The structures of proteins are directly related to their biological functions. The regulation of protein functions mainly depends on the dynamic regulation of their conformations and interactions. The study of protein structures and functions plays an essential role in the field of life science, which is also an important direction of proteomics development. The emergence of proteomics has led to major technological advances in mass spectrometry (MS). These advancements have not only benefitted MS-based high-throughput proteomics but have also increased the impact of MS in the fields of structural and molecular biology. This review briefly describes the principles, progresses and applications of the MS-based structural proteomics methods in recent years, including the native MS analysis of active proteins, limited proteolysis, chemical cross-linking, hydrogen-deuterium exchange, covalent labeling, and thermal proteome profiling. Finally, future development of the structural proteomics was summarized and prospected.
Construction of microbial cell factories is a feasible strategy for the sustainable production of chemicals, biofuels, and pharmaceutical molecules. However, the complex metabolism and rigid regulation of microbes hinders the efficient synthesis of the products of interest. Proteomics and metabolomics analyze enzymes and metabolites in terms of systems biology, thus unraveling complex biological systems and providing significant clues for microbial metabolic engineering. Here, the applications of proteomics and metabolomics in microbial metabolic engineering were reviewed, including the construction of genome-scale metabolic models, optimization of microbial product synthesis, guidance of microbial stress tolerance engineering, and prediction of rate-limiting steps. In addition, proteomics and metabolomics could be employed to explore secondary metabolic pathways in plants, to reveal novel genes or pathways for microbial synthesis of natural products. Finally, the development of bio-big data was discussed.
Endogenous phytohormone is a kind of trace organic small molecule compound synthesized in plants. It plays important roles in regulating the growth and development of plants throughout their life cycles, and responding to external stimuli. With the development of analytical methods for the detection of phytohormone, the amount of analytical samples is gradually reduced. Moreover, the differences in the types and contents of phytohormones in different plant tissues (or organs) are constantly presented. These developments greatly promote the study of the physiological effects of plant hormones. In recent years, the temporal and spatial distribution of endogenous phytohormones in several plant samples has attracted more and more attention in plant hormone analysis. This review summarizes the research progress made in the study of the spatial and temporal distribution of endogenous phytohormones in the last five years. Mainly, the review summarizes and discusses the difficulties associated with analysis, the development of analytical methods, and the spatial and temporal distribution of major plant hormones. The sampling methods, sample preparation and detection methods for spatial and temporal distribution studies are discussed in detail with a special focus on our work in plant hormone detection. Finally, the future development of the plant hormone analysis research field analysis is prospected.
Phthalates (PAEs) are a class of endocrine-disrupting chemicals. In recent years, the harmful effects of PAEs on human health, in particular their toxicity toward the reproductive system and development, have received significant attention because of their increasing production and usage. PAEs are ubiquitous in the environment and food products, resulting in unavoidable and long-term exposure in humans. Therefore, exposure and risk assessments of PAEs in humans are necessary. Screening and quantification of phthalate metabolites in urine is an important method for evaluating PAEs exposure, and establishing accurate analytical methods for this purpose has become a high priority. Phthalate monoesters and secondary metabolites are the most commonly targeted biomarkers of exposure to short-and long-chain PAEs, respectively. The combination of off-line or on-line solid phase extraction (SPE) with high performance liquid chromatography-tandem mass spectrometry has become the preferred method for the determination of phthalate metabolites. This paper reviews the analytical methods available for the detection of urinary phthalate metabolites and discusses the advantages, limitations, and challenges presented by each method in practical applications.
A metal-organic framework (MOF) MIL-101(Cr) incorporated polymer monolithic column was prepared, and a method was developed for the determination of 4-nitrophenol, 3-methylphenol, 2-nitrophenol and 2,4-dichlorophenol in water samples by on-line solid phase extraction (SPE) coupled with high performance liquid chromatography (HPLC). Effects of sample solution pH values, sample loading time, sample loading flow rates and desorption time on the on-line SPE of phenols were investigated. Under the optimized extraction conditions, large enhancement factors, wide linearity range, good precisions and low limits of detection were achieved. The results showed that the incorporation of MOFs in porous polymer monolith to fabricate MOF-incorporated monolith is one of the best ways to promote and expand the application of MOFs in on-line SPE.
