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Abstract Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid‐phase separations. Native proteomics should provide the most accurate bird's‐eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well‐purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra‐high mass range (UHMR) Orbitrap mass spectrometer. The nCZE‐MS technique enabled the measurement of a 115‐kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE‐MS analysis of anE.colicell lysate detected 72 proteoforms or protein complexes in a mass range of 30–400 kDa in a single run while consuming only 50‐ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes. 

Mass spectrometry (MS)-based top-down characterization of integral membrane proteins (IMPs) is crucial for understanding their functions in biological processes. However, it is technically challenging due to their low solubility in typical MS-compatible buffers. In this work, for the first time, we developed an efficient capillary zone electrophoresis (CZE)-tandem MS (MS/MS) method for the top-down proteomics (TDP) of IMPs enriched from mouse brains. Our technique employs a sample buffer containing 30% (v/v) formic acid and 60% (v/v) methanol for solubilizing IMPs and utilizes a separation buffer of 30% (v/v) acetic acid and 30% (v/v) methanol for maintaining the solubility of IMPs during CZE separation. Single-shot CZE-MS/MS identified 51 IMP proteoforms from the mouse brain sample. Coupling size exclusion chromatography (SEC) to CZE-MS/MS enabled the identification of 276 IMP proteoforms from the mouse brain sample containing 1-4 transmembrane domains. This proof-of-concept work demonstrates the high potential of CZE-MS/MS for the large-scale TDP of IMPs. 

Abstract Maize (Zea mays) production systems are heavily reliant on the provision of managed inputs such as fertilizers to maximize growth and yield. Hence, the effective use of nitrogen (N) fertilizer is crucial to minimize the associated financial and environmental costs, as well as maximize yield. However, how to effectively utilize N inputs for increased grain yields remains a substantial challenge for maize growers that requires a deeper understanding of the underlying physiological responses to N fertilizer application. We report a multiscale investigation of five field-grown maize hybrids under low or high N supplementation regimes that includes the quantification of phenolic and prenyl-lipid compounds, cellular ultrastructural features, and gene expression traits at three developmental stages of growth. Our results reveal that maize perceives the lack of supplemented N as a stress and, when provided with additional N, will prolong vegetative growth. However, the manifestation of the stress and responses to N supplementation are highly hybrid-specific. Eight genes were differentially expressed in leaves in response to N supplementation in all tested hybrids and at all developmental stages. These genes represent potential biomarkers of N status and include two isoforms of Thiamine Thiazole Synthase involved in vitamin B1 biosynthesis. Our results uncover a detailed view of the physiological responses of maize hybrids to N supplementation in field conditions that provides insight into the interactions between management practices and the genetic diversity within maize. 

Abstract Mass spectrometry (MS)‐based top‐down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post‐translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high‐resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS‐PAGE‐based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI‐MS) with capillary zone electrophoresis (CZE)‐MS/MS for high‐resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS‐PAGE gel and follow‐up cleanup as well as CZE‐MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high‐resolution separation and characterization of histone proteoforms. SDS‐PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high‐resolution separations of SDS‐PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems. 

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. 

Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) requires high-capacity separation and extensive gas-phase fragmentation of proteoforms. Herein, we coupled capillary zone electrophoresis (CZE) to electron-capture collision-induced dissociation (ECciD) on an Agilent 6545 XT quadrupole time-of-flight (Q-TOF) mass spectrometer for dTDP for the first time. During ECciD, the protein ions were first fragmented using ECD, followed by further activation and fragmentation by applying a CID potential. In this pilot study, we optimized the CZE-ECciD method for small proteins (lower than 20 kDa) regarding the charge state of protein parent ions for fragmentation and the CID potential applied to maximize the protein backbone cleavage coverage and the number of sequence-informative fragment ions. The CZE-ECciD Q-TOF platform provided extensive backbone cleavage coverage for three standard proteins lower than 20 kDa from only single charge states in a single CZE-MS/MS run in the targeted MS/MS mode, including ubiquitin (97%, +7, 8.6 kDa), superoxide dismutase (SOD, 87%, +17, 16 kDa), and myoglobin (90%, +16, 17 kDa). The CZE-ECciD method produced comparable cleavage coverage of small proteins (i.e., myoglobin) with direct-infusion MS studies using electron transfer dissociation (ETD), activated ion-ETD, and combinations of ETD and collision-based fragmentation on high-end orbitrap mass spectrometers. The results render CZE-ECciD a new tool for dTDP to enhance both separation and gas-phase fragmentation of proteoforms. 

Abstract We present a large‐scale top‐down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size‐exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post‐translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post‐translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N‐ and C‐terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub‐cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub‐cellular localization signal predictions. This large‐scale analysis illustrates the power of top‐down proteoform identification of post‐translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.