RESUMEN
Many important experiments in proteomics including protein digestion, enzyme substrate screening, enzymatic labeling, etc., involve the enzymatic reactions in a complex system where numerous substrates coexists with an enzyme. However, the enzyme kinetics in such a system remains unexplored and poorly understood. Herein, we derived and validated the kinetics equations for the enzymatic reactions in complex system. We developed an iteration approach to depict the enzymatic reactions in complex system. It was validated by 630 time-course points from 24 enzymatic reaction experiments and was demonstrated to be a powerful tool to simulate the reactions in the complex system. By applying this approach, we found that the ratio of substrate depletion is independent of other coexisted substrates under specific condition. This observation was then validated by experiments. Based on this striking observation, a simplified model was developed to determine the catalytic efficiencies of numerous competing substrates presented in the complex enzyme reaction system. When coupled with high-throughput quantitative proteomics technique, this simplified model enabled the accurate determination of catalytic efficiencies for 2369 peptide substrates of a protease by using only one enzymatic reaction experiment. Thus, this study provided, in the first time, a validated model for the large scale determination of specificity constants which could enable the enzyme substrate screening approach turned from a qualitative method of identifying substrates to a quantitative method of identifying and prioritizing substrates. Data are available via ProteomeXchange with identifier PXD004665.
Asunto(s)
Enzimas/metabolismo , Péptidos/análisis , Proteómica/métodos , Algoritmos , Ensayos Analíticos de Alto Rendimiento , Cinética , Modelos Teóricos , Especificidad por SustratoRESUMEN
We present a new strategy for systematic identification of phosphotyrosine (pTyr) by affinity purification mass spectrometry (AP-MS) using a Src homology 2 (SH2)-domain-derived pTyr superbinder as the affinity reagent. The superbinder allows for markedly deeper coverage of the Tyr phosphoproteome than anti-pTyr antibodies when an optimal amount is used. We identified â¼20,000 distinct phosphotyrosyl peptides and >10,000 pTyr sites, of which 36% were 'novel', from nine human cell lines using the superbinder approach. Tyrosine kinases, SH2 domains and phosphotyrosine phosphatases were preferably phosphorylated, suggesting that the toolkit of kinase signaling is subject to intensive regulation by phosphorylation. Cell-type-specific global kinase activation patterns inferred from label-free quantitation of Tyr phosphorylation guided the design of experiments to inhibit cancer cell proliferation by blocking the highly activated tyrosine kinases. Therefore, the superbinder is a highly efficient and cost-effective alternative to conventional antibodies for systematic and quantitative characterization of the tyrosine phosphoproteome under normal or pathological conditions.
Asunto(s)
Fosfotirosina/metabolismo , Proteómica , Línea Celular Tumoral , Humanos , Espectrometría de Masas , Fosforilación , Fosfotirosina/química , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Dominios Homologos srcRESUMEN
Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ', a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ' subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ' were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.
Asunto(s)
Complejo de Proteína del Fotosistema II/química , Proteínas de Plantas/química , Rhodophyta/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Datos de Secuencia Molecular , Complejo de Proteína del Fotosistema II/ultraestructura , Proteínas de Plantas/ultraestructura , Conformación Proteica , Multimerización de Proteína , Alineación de SecuenciaRESUMEN
During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein phosphatase 1 (PP1). However, the regulatory mechanisms underlying the aforementioned hierarchical signaling cascade during mitotic chromosome segregation have remained elusive. Sds22 is a conserved regulator of PP1 activity, but how it regulates PP1 activity in space and time during mitosis remains elusive. Here we show that Sds22 is a novel and cognate substrate of PLK1 in mitosis, and the phosphorylation of Sds22 by PLK1 elicited an inhibition of PP1-mediated dephosphorylation of Aurora B at threonine 232 (Thr232) in a dose-dependent manner. Overexpression of a phosphomimetic mutant of Sds22 causes a dramatic increase in mitotic delay, whereas overexpression of a non-phosphorylatable mutant of Sds22 results in mitotic arrest. Mechanistically, the phosphorylation of Sds22 by PLK1 strengthens the binding of Sds22 to PP1 and inhibits the dephosphorylation of Thr232 of Aurora B to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling hierarchy that orchestrates dynamic protein phosphorylation and dephosphorylation of critical mitotic regulators during chromosome segregation to guard chromosome stability.
