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

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Protein complexes are the functional machines in the cell and are heterogeneous due to protein sequence variations and post-translational modifications (PTMs). Here, we present an automated nondenaturing capillary isoelectric focusing-mass spectrometry (ncIEF-MS) methodology for uncovering the microheterogeneity of intact protein complexes. The method exhibited superior separation resolution for protein complexes than conventional native capillary zone electrophoresis (nCZE-MS). In our study, ncIEF-MS achieved liquid-phase separations and MS characterization of seven different forms of a streptavidin homotetramer with variations of N-terminal methionine removal, acetylation, and formylation and four forms of the carbonic anhydrase–zinc complex arising from variations of PTMs (succinimide, deamidation, etc.). In addition, ncIEF-MS resolved different states of an interchain cysteine-linked antibody–drug conjugate (ADC1) as a new class of anticancer therapeutic agents that bears a distribution of varied drug-to-antibody ratio (DAR) species. More importantly, ncIEF-MS enabled precise measurements of isoelectric points (pIs) of protein complexes, which reflect the surface electrostatic properties of protein complexes. We studied how protein sequence variations/PTMs modulate the pIs of protein complexes and how drug loading affects the pIs of antibodies. We discovered that keeping the N-terminal methionine residue of one subunit of the streptavidin homotetramer decreased its pI by 0.1, adding one acetyl group onto the streptavidin homotetramer reduced its pI by nearly 0.4, incorporating one formyl group onto the streptavidin homotetramer reduced its pI by around 0.3, and loading two more drug molecules on one ADC1 molecule increased its pI by 0.1. The data render the ncIEF-MS method a valuable tool for delineating protein complexes

Solid-Phase Microextraction-Aided Capillary Zone Electrophoresis-Mass Spectrometry: Toward Bottom-Up Proteomics of Single Human Cells

Capillary zone electrophoresis-mass spectrometry (CZE-MS) has been recognized as a valuable technique for the proteomics of mass-limited biological samples (i.e., single cells). However, its broad adoption for single cell proteomics (SCP) of human cells has been impeded by the low sample loading capacity of CZE, only allowing us to use less than 5% of the available peptide material for each measurement. Here we present a reversed-phase-based solid-phase microextraction (RP-SPME)-CZE-MS platform to solve the issue, paving the way for SCP of human cells using CZE-MS. The RP-SPME-CZE system was constructed in one fused silica capillary with zero dead volume for connection via in situ synthesis of a frit, followed by packing C8 beads into the capillary to form a roughly 2 mm long SPME section. Peptides captured by SPME were eluted with a buffer containing 30% (v/v) acetonitrile and 50 mM ammonium acetate (pH 6.5), followed by dynamic pH junction-based CZE-MS. The SPME-CZE-MS enabled the injection of nearly 40% of the available peptide sample for each measurement. The system identified 257 ± 24 proteins and 523 ± 69 peptides (N = 2) using a Q-Exactive HF mass spectrometer when only 0.25 ng of a commercial HeLa cell digest was available in the sample vial and 0.1 ng of the sample was injected. The amount of available peptide is equivalent to the protein mass of one HeLa cell. The data indicate that SPME-CZE-MS is ready for SCP of human cells

Integrated Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry System with an Immobilized Trypsin Microreactor for Online Digestion and Analysis of Picogram Amounts of RAW 264.7 Cell Lysate

A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE–ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide identifications (IDs) when 20 fmol was loaded. When only 1 fmol of BSA was injected, one BSA peptide was consistently detected. For the analysis of a standard protein mixture, the integrated system produced efficient protein digestion and confident identification for proteins with different molecular weights and isoelectric points when a low-femtomole amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 ± 1 and 7 ± 2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading corresponds to the protein content of three RAW 264.7 cells. In addition to high-sensitivity analysis, the integrated CZE–ESI-MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity

Physiological, Metabolic, and Transcriptomic Analyses Reveal the Responses of <i>Arabidopsis</i> Seedlings to Carbon Nanohorns