Core-shell magnetic nanoparticles, Fe3O4@MIL-100(Fe), were synthesized by layer-by-layer self-assembly. The final material, Fe3O4@MC, was obtained after high temperature calcination. Fe3O4@MC magnetic nanoparticles exhibited large pore size (17.78 nm) and high carbon content (6.79%). Thirty-three peptides of bovine serum albumin (BSA) digest were enriched by Fe3O4@MC magnetic nanomaterial. Fe3O4@MC magnetic nanomaterial also showed high selectivity for peptides when the mass ratio of BSA digest to BSA was 1:400. In addition, the nanomaterial material exhibited excellent enrichment performance for the endogenous peptides of human serum.
Protein persulfidation is an important oxidative translational modification which plays vital roles in many important processes including cellular senescence, endoplasmic reticulum stress, vasorelaxation, and apoptosis. The proteome-wide analysis of persulfidation is of great importance; therefore, this study combines filter-aided sample preparation with an iodoacetic acid functionalized polyamidoamine dendrimer to enrich persulfidated peptides (denoted as filter-aided dendrimer enrichment strategy, FADE). To evaluate the performance of this strategy, the synthetic persulfidated standard peptide was spiked into bovine serum albumin (BSA) digests at a mass ratio of 1:100, and was successfully identified by FADE. Moreover, in combination with stable isotope labelling by amino acids in cell culture technology, the FADE strategy was applied to enrich persulfidated peptides from NaHS-stimulated SHSY5Y cells over a concentration gradient, resulting in the identification of 163 persulfidated peptides. Bioinformatic analysis indicated that persulfidation might play important roles in the central nervous system.
The study of protein glycosylation is important to deepening the current understanding of its biological functions as well as to elucidate novel biomarkers of glycosylation. However, glycopeptides must be enriched prior to analysis due to their naturally low abundance. In this study, tryptophan functionalized polymer materials (denoted as Poly-Trp) were synthesized to serve as a novel biomimetic polymers. The resulting materials were characterized by various methods including scanning electron microscopy, thermal analysis, and infrared spectrometry, validating the successful synthesis of Poly-Trp. The retention of glycopeptides on Poly-Trp was investigated revealing that the retention ability decreased as the acetonitrile content of the mobile phase decreased and its acidity increased. Poly-Trp exhibited higher enrichment selectivity for glycopeptides from bovine fetuin than the commercial material ZIC-HILIC, and aminated silica which could defect the interference of 100-fold amount of substance ratios of bovine serum albumin. Poly-Trp is expected to have further applications in the purification of biological samples for the study of glycosylation.
Diabetes is a systemic metabolic disorder syndrome, mainly characterized by hyperglycemia, and is associated with the dysfunction of various organs, such as liver, pancreas, intestine, adipose muscle tissue, kidney and brain. It has become a global epidemic disease that seriously threatens human health. Therefore, mapping the global molecular signatures of diabetes-related disease spectrum can provide more comprehensive data to understand early clinical diagnosis, molecular typing, and pathological processes involved in diabetes mellitus. In this study, we performed a quantitative differential analysis on the endogenous peptidome of the serum samples obtained from healthy, prediabetes and type 2 diabetes groups to explore the peptidomics evolution in the development of diabetes. Partial least squares-discriminant analysis (PLS-DA) was used for pattern recognition. A nonparametric test was examined to find out the significantly changed endogenous peptides. As a result, 690 serum endogenous peptides were identified totally, among which 163 endogenous peptides were statistically different among the three groups. This could be promising quantitative peptidomics data for early screening, diagnosis and molecular typing of type 2 diabetes mellitus.