Asunto(s)
Anafase/fisiología , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica/fisiología , Cromosomas Humanos/metabolismo , Metafase/fisiología , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Aurora Quinasa B/genética , Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/genética , Inestabilidad Cromosómica/fisiología , Cromosomas Humanos/genética , Células HEK293 , Células HeLa , Humanos , Fosforilación , Proteína Fosfatasa 1/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Quinasa Tipo Polo 1RESUMEN
Temporal tyrosine phosphorylation (pTyr) plays a crucial role in numerous cellular functions. The characterization of the tyrosine phosphorylation states of cells is of great interest for understanding the underlying mechanisms. In this study, we developed sensitive and cost-effective methods for the assay of the global protein tyrosine phosphorylation in complex samples by using a novel engineered pTyr binding protein, Src SH2 domain triple-point mutant (Trm-SH2). Taking the advantage of the pan-specific interaction of Trm-SH2 to pTyr, a high throughput approach was developed to determine the total protein tyrosine phosphorylation level in a sample. This method allowed the detection of 0.025 ng of tyrosine phosphorylated proteins. The Trm-SH2 was further exploited to develop a method to profile the global tyrosine phosphorylation state. When this approach was applied to analyze the tyrosine phosphoproteome upon stimulation, distinct patterns were observed. This approach is readily used in many research and clinical fields for the analysis of tyrosine phosphorylated proteins in complex samples, including classifying aberrant phosphotyrosine-dependent signaling in cancer.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Fosfotirosina/análisis , Proteínas Tirosina Quinasas/metabolismo , Tirosina/metabolismo , Western Blotting , Factor de Crecimiento Epidérmico/farmacología , Células HeLa , Humanos , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutagénesis , Fosforilación/efectos de los fármacos , Fosfotirosina/inmunología , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Dominios Homologos src/genéticaRESUMEN
Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered as a hallmark in cellular signal transduction. However, its analysis at the proteome level remains challenging. The conventional immunopurification (IP) approach using antibodies specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. Our recent study indicated that SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we presented an efficient SH2 superbinder based workflow for the sensitive analysis of tyrosine phosphoproteome. This new method can identify 41% more pTyr peptides than the previous method. Its excellent performance was demonstrated by the analysis of a variety of different samples. For the highly tyrosine phosphorylated sample, for example, pervanadate-treated Jurkat T cells, it identified over 1800 high confident pTyr sites from only 2 mg of proteins. For the unstimulated Jurkat cells, where the pTyr events rarely occurred, it identified 343 high confident pTyr sites from 5 mg of proteins, which was 31% more than that obtained by the antibody-based method. For the heterogeneous sample of tissue, it identified 197 high confident pTyr sites from 5 mg protein digest of mouse skeletal muscle. In general, it is a sensitive, robust and cost-effective approach and would have wide applications in the study of the regulatory role of tyrosine phosphorylation in diverse physiological and pathological processes.
Asunto(s)
Proteoma/análisis , Tirosina/química , Tirosina/metabolismo , Secuencia de Aminoácidos , Humanos , Células Jurkat , Espectrometría de Masas/métodos , Péptidos/química , Péptidos/metabolismo , Fosforilación , Proteómica/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.
