Proteomic analysis reveals that the co-ordination of cytosolic and mitochondrial pathways is beneficial for sabinene biosynthesis in engineered Saccharomyces cerevisiae.

Reconstruction and optimization of biosynthetic pathways can help to overproduce target chemicals in microbial cell factories based on genetic engineering. However, the perturbation of biosynthetic pathways on cellular metabolism is not well investigated and profiling the engineered microbes remains challenging. The rapid development of omics tools has the potential to characterize the engineered microbial cell factory. Here, we performed label-free quantitative proteomic analysis and metabolomic analysis of engineered sabinene overproducing Saccharomyces cerevisiae strains. Combined metabolic analysis andproteomic analysis of targeted mevalonate (MVA) pathway showed that co-ordination of cytosolic and mitochondrial pathways had balanced metabolism, and genome integration of biosynthetic genes had higher sabinene production with less MVA enzymes. Furthermore, comparative proteomic analysis showed that compartmentalized mitochondria pathway had perturbation on central cellular metabolism. This study provided an omics analysis example for characterizing engineered cell factory, which can guide future regulation of the cellular metabolism and maintaining optimal protein expression levels for the synthesis of target products.

Metal organic layers enabled cell surface engineering coupling biomembrane fusion for dynamic membrane proteome profiling.

Systematically dissecting the highly dynamic and tightly communicating membrane proteome of living cells is essential for the system-level understanding of fundamental cellular processes and intricate relationship between membrane-bound organelles constructed through membrane traffic. While extensive efforts have been made to enrich membrane proteins, their comprehensive analysis with high selectivity and deep coverage remains a challenge, especially at the living cell state. To address this problem, we developed the cell surface engineering coupling biomembrane fusion method to map the whole membrane proteome from the plasma membrane to various organelle membranes taking advantage of the exquisite interaction between two-dimensional metal-organic layers and phospholipid bilayers on the membrane. This approach, which bypassed conventional biochemical fractionation and ultracentrifugation, facilitated the enrichment of membrane proteins in their native phospholipid bilayer environment, helping to map the membrane proteome with a specificity of 77% and realizing the deep coverage of the HeLa membrane proteome (5087 membrane proteins). Furthermore, membrane N-phosphoproteome was profiled by integrating the N-phosphoproteome analysis strategy, and the dynamic membrane proteome during apoptosis was deciphered in combination with quantitative proteomics. The features of membrane protein N-phosphorylation modifications and many differential proteins during apoptosis associated with mitochondrial dynamics and ER homeostasis were found. The method provided a simple and robust strategy for efficient analysis of membrane proteome, offered a reliable platform for research on membrane-related cell dynamic events and expanded the application of metal-organic layers.

Improved Cross-Linking Coverage for Protein Complexes Containing Low Levels of Lysine by Using an Enrichable Photo-Cross-Linker.

Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.

High-Sensitivity Detection toward SARS-CoV-2 S1 Glycoprotein by Parallel Reaction Monitoring Mass Spectrometry.

The outbreak of coronavirus disease 2019 (COVID-19) has overwhelmed the global economy and human well-being. On account of the sharp increase in test demand, there is a need for an accurate and alternative diagnosis method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, with the aim to specifically identify the trace SARS-CoV-2 S1 glycoprotein, we developed a high-sensitivity and high-selectivity diagnostic method based on the targeted parallel reaction monitoring (PRM) assay of eight selected peptides. This study emphasizes the outstanding detection sensitivity of 0.01 pg of the SARS-CoV-2 S1 glycoprotein even in the interference of other structural proteins, which to our knowledge is the current minimum limit of detection for the SARS-CoV-2 S1 glycoprotein. This technology could further identify 0.01 pg of the SARS-CoV-2 S1 glycoprotein in a spike pseudovirus, revealing its practical effectiveness. All our preliminary results throw light on the capability of the mass spectrometry-based targeted PRM assay to identify SARS-CoV-2 as a practicable orthogonal diagnostic tool. Furthermore, this technology could be extended to other pathogens (e.g., MERS-CoV S1 protein or SARS-CoV S1 protein) by quickly adjusting the targeted peptides of MS data acquisition. In summary, this strategy is universal and flexible and could be quickly adjusted to detect and discriminate different mutants and pathogens.

