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1.
Yamamoto T  Izumi S  Gekko K 《FEBS letters》2006,580(15):3638-3642
The 70S ribosome from Escherichia coli is a supermacro complex (MW: 2.7MDa) comprising three RNA molecules and more than 50 proteins. We have for the first time successfully analyzed the flexibility of 70S ribosomal proteins in solution by detecting the hydrogen/deuterium exchange with mass spectrometry. Based on the deuterium incorporation map of the X-ray structure obtained at the time of each exchange, we demonstrate the structure-flexibility-function relationship of ribosome focusing on the deuterium incorporation of the proteins binding ligands (tRNA, mRNA, and elongation factor) and the relation with structural assembly processes.  相似文献   

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Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was to achieve a detailed structural characterization of modifications induced by transglutaminase on gliadin peptides. Therefore, structural analyses were carried out on a recombinant alpha-gliadin and on a panel of 26 synthetic peptides, overlapping the complete protein sequence. Modified glutamine residues were identified by means of advanced mass-spectrometric methodologies on the basis of MALDI-TOF-MS and tandem mass spectrometry. Results led to the identification of 19 of 94 glutamine residues present in the recombinant alpha-gliadin, which were converted into glutamic acid residues by a transglutaminase-mediated reaction. This allowed us to achieve a global view of the modifications induced by the enzyme on this protein. Furthermore, results gathered could likely be utilized as relevant information for a better understanding of processes leading to T-cell recognition of gliadin peptides involved in celiac disease.  相似文献   

4.
Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo, proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.  相似文献   

5.
O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.  相似文献   

6.
Amide hydrogen/deuterium exchange detected by mass spectrometry (HXMS) is seeing wider use for the identification of intrinsically disordered parts of proteins. In this review, we discuss examples of how discovery of intrinsically disordered regions and their removal can aid in structure determination, biopharmaceutical quality control, the characterization of how post-translational modifications affect weak structuring of disordered regions, the study of coupled folding and binding, and the characterization of amyloid formation. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.  相似文献   

7.
Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein. Toward this end, we have utilized a combination of chemical cross-linking/mass spectrometry and computational modeling to construct a pseudo-atomic model of the lambda major capsid protein as a monomer, as well as in the context of the assembled procapsid shell. The approach described here is generalizable and can be used to provide structural models for any biological complex of interest. The procapsid structural model is in good agreement with published biochemical data indicating that procapsid expansion exposes hydrophobic surface area and that this serves to nucleate assembly of capsid decoration protein, gpD. The model further implicates additional molecular interactions that may be critical to the assembly of the capsid shell and for the stabilization of the structure by the gpD decoration protein.  相似文献   

8.
Despite the great economical interest of locusts in agriculture, knowledge on their chemoreception systems is still poor. Phenylacetonitrile is recognised as a pheromone of the desert locust Schistocerca gregaria, triggering gregarization, promoting aggregation and inhibiting courtship. However, in the other major locust species, Locusta migratoria, pheromones have not been reported. We have identified the two isomers of naphthylpropionitrile from the male reproductive organs of L. migratoria. Chemical synthesis has confirmed the identity of the two compounds. Both isomers show significant affinity to CSP91, a protein reported in the testis, but not to three other proteins of the same family (CSP180, CSP540 and CSP884) expressed in female accessory glands. The striking similarity of these compounds with phenylacetonitrile and the unusual nature of such chemicals strongly suggest that naphthylpropionitrile could be pheromones for L. migratoria, while their site of expression and binding activity indicate a role in communication between sexes.  相似文献   

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Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC–MS/MS system. The quantification limits were between 0.17 nmol/L (5-formylTHF) and 1.79 nmol/L (THF), and the assay was linear up to 100 nmol/L (5-methylTHF) and 10 nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r = 0.939; p < 0.001). The mean total folate from 32 apparently healthy subjects was 18.09 nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.  相似文献   

11.
Three common urological diseases are bladder cancer, urinary tract infection, and hematuria. Seventeen bladder cancer biomarkers were previously discovered using iTRAQ - these findings were verified by MRM-MS in this current study. Urine samples from 156 patients with hernia (n=57, control), bladder cancer (n=76), or urinary tract infection/hematuria (n=23) were collected and subjected to multiplexed LC-MRM/MS to determine the concentrations of 63 proteins that are normally considered to be plasma proteins, but which include proteins found in our earlier iTRAQ study. Sixty-five stable isotope-labeled standard proteotypic peptides were used as internal standards for 63 targeted proteins. Twelve proteins showed higher concentrations in the bladder cancer group than in the hernia and the urinary tract infection/hematuria groups, and thus represent potential urinary biomarkers for detection of bladder cancer. Prothrombin had the highest AUC (0.796), with 71.1% sensitivity and 75.0% specificity for differentiating bladder cancer (n=76) from non-cancerous (n=80) patients. The multiplexed MRM-MS data was used to generate a six-peptide marker panel. This six-peptide panel (afamin, adiponectin, complement C4 gamma chain, apolipoprotein A-II precursor, ceruloplasmin, and prothrombin) can discriminate bladder cancer subjects from non-cancerous subjects with an AUC of 0.814, with a 76.3% positive predictive value, and a 77.5% negative predictive value. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.  相似文献   