Asunto(s)
Cromatografía de Afinidad/métodos , Fosfopéptidos/análisis , Proteoma/análisis , Avidina/química , Avidina/metabolismo , Humanos , Células Jurkat , Fosfopéptidos/aislamiento & purificación , Fosforilación , Proteoma/metabolismo , Tirosina/metabolismo , Dominios Homologos srcRESUMEN
Sialylation typically occurs at the terminal of glycans, and its aberration often correlates with diseases including neurological diseases and cancer. However, the analysis of glycoprotein sialylation in complex biological samples is still challenging due to their low abundance. Herein, a histidine-bonded silica (HBS) material with a hydrophilic interaction and switchable surface charge was fabricated to enrich sialylated glycopeptides (SGPs) from the digest of proteomics samples. High selectivity toward SGPs was obtained by combining the superior hydrophilicity and switchable-charge characteristics. During the enrichment of sialylated glycopeptides from bovine fetuin digest, seven glycopeptides were detected even at the ratio of 1:5000 with the nonsialylated glycopeptides, demonstrating the high specificity of SGP enrichment by using HBS material. Then, HBS material was further utilized to selectively enrich SGPs from the protein digest of human serum, and 487 glycosites were identified from only 2 µL of human serum; 92.0% of the glycopeptides contained at least one sialic acid, indicating good performance for SGP enrichment by using HBS material. Furthermore, the prepared HBS material also has great potential applications in the analysis of glycoprotein sialylation from other complex biological samples.
Asunto(s)
Glicoproteínas/sangre , Histidina/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Ácido N-Acetilneuramínico/metabolismo , Dióxido de Silicio/metabolismo , Glicoproteínas/metabolismo , Histidina/química , Estructura Molecular , Ácido N-Acetilneuramínico/sangre , Dióxido de Silicio/química , Propiedades de SuperficieRESUMEN
The diversity of O-linked glycan structures has drawn increasing attention due to its vital biological roles. However, intact O-glycopeptides with different glycans are typically not well elucidated using the current methods. In this work, an integrated strategy was developed for comprehensive analysis of O-GalNAc glycosylation by combining hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type collision induced decomposition (beam-CID) detection, and in silico deglycosylation method for spectra interpretation. In this strategy, the intact O-GalNAc glycopeptides were selectively enriched and the original spectra obtained by time-of-flight (TOF)-CID were preprocessed using an in silico deglycosylation method, enabling direct searching without setting multiple glycosylation modifications, which could significantly decrease the search space. This strategy was applied to analyze the O-GalNAc glycoproteome of human serum, leading to identification of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About 81% of the glycopeptides contained at least one sialic acid, which could reveal the microheterogeneity of O-GalNAc glycosylation. Up until now, this is the largest data set of intact O-GalNAc glycoforms from complex biological samples at the proteome level. Furthermore, this method is readily applicable to study O-glycoform heterogeneity in other complex biological systems.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Glicopéptidos/sangre , Proteómica/métodos , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Fetuínas/metabolismo , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas en TándemRESUMEN
Micropattern topography is widely investigated for its role in mediating stem cell differentiation, but remains unexplored for phenotype switch between mature cell types. This study investigated the potential of inducing tenogenic phenotype in human dermal fibroblasts (hDFs) by artificial elongation of cultured cells. Our results showed that a parallel microgrooved topography could convert spread hDFs into an elongated shape and induce a predominant tenogenic phenotype as the expression of biomarkers was significantly enhanced, such as scleraxis, tenomodulin, collagens I, III, VI, and decorin. It also enhanced the expression of transforming growth factor (TGF)-ß1, but not α-smooth muscle actin. Elongated hDFs failed to induce other phenotypes, such as adiopogenic, chondrogenic, neurogenic, and myogenic lineages. By contrast, no tenogenic phenotype could be induced in elongated human chondrocytes, although chondrogenic phenotype was inhibited. Exogenous TGF-ß1 could enhance the tenogenic phenotype in elongated hDFs at low dose (2 ng/ml), but promoted myofibroblast transdifferentiation of hDFs at high dose (10 ng/ml), regardless of cell shape. Elongated shape also resulted in decreased RhoA activity and increased Rho-associated protein kinase (ROCK) activity. Antagonizing TGF-ß or inhibiting ROCK activity with Y27632 or depolymerizing actin with cytochalasin D could all significantly inhibit tenogenic phenotype induction, particularly in elongated hDFs. In conclusion, elongation of cultured dermal fibroblasts can induce a predominant tenogenic phenotype likely via synergistic effect of TGF-ß and cytoskeletal signaling.
