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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.

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.

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 (μ(ef)) using large-scale top-down proteomics data sets from CZE–MS/MS. We achieved a linear correlation between experimental and predicted μ(ef) of E. coli proteoforms (R(2) = 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 μ(ef). Our results also indicate that N-terminal acetylation and phosphorylation both decrease the proteoforms’ charge by roughly one charge unit.

In this study, total ozone columns collected from nine sites of the AErosol RObotic NETwork (AERONET) in China are used to evaluate total ozone column monthly mean products of the Ozone Monitoring Instrument (OMI). The results show the correlated coefficient of the two datasets is 0.95. The long temporal variations and spatial distributions of the monthly mean products of the total ozone columns and tropospheric ozone columns in spring, summer, autumn and winter were plotted. The result shown in the pictures indicate that the total ozone columns gradually increase from low latitude to high latitude, reach the maximum value in winter and spring in Northeast China, and the values in Qinghai-Tibet Plateau are lower than those of other regions of the same latitude. The monthly mean of total ozone columns at low latitude have no obvious seasonal variation. With the increase of latitude, the seasonal variation of monthly mean products of total ozone column becomes more and more obvious. Tropospheric ozone columns are the highest in summer, followed by spring and autumn, and the lowest in winter, which are mainly concentrated in the more developed areas in eastern China. The lowest value of tropospheric ozone column in China occurs in the Qinghai-Tibet Plateau in winter and the highest value in North China in summer. The study indicates that the total ozone columns vary with latitude while tropospheric ozone columns are more susceptible to human activities and natural conditions.

Eco-friendly poly(butylenes succinate) (PBS)/bamboo carbon lubricant composites were designed and prepared by hot pressing. The hardness and wear properties of the designed PBS composites were studied. The results showed that the hardness of PBS composite containing 50% of the bamboo carbon lubricant increased by more than 150%. The composite material with 50% bamboo carbon lubricant had the lowest wear rate, which was up to 72% lower than that of pure PBS. The wear of neat PBS was harsh with severe cut and even partly melted points appearing on the worn surface. The wear modes of PBS bamboo carbon composite were abrasive wear and fatigue wear. While the worn surface of PBS bamboo carbon lubricant composite was with slight exfoliation, its wear mode was slight adhesive wear. It is concluded that the addition of bamboo carbon lubricant can broaden the application range of eco-friendly polymer of PBS in the engineering field of anti-wear requirements.

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.

In order to control the air pollution in China, the Chinese government issued an air pollution control action plan in Sep. 2013. The goal of the first phase of the plan is by 2017, the air quality was to improve significantly compared to 2012. In this study, the satellite aerosol product derived from the MODerate-resolution Imaging Spectroradiometer (MODIS) over East China were collected and processed to evaluate the effectiveness of the plan. The results show that the mean aerosol optical depth (AOD) over East China in 2017 has reduced about 23.17% compared to 2012, and the trends of the MODIS AOD during the period from 2010 to 2019 over the domain is −0.012 per year, which indicates that the air pollution plan has been well implemented to achieve the expected goals.

In this study, a novel strategy to prepare sodium alginate (SA)/nano fibrillated cellulose (NFC) double network (DN) hydrogel beads with the aid of graphene oxide (GO) was developed. In comparison with the multi-step freezing-thawing method, this study employs a facile one-step freeze drying method with the presence of GO sheets. The crucial roles of GO were highlighted as an efficient nucleating agent of NFC and a reinforcer for the hydrogel. The adsorption property of the DN hydrogel towards crystal violet (CV) was also studied. Results indicated that the introduction of GO could greatly facilitate the formation of double networks. Furthermore, the as-prepared DN hydrogel beads exhibited an efficacious adsorption property towards CV. The maximum adsorption capacity of the hydrogels for CV was observed as 665 mg g−1. Therefore, our approach here represents a facile method for the preparation of crystalline polymer based DN hydrogels to replace the awkward freezing-thawing process, giving inspiration for DN hydrogels design and preparation. Moreover, due to its efficient adsorption capacity, the hydrogels hold great promise for the water pollution control materials.

In this study, the ground measured nitrogen dioxide (NO 2 ) column at six sites of the AErosol RObotic NETwork (AERONET) in China were collected to evaluate the Global Ozone Monitoring Experiment-2 (GOME-2) tropospheric NO 2 product. The results show the correlated coefficient of the two datasets is 0.851. The long temporal variations and spatial distributions of the GOME-2 tropospheric (trop) NO 2 in the heating season (Nov, Dec, Jan and Feb) and the non-heating season over China were plotted. The results indicate that in the heating season, the trop NO 2 column values in the North and East China are much higher in the non-heating seasons. Although the trends of the trop NO 2 column in the non-heating season from 2013 to 2017 are significantly decreased, the variations in the heating are not significant. It proved that the coal heating can have a great impact on trop NO 2 column variations in China.

Environmental stresses influence the growth of plants and the productivity of crops. Salinity is one of the most important abiotic stresses for agricultural crops. Programmed cell death (PCD) is induced by a variety of biotic and abiotic stresses in plants, including high salinity treatment. OsPDCD5, an ortholog of the mammalian-programmed cell death 5 gene, is up-regulated under low temperature and salt treatments in rice (Oryza sativa). In this study, transgenic rice that constitutively expressed antisense-OsPDCD5 increased salt stress tolerance in unique ways by inhibiting PCD pathways and regulating specific groups of stress-related genes. Real-time quantitative PCR analysis showed that PCD pathways were inhibited under both normal conditions and salt stress in transgenic rice. Using transcript microarray analysis, 38 stress-related genes regulated in transgenic rice were identified, which appeared to render the transgenic rice adapted for salt stress conditions. Our results suggested that PCD pathways were inhibited and salt stress signaling pathways were regulated in transgenic rice even under normal growth conditions without stress. PCD was further inhibited and these stress-related genes were further regulated to increase tolerance to high salinity under salt stress conditions, thereby leading to build-up of stress protection mechanisms in transgenic rice.