Alkynyl-Enrichable Carboxyl-Selective Crosslinkers to Increase the Crosslinking Coverage for Deciphering Protein Structures.

The coverage of chemical crosslinking coupled with mass spectrometry (CXMS) is of great importance to determine its ability for deciphering protein structures. At present, N-hydroxysuccinimidyl (NHS) ester-based crosslinkers targeting lysines have been predominantly used in CXMS. However, they are not always effective for some proteins with few lysines. Other amino acid residues such as carboxyl could be crosslinked to complement lysines and improve the crosslinking coverage of CXMS, but the low intrinsic chemical reactivity of carboxyl compromises the application of carboxyl-selective crosslinkers for complex samples. To enhance the crosslinking efficiency targeting acidic residues and realize in-depth crosslinking analysis of complex samples, we developed three new alkynyl-enrichable carboxyl-selective crosslinkers with different reactive groups such as hydrazide, amino, and aminooxy. The crosslinking efficiencies of the three crosslinkers were systematically evaluated, giving the best reactivity of the amino-functionalized crosslinker BAP. Furthermore, BAP was extended to the crosslinking analysis of Escherichia coli lysate in combination with efficient crosslink enrichment. A total of 1291 D/E-D/E crosslinks involved in 392 proteins were identified under a false discovery rate (FDR) of ≤1%. Obvious structural complementarity of BAP was exhibited to the lysine-targeting crosslinker, facilitating the capability of CXMS for protein structure elucidation. To the best of our knowledge, this was the first time for the carboxyl-selective crosslinker to achieve proteome-wide crosslinking analysis of the whole cell lysate. Collectively, we believe that this work not only expands on a promising toolkit of CXMS targeting acidic residues but also provides a valuable guideline to advance the performance of carboxyl-selective crosslinkers.

The cytotoxicity of PM2.5 and its effect on the secretome of normal human bronchial epithelial cells.

Exposure to airborne fine particulate matter (PM2.5) induced various adverse health effects, such as metabolic syndrome, systemic inflammation, and respiratory disease. Many works have studied the effects of PM2.5 exposure on cells through intracellular proteomics analyses. However, changes of the extracellular proteome under PM2.5 exposure and its correlation with PM2.5-induced cytotoxicity still remain unclear. Herein, the cytotoxicity of PM2.5 on normal human bronchial epithelia cells (BEAS-2B cells) was evaluated, and the secretome profile of BEAS-2B cells before and after PM2.5 exposure was investigated. A total of 83 proteins (58 upregulated and 25 downregulated) were differentially expressed in extracellular space after PM2.5 treatment. Notably, we found that PM2.5 promoted the release of several pro-apoptotic factors and induced dysregulated secretion of extracellular matrix (ECM) constituents, showing that the abnormal extracellular environment attributed to PM2.5-induced cell damage. This study provided a secretome data for the deep understanding of the molecular mechanism underlying PM2.5-caused human bronchial epithelia cell damage.

Lipopolysaccharide-pretreated mesenchymal stem cell-conditioned medium optimized with 10 kDa filter attenuates the injury of H9c2 cardiomyocytes in a model of hypoxia/reoxygenation.