Exosomes are tiny vesicles secreted by cells, can be important mediators of cell-to-cell communication, and play unique roles in disease diagnosis and treatment. Osteoporosis is a metabolic bone disease with high incidence in the elderly, characterized by low bone mineral density and deterioration of bone microstructure. Highly specific diagnostic methods capable of identifying early-stage osteoporosis are urgently needed. In this study, serum exosomes were comprehensively enriched and characterized. In total, 179 exosomal proteins were identified using liquid chromatography-mass spectrometry, most of which are involved in important biological processes such as defense and immune responses. Through label free quantification of serum exosomes, 188, 224, and 185 proteins were identified in the normal, osteopenia, and osteoporosis groups, respectively. Quantitative results also showed that 17 proteins were significantly (p <0.05) dysregulated in the osteoporosis and osteopenia groups, including Integrin β 3, Integrin α 2 β 1, Talin 1, and Gelsolin. This study provides potential molecular markers for osteoporosis studs and will contributes to the elucidating the pathogenesis of osteoporosis.
The visualization of the microcosmic behavior of proteins in vivo is the key to real-time monitoring of proteins. A series of wash-free SNAP-tag probes were designed and synthesized based on the combination of SNAP-tag and small organic molecule fluorescent dyes. SNAP-tag, which specifically recognized O6-benzylguanine, could be labeled with a fluorophore (e. g., 1,8-naphthalimide) through the formation of covalent bonds. Furthermore, the change from a hydrophilic environment to the hydrophobic cavum of SNAP-tag realized a 2-13-fold enhancement in fluorescence. Through the fusion of SNAP-tag and the target protein, the probes could recognize the mitochondrial proteins (e. g., cytochrome oxidase, Cox8A) and nuclear proteins (e. g., H2B) in living cells. Besides, the fluorescent probes allowed the in-situ real-time monitoring of proteins without washing.
One of the major shortcomings in top-down proteomics is the lack of efficient separations for intact proteins that can be effectively coupled to mass spectrometry. Capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (nanoRPLC) are two methods that can be coupled to mass spectrometry directly and have been recently advanced in terms of their ability to separate intact proteins in complex biological mixtures. In this work, for the first time, we compared the state-of-the-art nanoRPLC-MS/MS and CZE-MS/MS platforms for top-down characterization of a standard protein mixture and an Escherichia coli (E. coli) proteome sample. CZE-MS produced comparable signals of standard proteins to RPLC-MS with 10-times less sample consumption. Interestingly, the proteins in RPLC-MS tended to have higher charge states than in CZE-MS, most likely due to the high acetonitrile concentration in RPLC mobile phase, leading to the more extensive unfolding of proteins in RPLC compared to in CZE. CZE-MS/MS identified 159 proteins and 513 proteoforms using 1-μg E. coli proteins in a single run and outperformed RPLC-MS/MS using 1-μg E. coli proteins in terms of protein and proteoform identifications (159 vs. 105 proteins and 513 vs. 277 proteoforms). The RPLC-MS/MS using 8-μg E. coli proteins identified 245 proteins and 1004 proteoforms in a single run, and the data was much better than that from CZE-MS/MS (1-μg E. coli proteins) regarding the number of identifications because of the 8-times higher sample loading amount and significantly wider separation window of RPLC-MS/MS compared to CZE-MS/MS.
Speckle type BTB/POZ protein (SPOP) is one of the most frequently mutated protein in prostate cancer. In this study, proteomics and metabolomics were integrated to study the effects of SPOP mutation on metabolism. First, LNCaP control (CON), SPOP wild-type (SPOP_WT), and SPOP mutation (SPOP_Y87N and SPOP_F133L) cells were subjected to a metabolomics study. The metabolomics data of LNCaP CON, SPOP_WT, SPOP_Y87N, and SPOP_F133L cells were evaluated by partial least squares-discriminant analysis (PLS-DA). Four groups could be clearly differentiated with an explanation ability of R2X=0.512, R2Y=0.616 and predictive ability of Q2=0.475. Totally, 36 differential metabolites were defined with variable importance for the projection (VIP) value > 1. Then, the 36 metabolites were subjected to one-way ANOVA analysis. Fumaric acid, malic acid, citric acid, aspartic acid, and asparagine were increased in LNCaP SPOP mutation cells compared to that in LNCaP SPOP_WT cells. Using a proteomics study, 909 differential proteins were found in LNCaP SPOP_Y87N and SPOP_F133L cells. MetaboAnalyst 3.0 was used to enrich metabolic pathways by using differential metabolites. KOBAS 3.0 was used to enrich metabolic pathways by using differential proteins. Both metabolomics and proteomics analysis showed that the tricarboxylic acid (TCA) cycle and aminoacyl-tRNA biosynthesis were significantly changed. To validate these findings, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was performed in Du145 SPOP knock-out cells. The results indicated that the TCA cycle was activated in Du145 SPOP knock-out cells. Collectively, this study found that SPOP mutation significantly promoted TCA cycle in prostate cancer cells.
