells

Deep Top-Down Proteomics Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry: Identification of 5700 Proteoforms from the <i>Escherichia coli</i> 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 <i>Escherichia coli</i>. The platform generated high peak capacity (∼4000) for separation of intact proteins, leading to the identification of 5700 proteoforms from the <i>Escherichia coli</i> 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

Deep Top-Down Proteomics Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry: Identification of 5700 Proteoforms from the <i>Escherichia coli</i> 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 <i>Escherichia coli</i>. The platform generated high peak capacity (∼4000) for separation of intact proteins, leading to the identification of 5700 proteoforms from the <i>Escherichia coli</i> 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

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