Asunto(s)
Diferenciación Celular/fisiología , Forma de la Célula/fisiología , Fibroblastos/citología , Miofibroblastos/citología , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular , Transdiferenciación Celular , Células Cultivadas/metabolismo , Humanos , Fenotipo , Transducción de Señal/efectos de los fármacosRESUMEN
Large-scale N-glycoproteome studies have been hindered by poor solubility of hydrophobic membrane proteins and the complexity of proteome samples. Herein, we developed a detergent-assisted glycoprotein capture method to reduce these issues by conducting hydrazide chemistry-based glycoprotein capture in the presence of strong detergents such as sodium dodecyl sulfate and Triton X-100. The strong detergents helped to solubilize hydrophobic membrane proteins and then increased the access of hydrazide groups to oxidized glycoproteins, thus increasing the coverage of the N-glycoproteome. Compared with the conventional glycopeptide capture method, the detergent-assisted glycoprotein capture approach nearly doubled the number of N-glycosylation sites identified from HEK 293T cells with improved specificity. Application of this approach in the larger scale N-glycoproteomics analysis of the HEK 293T cell membrane led to the identification of 2253 unique N-glycosites from 953 proteins. Furthermore, the application of this approach to human serum resulted in the identification of 850 N-glycosylation sites without any immunodepletion or fractionation. Overall, the detergent-assisted glycoprotein capture method simplified the capture process, and it increased the number of sites observed on both hydrophobic membrane proteins and hydrophilic secreted proteins.
Asunto(s)
Detergentes/química , Glicoproteínas/análisis , Proteínas de la Membrana/análisis , Proteómica/métodos , Células HEK293 , Humanos , Proteoma/análisis , Suero/químicaRESUMEN
Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochores. During chromosome alignment, kinetochore-bound microtubules undergo dynamic cycles between growth and shrinkage, leading to an oscillatory movement of chromosomes along the spindle axis. Although kinetochore protein CENP-H serves as a molecular control of kinetochore-microtubule dynamics, the mechanistic link between CENP-H and kinetochore microtubules (kMT) has remained less characterized. Here, we show that CSPP1 is a kinetochore protein essential for accurate chromosome movements in mitosis. CSPP1 binds to CENP-H in vitro and in vivo. Suppression of CSPP1 perturbs proper mitotic progression and compromises the satisfaction of spindle assembly checkpoint. In addition, chromosome oscillation is greatly attenuated in CSPP1-depleted cells, similar to what was observed in the CENP-H-depleted cells. Importantly, CSPP1 depletion enhances velocity of kinetochore movement, and overexpression of CSPP1 decreases the speed, suggesting that CSPP1 promotes kMT stability during cell division. Specific perturbation of CENP-H/CSPP1 interaction using a membrane-permeable competing peptide resulted in a transient mitotic arrest and chromosome segregation defect. Based on these findings, we propose that CSPP1 cooperates with CENP-H on kinetochores to serve as a novel regulator of kMT dynamics for accurate chromosome segregation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/fisiología , Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Células HeLa , Humanos , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Mitosis/genética , Unión Proteica , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Protein methylation is receiving more and more attention for its important regulating role in diverse biological processes including epigenetic regulation of gene transcription, RNA processing, DNA damage repair, and signal transduction. Global analysis of protein methylation at the proteome level requires the enrichment of methylated peptides with various forms; unfortunately, the immunoaffinity purification method can only enrich a subset of them due to lacking of pan specific antibody. Because methylation does not significantly alter the physicochemical properties of arginine or lysine residues, chemical approach for global methylome analysis is still at infancy. In this study, by exploiting the fact that the methylation on Arg and Lys prohibiting the cleavage by proteases for these sites, we developed an antibody-free method to enrich methylated peptides, which enabled the identification of 887 methylation forms on 768 sites from HepG2 cells. This technique allows the simultaneous analysis of both Lys and Arg methylation while it has better performance for the identification of Arg methylation. It should find broad applications in studying methylation regulated biological processes.