Plant height is a decisive factor in plant architecture. Rice (Oryza sativa) plants have the potential for rapid internodal elongation, which determines plant height. A large body of physiological research has shown that ethylene and gibberellin are involved in this process. The APETALA2 (AP2)/Ethylene-Responsive Element Binding Factor (ERF) family of transcriptional factors is only present in the plant kingdom. This family has various developmental and physiological functions. A rice AP2/ERF gene, OsEATB (for ERF protein associated with tillering and panicle branching) was cloned from indica rice variety 9311. Bioinformatic analysis suggested that this ERF has a potential new function. Ectopic expression of OsEATB showed that the cross talk between ethylene and gibberellin, which is mediated by OsEATB, might underlie differences in rice internode elongation. Analyses of gene expression demonstrated that OsEATB restricts ethylene-induced enhancement of gibberellin responsiveness during the internode elongation process by down-regulating the gibberellin biosynthetic gene, ent-kaurene synthase A. Plant height is negatively correlated with tiller number, and higher yields are typically obtained from dwarf crops. OsEATB reduces rice plant height and panicle length at maturity, promoting the branching potential of both tillers and spikelets. These are useful traits for breeding high-yielding crops.

OsPDCD5 is the rice (Oryza sativa L.) homolog of PDCD5, a gene involved in programmed cell death (PCD) regulation and highly conserved over time. PDCD5 overexpression in tumor cells enhances apoptosis triggered by growth factor or serum deprivation. In our previous research, constitutive OsPDCD5 overexpression induces early death in transgenic plants. In this study, an inducible OsPDCD5 expression strategy was employed to systematically study the role of OsPDCD5 in rice. With this system, OsPDCD5 was found to be capable of independently inducing cell death in three-leaf stage and older seedlings. These altered plants exhibited lesion mimic phenotype, abnormal leaf morphology, DNA fragmentation, hydrogen peroxide production, and mitochondrial distortion. In nature, RNA hybridization in situ has shown that OsPDCD5 expression was predominantly localized to the tapetal layer where PCD occurred. Transcript microarray analyses here revealed that many PCD-related genes were involved. These data, taken together, indicated that OsPDCD5-induced cell death is a kind of PCD. Nevertheless, OsPDCD5 failed to induce any visible morphological phenotypes in two-leaf stage and younger seedlings. Transcript microarray analyses and quantitative real-time polymerase chain reaction showed that the Bax inhibitor-1 (BI-1) mRNA concentration and the activity of an ubiquitin gene were specifically changed. Endogenous OsPDCD5 upregulation, induced by ectopic OsPDCD5 expression in three-leaf stage seedlings, was also absent in two-leaf stage seedlings, suggesting strongly that young seedlings could inhibit PCD at some level.

Using laser 40Ar/39Ar dating method, we have gotten the metamorphic ages of lawsonite blueschist and epidote blueschist from Jiuquan, northern Qilian Mountain, NW China. The high quality laser 40Ar/39Ar dating of glaucophane from lawsonite blueschist gives an isochron age of 413±5 Ma. The isochron age obtained from phengite in epidote blueschist is 415±7 Ma. These data, combining with peak metamorphic P-T conditions and regional geological setting, allow us to infer that the lower limit of the ages of the prograde subduction metamorphism from lawsonite blueschist facies to epidote blueschist facies occurred at ca. 413–415 Ma, which also suggests that the formation of lawsonite blueschist in the northern Qilian Mountain maybe resulted from the corner flow in the cold subduction zone. This study shows that the final closing time of the northern Qilian remnant oceanic basin is about 413–415 Ma, which also represents the convergent age between the North China Craton and the Qaidam block.

Cathodoluminescence (CL) imaging and ion microprobe (SHRIMP) U-Pb dating were carried out for zircons from eclogites in the North Qilian Mountains. The results show weighted mean ages of 463±6 Ma and 468±13 Ma for two samples, respectively. These ages are the earliest record of Caledonian high-pressure metamorphism in the North Qilian Mountains, and they may represent the timing of eclogite-facies metamorphism when the oceanic crust was subducted to mantle depths in this orogenic belt.

SummaryAllelic expression of the rice yield-related gene, leucine-rich receptor-like kinase 6 (LRK6), in the hybrid of 93-11 (Oryza sativa L. subsp. Indica var. 93-11) and Nipponbare (O. sativa L. subsp. Japonica var. Nipponbare) is determined by allelic promoter cis-elements. Using deletion analysis of the LRK6 promoter, we identified two distinct regions that might contribute to LRK6 expression. Sequence alignment revealed differences in these LRK6 promoter regions in 93-11 and Nipponbare. One of the segments, named differential sequence of LRK6 promoter 2 (DSLP2), contains potential transcription factor binding sites. Using a yeast one-hybrid assay, we isolated an ethylene-responsive factor (ERF) protein that binds to DSLP2. Sequence analysis and a GCC-box assay showed that the ERF gene, O. sativa ERF 3 (OsERF3), which belongs to ERF subfamily class II, has a conserved ERF domain and an ERF-associated amphiphilic repression repressor motif. We used an in vivo mutation assay to identify a new motif (5′-TAA(A)GT-3′) located in DSLP2, which interacts with OsERF3. These results suggest that OsERF3, an AP2 (APETALA 2 Gene)/ERF transcription factor, binds the LRK6 promoter at this new motif, which might cause differential expression of LRK6 in the 93-11/Nipponbare hybrid.