Mesenchymal stem cell-derived conditioned medium (MSC-CM) improves cardiac function, which is partly attributed to the released paracrine factors. Since such cardioprotection is moderate and transient, it is essential that MSC-CM's effective components are optimized to alleviate myocardial injury. To optimize MSC-CM, MSCs were treated with or without lipopolysaccharides (LPSs) for 48 h (serum-free), and the supernatant was collected. Then, LPS-CM (MSC stimulated by LPS) was further treated with LPS remover (LPS Re-CM) or was concentrated with a 10 kDa cutoff filter (10 kDa-CM). Enzyme-linked immunosorbent assay showed that all the pretreatments increased the levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin growth factor (IGF) except LPS Re-CM; 10 kDa-CM was superior to the other CMs. Cell Counting Kit-8 displayed that the viability of injured H9c2 cells was enhanced with the increase in the MSC-CM concentration. We also found that the 10 kDa-CM significantly alleviated H9c2 hypoxia/reoxygenation (H/R) injury, as evidenced by the increased Bcl-2/Bax ratio, and decreased the levels of lactate dehydrogenase and cardiac troponin. Transmission electron microscopy (TEM), TdT-mediated dUTP nick-end labelling (TUNEL), and hematoxylin and eosin staining (H&E) confirmed that 10 kDa-CM inhibited H/R-induced H9c2 morphological changes. Proteomic analysis identified 41 differentially expressed proteins in 10 kDa-CM, among which anti-inflammation, proangiogenesis, and antiapoptosis were related to cardiac protection. This study indicates that 10 kDa-CM protects H9c2 cardiomyocytes from H/R injury by preserving most of the protective factors, such as VEGF, HGF, and IGF, in MSC-CM.

A1 Ions: Peptide-Specific and Intensity-Enhanced Fragment Ions for Accurate and Multiplexed Proteome Quantitation.

Accurate proteome quantitation is of great significance to deeply understand various cellular and physiological processes. Since a1 ions, generated from dimethyl-labeled peptides, exhibited high formation efficiency (up to 99%) and enhanced intensities (2.34-fold by average) in tandem mass spectra, herein, we proposed an a1 ion-based proteome quantitation (APQ) method, which showed high quantitation accuracy (relative errors < 7%) and precision (median coefficients of variation ≤ 11%) even in a 20-fold dynamic range. Notably, due to the mass differences of a1 ions from peptides with different N-terminal amino acids, APQ demonstrated interference-free capacity by distinguishing target peptides from the coisolated ones. By designing an isobaric dimethyl labeling strategy, we achieved simultaneous proteome-wide measurements across up to eight samples. Using APQ to quantify the time-resolved proteomic profiles during a TGF-ß-induced epithelial-mesenchymal transition, we found many differentially expressed proteins associated with fatty acid degradation, indicating that fatty acid metabolism reprogramming occurred during the process. The APQ method combines high quantitation accuracy with multiplexing capacity, which is suitable for deep mining and understanding of dynamic biological processes.

An artificial antibody for exosome capture by dull template imprinting technology.

Exosomes are extracellular vesicles with unique size distribution derived from the parent cells. They are involved in intercellular communication and transport, and are also biomarkers for early diagnosis and prognosis of disease. However, the isolation and characterization of exosomes face the challenges of large sample requirements and low enrichment efficiency of traditional methods. Herein, a simple method is proposed for the preparation of an artificial antibody that has a synergistic effect by featuring nanocavities obtained by dull template imprinting and molecular recognition conferred by electrostatic interaction. With this artificial antibody, highly efficient capture and proteome analysis of exosomes from urine and cell culture media are achieved: for urine, the abundance of Top100 exosomal proteins enriched by this artificial antibody increased from 1.85% to 9.66%. For the cell culture medium, the abundance of the Top100 proteins enriched by this artificial antibody was 28.4%. Moreover, this artificial antibody has a comparable effect to the commercial precipitation-based method in capturing exosomes and has advantages in removing contaminants such as prothymosin alpha (PTMA), demonstrating the superior selectivity of the artificial antibody. Overall, this artificial antibody holds promise to capture exosomes from biofluids and is compatible with subsequent proteome analysis.

All-Ion Monitoring-Directed Low-Abundance Protein Quantification Reveals CALB2 as a Key Promoter in Hepatocellular Carcinoma Metastasis.