Carbon-based nanomaterials have potential applications in nanoenabled agriculture. However, the physiological and molecular mechanisms underlying single-walled carbon nanohorn (SWCNH)-mediated plant growth remain unclear. Here, we investigated the effects of SWCNHs on Arabidopsis grown in 1/4-strength Murashige and Skoog medium via physiological, genetic, and molecular analyses. Treatment with 0.1 mg/L SWCNHs promoted primary root (PR) growth and lateral root (LR) formation; 50 and 100 mg/L SWCNHs inhibited PR growth. Treatment with 0.1 mg/L SWCNHs increased the lengths of the meristematic and elongation zones, and transcriptomic and genetic analyses confirmed the positive effects of SWCNHs on root tip stem cell niche activity and meristematic cell division potential. Increased expression of YUC3 and YUC5 and increased PIN2 abundance improved PR growth and LR development in 0.1 mg/L SWCNH-treated seedlings. Metabolomic analyses revealed that SWCNHs altered the levels of sugars, amino acids, and organic acids, suggesting that SWCNHs reprogrammed carbon/nitrogen metabolism in plants. SWCNHs also regulate plant growth and development by increasing the levels of several secondary metabolites; transcriptomic analyses further supported these results. The present results are valuable for continued use of SWCNHs in agri-nanotechnology, and these molecular approaches could serve as examples for studies on the effects of nanomaterials in plants

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

Integrated Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry System with an Immobilized Trypsin Microreactor for Online Digestion and Analysis of Picogram Amounts of RAW 264.7 Cell Lysate

A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE–ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide identifications (IDs) when 20 fmol was loaded. When only 1 fmol of BSA was injected, one BSA peptide was consistently detected. For the analysis of a standard protein mixture, the integrated system produced efficient protein digestion and confident identification for proteins with different molecular weights and isoelectric points when a low-femtomole amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 ± 1 and 7 ± 2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading corresponds to the protein content of three RAW 264.7 cells. In addition to high-sensitivity analysis, the integrated CZE–ESI-MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity

TopFD: A Proteoform Feature Detection Tool for Top–Down Proteomics

Top-down liquid chromatography-mass spectrometry (LC-MS) analyzes intact proteoforms and generates mass spectra containing peaks of proteoforms with various isotopic compositions, charge states, and retention times. An essential step in top-down MS data analysis is proteoform feature detection, which aims to group these peaks into peak sets (features), each containing all peaks of a proteoform. Accurate protein feature detection enhances the accuracy in MS-based proteoform identification and quantification. Here, we present TopFD, a software tool for top-down MS feature detection that integrates algorithms for proteoform feature detection, feature boundary refinement, and machine learning models for proteoform feature evaluation. We performed extensive benchmarking of TopFD, ProMex, FlashDeconv, and Xtract using seven top-down MS data sets and demonstrated that TopFD outperforms other tools in feature accuracy, reproducibility, and feature abundance reproducibility

Coupling High-Field Asymmetric Waveform Ion Mobility Spectrometry with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has emerged as an essential technique for top-down proteomics (TDP), providing superior separation efficiency and high detection sensitivity for proteoform analysis. Here, we aimed to further enhance the performance of CZE-MS/MS for TDP via coupling online gas-phase proteoform fractionation using high-field asymmetric waveform ion mobility spectrometry (FAIMS). When the compensation voltage (CV) of FAIMS was changed from −50 to 30 V, the median mass of identified proteoforms increased from less than 10 kDa to about 30 kDa, suggesting that FAIMS can efficiently fractionate proteoforms by their size. CZE-FAIMS-MS/MS boosted the number of proteoform identifications from a yeast sample by nearly 3-fold relative to CZE-MS/MS alone. It particularly benefited the identification of relatively large proteoforms, improving the number of proteoforms in a mass range of 20–45 kDa by 6-fold compared to CZE-MS/MS alone. FAIMS fractionation gained nearly 20-fold better signal-to-noise ratios of randomly selected proteoforms than no FAIMS. We expect that CZE-FAIMS-MS/MS will be a useful tool for further advancing the sensitivity and coverage of TDP. This work shows the first example of coupling CE with ion mobility spectrometry (IMS) for TDP