Asunto(s)
Péptidos/análisis , Péptidos/química , Arginina/química , Arginina/metabolismo , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Lisina/química , Lisina/metabolismo , Metilación , Péptidos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In this study, we developed a Ti(IV) monolithic spin tip for phosphoproteome analysis of a minute amount of biological sample for the first time. The surface of polypropylene pipet tip was activated by the photoinitiator benzophenone under UV light radiation followed by polymerization of ethylene glycol methacrylate phosphate and bis-acrylamide in the tip to form a porous monolith with reactive phosphate groups. The as-prepared tips grafted with monolithic adsorbent were then chelated with titanium(IV) ion for phosphopeptide enrichment. It was found that the tips enabled fast and efficient capture of phosphopeptides from microscale complex samples. The monolithic tip was demonstrated to have a detection limit as low as 5 fmol ß-casein tryptic digest, along with an exceptionally high specificity to capture phosphopeptides from complex tryptic digest mixed with an unphosphorylated protein and a phosphorylated protein at a molar ratio up to 1000:1. When the tip was applied to enrich phosphopeptides from 5 µg of tryptic digest of complex HeLa cell proteins, 1185 high confidence of phosphorylated sites were successfully identified with the specificity as high as 92.5%. So far, this is the most sensitive phosphoproteomics analysis using a standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for proteome-wide phosphorylation analysis in mammalian cells.
Asunto(s)
Cromatografía Liquida/instrumentación , Fosfoproteínas/análisis , Polipropilenos/química , Proteoma/análisis , Espectrometría de Masas en Tándem/instrumentación , Titanio/química , Acrilamidas/química , Adsorción , Benzofenonas/efectos de la radiación , Caseínas/análisis , Cromatografía Liquida/métodos , Células HeLa , Humanos , Límite de Detección , Metacrilatos/química , Fragmentos de Péptidos/análisis , Fosfatos/química , Porosidad , Proteómica , Espectrometría de Masas en Tándem/métodos , Rayos UltravioletaRESUMEN
In situ and timed extraction of cellular peptides is a great challenge for dynamic and global proteomic investigation of live cells. In this work, a mesoporous silica nanocarrier with photoswitchable off/on coumarin gates (MSNcg) was developed for capturing peptides from the cytosol of living HeLa cells. The MSNcg was constructed from mesoporous silica nanoparticle (MSN) and its subsequent modifications with TAT peptides and coumarin, to endow the features of the size-exclusion effect of the mesoporous silica and the localization of nanocarrier at cytosol by TAT peptide and to control the closing and opening of the coumarin gates by reversible photodimerization and photocleavage. With the pre-endocytosing of MSNcg, 126 cytosol peptides were harvested and identified from living HeLa cells. Moreover, 3 peptides were captured containing dynamic and changeable information. The extraction strategy of using MSNcg exhibited promising potentials in the in situ and dynamic extraction of endogenous peptides and/or proteins from living systems.
Asunto(s)
Portadores de Fármacos/química , Nanopartículas/química , Péptidos/aislamiento & purificación , Procesos Fotoquímicos , Dióxido de Silicio/química , Supervivencia Celular , Células HeLa , Humanos , Tamaño de la Partícula , Péptidos/química , Porosidad , Propiedades de Superficie , Factores de TiempoRESUMEN
Cyclodextrins and their derivatives are one of the most common and successful chiral selectors. However, there have been few publications about the use of cyclodextrin-modified monoliths. In this study, organic hybrid monoliths were prepared by the immobilization of derivatized ß-cyclodextrin alone or with l-2-allylglycine hydrochloride to the polyhedral oligomeric silsesquioxane methacryl substituted monolith. The main topic of this study is a combined system with dual chiral selectors (l-2-allylglycine hydrochloride and ß-cyclodextrin) as monolithic chiral stationary phase. The effect of l-2-allylglycine hydrochloride concentration on enantioseparation was investigated. The enantioseparation of the four acidic compounds with resolutions up to 2.87 was achieved within 2.5 min on the prepared chiral monolithic column in capillary liquid chromatography. Moreover, the possible mechanism of enantioseparation was discussed.