Because of the wide abundance range of the proteome, achieving high-coverage quantification of low-abundance proteins is always a major challenge. In this study, a complete pipeline focused on all-ion monitoring (AIM) is first constructed with the concept of untargeted parallel-reaction monitoring, including the seamless connection of protein sample preparation, liquid chromatography mass spectrometry (LC-MS) acquisition, and algorithm development to enable the in-depth quantitative analysis of low-abundance proteins. This pipeline significantly improves the reproducibility and sensitivity of sample preparation and LC-MS acquisition for low-abundance proteins, enabling all the precursors ions fragmented and collected. Contributed by the advantages of the AIM method with all the target precursor acquisition by the data-dependent acquisition (DDA) approach, together with the ability of data-independent acquisition to fragment all precursor ions, the quantitative accuracy and precision of low-abundance proteins are greatly enhanced. As a proof of concept, this pipeline is employed to discover the key differential proteins in the mechanism of hepatocellular carcinoma (HCC) metastasis. On the basis of the superiority of AIM, an extremely low-abundance protein, CALB2, is proposed to promote HCC metastasis in vitro and in vivo. We also reveal that CALB2 activates the TRPV2-Ca2+-ERK1/2 signaling pathway to induce HCC cell metastasis. In summary, we provide a universal AIM pipeline for the high-coverage quantification of low-abundance functional proteins to seek novel insights into the mechanisms of cancer metastasis.

Zn(II)-DPA functionalized graphene oxide two-dimensional nanocomposites for N-phosphoproteins enrichment.

Protein N-phosphorylation is increasingly recognized as a critical post-translational modification in central metabolism and cell signaling. However, other functions of N-phosphorylation in organism are largely unexplored which is attributed to lack high efficient enrichment methods. To address this, Bis(zinc(II)-dipicolylamine) functionalized graphene oxide nanocomposites (Zn(II)-DPA-GO) was designed via loading bis(zinc(II)-dipicolylamine) (Zn(II)-DPA) on graphene oxide surface by π-π stacking, with the advantages of large immobilization amount of Zn(II)-DPA, layered structure, excellent solubility and low non-special adsorption. The material was firstly verified excellent enrichment ability for standard proteins and E.coli lysates. By integrating the enrichment method into proteomics workflow, 36 N-phosphoproteins functioned in metabolic process, biological regulation, localization, cellular processes were identified from Bacillus subtilis. Furthermore, motif analysis showed that leucine and isoleucine were enriched near the sites, providing potential clues for the discovery of new sites. The method provides an alternative for the discovery of N-phosphoproteins with significant biological functions.

Targeted killing of tumor cells based on isoelectric point suitable nanoceria-rod with high oxygen vacancies.

Nanozymes have great potential applications in tumor treatment due to their good stability, high biocompatibility, easy preparation and versatility. However, it remains a challenge to design highly active nanozymes with tumor cell targeting. Herein, three nanoceria structures (nanoceria-rod; nanoceria polyhedra, abbreviated as nanoceria-poly.; and nanoceria-cube) with different surface oxygen vacancy concentrations are designed. Among them, nanoceria-rod shows the highest enzyme activity and tumor cell toxicity because of its highest concentration of oxygen vacancies on the surface. Further study shows that nanoceria-rod can selectively enter tumor cells because nanoceria-rod with a suitable isoelectric point (IEP) remains positively charged in the acidic microenvironment of the tumor but negatively charged in the physiological microenvironment of normal cells. Nanoceria-rod distributes in lysosomes and phagosomes to produce reactive oxygen species (ROS) in tumor cells. Finally, the mitochondrial membrane potential (MMP) was reduced, which caused cell apoptosis. This study provides an interesting new tumor-targeting therapy method, which could also be extended to other drug nanocarriers and diagnostic imaging nanomaterials for tumors.

Bone marrow mesenchymal stem cell-derived exosomal miR-34c-5p ameliorates RIF by inhibiting the core fucosylation of multiple proteins.