The global increase in the prevalence of gestational diabetes mellitus(GDM)in recent years has prompted the study of the effect of GDM on the metabolism between mother and fetus. In this study, the metabolomic investigation of the umbilical cord blood of mothers presenting GDM was performed using liquid chromatography-mass spectrometry (LC-MS), orthogonal projections to latent structures discriminant analysis (OPLS-DA), and network analysis to assess GDM-related metabolic biomarkers. The results showed that arachidonic acid (AA) played an important role in the key metabolic network while further pathway analysis suggested that GDM induced unsaturated fatty acid metabolic disorder. This study provides the underlying metabolic mechanism of GDM-induced metabolic abnormalities between mother and fetus.
One of the most abundant biological volatile organic compounds (BVOCs) in the atmosphere, monoterpene, is characterized by its short lifetime, low concentration, fast temporal and spatial variations, and wide variety of isomers. In this study, a multi-capillary column (MCC) was combined with high-pressure photoionization time-of-flight mass spectrometry (HPPI-TOF MS) and employed to develop an MCC-HPPI-TOF MS combination instrument as an online two-dimensional gas chromatography-mass spectrometry (GC-MS) method for the rapid qualitative and quantitative analysis of monoterpene isomers. As a result, six monoterpene isomers, α -pinene, β -pinene, α -terpinene, γ -terpinene, 3-carene, and limonene, were successfully isolated in 180 s with limits of detection (LODs) as low as 6 μg/m3 without sample pre-enrichment. This method was successfully applied to the rapid online analysis of monoterpenes released from the branches and leaves of Cedrus atlantica and Sabina chinensis, which shows the capability and potential application of the method for the online detection of complex sample mixtures in environmental monitoring, process analysis, and other fields.
A liquid chromatography method was established for the determination of zearalanone (ZAN) raw material. The qualitative analysis of ZAN and its trace impurities was performed by ultra performance liquid chromatography-diode array detector (UPLC-DAD) and ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS), and the response factors of each impurity were calculated. The three main organic impurities in the ZAN raw material were identified as β -zearalanol, α -zearalanol and a dehydration product of zearalanol with relative response factors of 0.5352, 0.8594 and 0.6973, respectively. The main component of the ZAN raw material was determined by the calibration factor normalization method. The purity of zearalanone was determined to be 99.6% with a standard deviation of 0.01%. This method can provide a technical support for the development of ZAN standard materials.
A method for the determination of organic ultravioletfilters (OUFs) in water samples was developed by the combination of polymeric ionic liquid-based magnetic solid-phase extraction (PIL-MSPE) and high performance liquid chromatography-diode array detection (HPLC-DAD). To achieve the optimum extraction performance for the target analytes, several key variables, including desorption solvent, adsorption and desorption time, sample pH value and ionic strength in the sample matrix, were investigated in detail. Under the most favorable extraction conditions, linear behaviors were observed in the ranges of 0.5-200.0 μg/L for octyl salicylate and 0.2-200.0 μg/L for the other analytes. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) for the target OUFs were in the ranges of 0.009-0.13 μg/L and 0.031-0.43 μg/L, respectively. The developed PIL/MSPE-HPLC-DAD method was successfully applied to detect trace OUFs in real water samples. The spiked recoveries at different spiked levels were 71.4%-120%, and the RSDs were below 10% for all the compounds in all the samples. The proposed method has some unique merits including simple operation, fast extraction procedure, high sensitivity and environmental friendliness. It can become a useful method for the monitoring of trace OUFs in water samples.
byDalian Institute of Chemical
Physics,CASNational Chromatographic R. and A.
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