RESUMEN
A novel silica-based stationary phase with branched octadecyl groups was prepared by the sequential employment of the Michael addition reaction and photoinduced thiol-yne click chemistry with 3-aminopropyl-functionalized silica microspheres as the initial material. The resulting stationary phase denoted as SiO2 -N(C18)4 was characterized by elemental analysis, FTIR spectroscopy and Raman spectroscopy, demonstrating the existence of branched octadecyl groups in silica microspheres. The separations of benzene homologous compounds, acid compounds and amine analogues were conducted, demonstrating mixed-mode separation mechanism on SiO2 -N(C18)4 . Baseline separation of basic drugs mixture was acquired with the mobile phase of acetonitrile/H2 O (5%, v/v). SiO2 -N(C18)4 was further applied to separate Corydalis yanhusuo Wang water extracts, and more baseline separation peaks were obtained for SiO2 -N(C18)4 than those on Atlantis dC18 column. It can be expected that this new silica-based stationary phase will exhibit great potential in the analysis of basic compounds.
RESUMEN
Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.
Asunto(s)
Gotas Lipídicas/metabolismo , Proteoma/metabolismo , Ustilaginales/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Metabolismo de los Lípidos , Proteoma/genética , Ustilaginales/genéticaRESUMEN
Nano-bio interface has been paid much attention recently, though with the lack of methodology to predict the potential responses in biological systems such as cells induced by nanomaterials. In this study, we described a proteomic approach to investigate the proteome change in K562 cells exposed to oxidized single-walled carbon nanotubes (o-SWCNTs). 605 proteins were identified by semi-quantitative proteomic analysis (SQPA), including 29 significantly changed proteins with spectra count (SpC) ratios lager than 2 or less than 0.5. Three of them including HBA, CFL1 and LMAN2 were further validated by western blotting. The differential proteins were further classified by Ingenuity Pathways Analysis (IPA) to integrate them into a signaling network. Based on the information by this network, we predict that o-SWCNT treatment activated cell aggregation, decreased cell migration, but had no effect on cell death. And these cellular responses were further experimentally demonstrated. The protein signaling network established in this study would greatly benefit the studies on the bio-applications of o-SWCNTs and their toxicity studies. Our study demonstrated that proteomics could be used as a predicting tool to study nano-bio interface at cellular level.
Asunto(s)
Cromatografía Liquida/métodos , Nanotecnología , Nanotubos de Carbono , Proteómica , Espectrometría de Masas en Tándem/métodos , Muerte Celular/efectos de los fármacos , Humanos , Células K562 , Nanotubos de Carbono/toxicidad , Transducción de SeñalRESUMEN
Selective enrichment of specific peptides is an effective way to identify low abundance proteins. Fractionation of peptides prior to mass spectrometry is another widely used approach to reduce sample complexity in order to improve proteome coverage.In this study, we designed a multi-stage digestion strategy to generate peptides with different trypsin cleavage kinetics. It was found that each of the collected peptide fractions yielded many new protein identifications compared to the control group due to the reduced complexity. The overlapping peptides identified between adjacent fractions were very low, indicating that each fraction had different sets of peptides. The multi-stage digestion strategy separates tryptic peptides with different cleavage kinetics while RPLC separates peptides with different hydrophobicity. These two separation strategies were highly orthogonal, and showed an effective multidimensional separation to improve proteome coverage.