Renal interstitial fibrosis (RIF) is an incurable pathological lesion in chronic kidney diseases. Pericyte activation is the major pathological characteristic of RIF. Fibroblast and macrophage activation are also involved in RIF. Studies have revealed that core fucosylation (CF), an important post-translational modification of proteins, plays a key role in pericyte activation and RIF by regulating multiple profibrotic signaling pathways as a hub-like target. Here, we reveal that mesenchymal stem cell (MSC)-derived exosomes reside specifically in the injured kidney and deliver microRNA (miR)-34c-5p to reduce cellular activation and RIF by inhibiting CF. Furthermore, we showed that the CD81-epidermal growth factor receptor (EGFR) ligand-receptor complex aids the entry of exosomal miR-34c-5p into pericytes, fibroblasts, and macrophages. Altogether, our findings reveal a novel role of MSC-derived exosomes in inhibiting multicellular activation via CF and provide a potential intervention strategy for renal fibrosis.

Surface Nanosieving Polyether Sulfone Particles with Graphene Oxide Encapsulation for the Negative Isolation toward Extracellular Vesicles.

Extracellular vesicles (EVs) contain specific biomarkers for disease diagnosis. Current EV isolation methods are hampered in important biological applications due to their low recovery and purity. Herein, we first present a novel EV negative isolation strategy based on surface nanosieving polyether sulfone particles with graphene oxide encapsulation (SNAPs) by which the coexisting proteins are irreversibly adsorbed by graphene oxide (GO) inside the particles, while EVs with large sizes are excluded from the outside due to the well-defined surface pore sizes (10-40 nm). By this method, the purity of the isolated EVs from urine could be achieved 4.91 ± 1.01e10 particles/µg, 40.9-234 times higher than those obtained by the ultracentrifugation (UC), size-exclusion chromatography (SEC), and PEG-based precipitation. In addition, recovery ranging from 90.4 to 93.8% could be obtained with excellent reproducibility (RSD < 6%). This was 1.8-4.3 times higher than those obtained via SEC and UC, comparable to that obtained by PEG-based precipitation. Taking advantage of this strategy, we further isolated urinary EVs from IgA nephropathy (IgAN) patients and healthy donors for comparative proteome analysis, by which significantly regulated EV proteins were found to distinguish IgAN patients from healthy donors. All of the results indicated that our strategy would provide a new avenue for highly efficient EV isolation to enable many important clinical applications.

Bis(zinc(II)-dipicolylamine)-functionalized sub-2 µm core-shell microspheres for the analysis of N-phosphoproteome.

Protein N-phosphorylation plays a critical role in central metabolism and two/multicomponent signaling of prokaryotes. However, the current enrichment methods for O-phosphopeptides are not preferred for N-phosphopeptides due to the intrinsic lability of P-N bond under acidic conditions. Therefore, the effective N-phosphoproteome analysis remains challenging. Herein, bis(zinc(II)-dipicolylamine)-functionalized sub-2 µm core-shell silica microspheres (SiO2@DpaZn) are tailored for rapid and effective N-phosphopeptides enrichment. Due to the coordination of phosphate groups to Zn(II), N-phosphopeptides can be effectively captured under neutral conditions. Moreover, the method is successfully applied to an E.coli and HeLa N-phosphoproteome study. These results further broaden the range of methods for the discovery of N-phosphoproteins with significant biological functions.

Carboxypeptidase B-Assisted Charge-Based Fractional Diagonal Chromatography for Deep Screening of C-Terminome.

The determination of protein C-termini is of great significance for protein function annotation and proteolysis research. However, the progress of C-terminomics is still far behind its counterpart, N-terminomics, because of the low reactivity of the carboxyl group. Herein, we developed a negative selection strategy, termed carboxypeptidase B-assisted charge-based fractional diagonal chromatography (CPB-ChaFRADIC), to achieve a global C-terminome analysis. The highly reactive carboxypeptidase B cleavage was utilized to reduce the charge state of non-C-terminal peptides. Together with high-performance charge-based fractional diagonal chromatography, the C-terminal peptides could be isolated. Such a strategy was applied for profiling C-termini from Escherichia coli cell lysates and 441 canonical C-termini and 510 neo-C-termini originating from proteolytic processing were identified. These findings represent 2-fold and 5.8-fold that of identified C-termini via direct analysis, respectively. Using parallel digestion with trypsin and LysC, such a strategy enabled the identification of 604 canonical C-termini and 818 neo-C-termini, representing the largest C-terminome data set of E. coli, and no deficiency in His/Lys/Arg-containing C-terminal peptides was observed. The presented CPB-ChaFRADIC strategy is therefore a highly efficient and unbiased strategy for large-scale C-terminome analysis. Furthermore, using the CPB-ChaFRADIC strategy, we identified 107 cleavage sites and 102 substrates of caspase-3 in Jurkat cells, demonstrating that the CPB-ChaFRADIC strategy shows great promise in promoting proteolysis research. Data are available via ProteomeXchange with identifier PXD018520.

Ampholine immobilized polymer microspheres for increasing coverage of human urinary proteome.

Urinary proteome, as an important component of body fluid proteome, could reflect kidney, urogenital tract function and pathological changes of human organs. This study reports a convenient strategy for urine proteome analysis through ampholine immobilized polymer microsphere (ampholine@PM) fractionation strategy. After ampholine@PM treatment, 16,543 unique peptides corresponding to 2173 non-redundant urinary proteins were identified, while only 856 proteins, corresponding to 3524 peptides were identified in the crude urine sample. The number of proteins and peptides was increased by 1.54 and 3.69 times, respectively. 31 urinary candidate biomarkers have also been identified (17 candidate biomarkers of glomerular injury and 14 candidate biomarkers of tubular injury), showing the potential of our strategy in urinary biomarker discovery study. In additional to the urine proteome, N-glycoproteome analysis was also performed after ampholine@PM fractionation followed by the N-glycopeptides enrichment. The number was increased from 144 to 281 for N-glycoproteins, 261 to 709 for N-glycopeptides, and 226 to 493 for N-glycosylation sites, after ampholine@PM treatment. Based on the significant increase on the identified N-glycoprotein number, ampholine@PM fractionation strategy also offered a beneficial tool for the post translational modification analysis of urine proteome.

Ionic liquid-assisted protein extraction method for plant phosphoproteome analysis.

Protein phosphorylation is one of the most important post-translational modifications (PTM) and plays critical roles in maintaining many biological processes of plant species, such as being a significant signal related to resistance to tobacco mosaic virus (TMV) infection in tobacco. Compared to other organisms, in-depth profiling of plant phosphoproteome remains challenging due to the harsh extraction environment of plant proteins and low abundance of plant phosphorylation, generally requiring large amount of plant materials. Herein, we developed an integrated strategy for efficient sample preparation of amounts of plant tissues, by integrating ionic liquid (IL)-assisted protein extraction, in-solution digestion, precipitation-assisted IL removal, as well as immobilized metal ion affinity chromatography (IMAC) enrichment of phosphopeptides together. In this strategy, to improve the efficiency of protein extraction and enzymatic digestion, IL of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was used as the solubilizer due to its excellent solubilizing ability and enzyme compatibility demonstrated in our previous work. Briefly, the extraction capability of C12Im-Cl for protein amount from tobacco leaves was improved 1.9-fold compared to the commonly used urea-assisted method. Notably, to avoid its interference with subsequent LC-MS analysis, the IL was easily removed from the peptide solution by our proposed ion substitution-mediated C12Im + precipitation strategy with high efficiency. By handling 10 mg of starting protein materials of tobacco leaves, 14,441 unique phosphopeptides, assigned to 5153 unique phosphoproteins were confidently identified. To the best of our knowledge, this was the most comprehensive phosphorylation dataset for tobacco so far. All the results demonstrated our strategy was of great potential to promote the large-scale analysis of plant phosphoproteome.