12.
孙伟  许杰  张薇  罗倩  台萃 《微生物学报》2023,63(12):4800-4813
【目的】糖丝菌属(Saccharothrix)是一类丝状稀有放线菌,在生物医药、工业酶制剂和环境修复等领域展现出应用价值。本研究尝试建立以核糖体蛋白质为标志物,利用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization-time of flight mass spectrometry,MALDI-TOF MS)技术鉴定糖丝菌属放线菌的方法。【方法】检索基因组数据库,提取糖丝菌属测序菌株15种核糖体蛋白质的序列并计算理论分子量;通过分子量比对分析糖丝菌属不同菌种之间及其模式菌株与邻近属菌种模式菌株之间15种核糖体蛋白质的匹配度,提出鉴定至菌种及属的核糖体蛋白质匹配数标准;选取目标属和非目标属菌种进行MALDI-TOF MS测试和分析并修正鉴定标准。【结果】将待测菌株的MALDI-TOF质谱峰与糖丝菌属各菌种模式菌株的15种核糖体蛋白质分别匹配,通过最大匹配数、质谱峰强度模式及特征质谱峰鉴定至属或种。【结论】本研究建立了基于15种核糖体蛋白质标志物及MALDI-TOF MS技术鉴定糖丝菌属放线菌的方法,可用于定向筛选和快速鉴...  相似文献   

13.
Cytokinin oxidase/dehydrogenase (CKO; EC 1.5.99.12) irreversibly degrades the plant hormones cytokinins. A recombinant maize isoenzyme 1 (ZmCKO1) produced in the yeast Yarrowia lipolytica was subjected to enzymatic deglycosylation by endoglycosidase H. Spectrophotometric assays showed that both activity and thermostability of the enzyme decreased after the treatment at non-denaturing conditions indicating the biological importance of ZmCKO1 glycosylation. The released N-glycans were purified with graphitized carbon sorbent and analyzed by MALDI-TOF MS. The structure of the measured high-mannose type N-glycans was confirmed by tandem mass spectrometry (MS/MS) on a Q-TOF instrument with electrospray ionization. Further experiments were focused on direct analysis of sugar binding. Peptides and glycopeptides purified from tryptic digests of recombinant ZmCKO1 were separated by reversed-phase chromatography using a manual microgradient device; the latter were then subjected to offline-coupled analysis on a MALDI-TOF/TOF instrument. Glycopeptide sequencing by MALDI-TOF/TOF MS/MS demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338. The bound glycans contained 3-14 mannose residues. Interestingly, Asn134 was found only partially glycosylated. Asn338 was the sole site to carry large glycan chains exceeding 25 mannose residues. This observation demonstrates that contrary to a previous belief, the heterologous expression in Y. lipolytica may lead to locally hyperglycosylated proteins.  相似文献   

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Biotransformation of chemically stable compounds to reactive metabolites which can bind covalently to macromolecules, such as proteins and DNA, is considered as an undesirable feature of drug candidates. As part of an overall assessment of absorption, distribution, metabolism and excretion (ADME) properties, many pharmaceutical companies have put methods in place to screen drug candidates for their tendency to generate reactive metabolites and as well characterize the nature of the reactive metabolites through in vitro and in vivo studies. After identification of the problematic compounds, steps can be taken to minimize the potential of bioactivation through appropriate structural modifications. For these reasons, detection, structural characterization and quantification of reactive metabolites by mass spectrometry have become an important task in the drug discovery process. Triple quadrupole mass spectrometry is traditionally employed for the analysis of reactive metabolites. In the past 3 years, a number of new mass spectrometry methodologies have been developed to improve the sensitivity, selectivity and throughput of the analysis. This review focuses on the recent advances in the detection and characterization of reactive metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in drug discovery and development, especially through the use of linear ion trap (LTQ), hybrid triple quadrupole-linear ion trap (Q-trap) and the high resolution LTQ-Orbitrap instruments.  相似文献   

16.
Molecular chaperones Hsp70 and Hsp90 are in part responsible for maintaining the viability of cells by facilitating the folding and maturation process of many essential client proteins. The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) has been shown in vitro and in vivo to associate with Hsp70 and Hsp90 and ubiquitinate them, thus targeting them to the proteasome for degradation. Here, we study one facet of this CHIP-mediated turnover by determining the lysine residues on human Hsp70 and Hsp90 ubiquitinated by CHIP. We performed in vitro ubiquitination reactions of the chaperones using purified components and analyzed the samples by tandem mass spectrometry to identify modified lysine residues. Six such ubiquitination sites were identified on Hsp70 (K325, K451, K524, K526, K559, and K561) and 13 ubiquitinated lysine residues were found on Hsp90 (K107, K204, K219, K275, K284, K347, K399, K477, K481, K538, K550, K607, and K623). We mapped the ubiquitination sites on homology models of almost full-length human Hsp70 and Hsp90, which were found to cluster in certain regions of the structures. Furthermore, we determined that CHIP forms polyubiquitin chains on Hsp70 and Hsp90 linked via K6, K11, K48, and K63. These findings clarify the mode of ubiquitination of Hsp70 and Hsp90 by CHIP, which ultimately leads to their degradation.  相似文献   

17.
A number of viruses contain lipid membranes, which are in close contact with capsid proteins and/or nucleic acids and have an important role in the viral infection process. In this study membrane lipids of intact viruses have been analysed by MALDI-TOF/MS with a novel methodology avoiding lipid extraction and separation steps. To validate the novel method, a wide screening of viral lipids has been performed analysing highly purified intact bacterial and archaeal viruses displaying different virion architectures. Lipid profiles reported here contain all lipids previously detected by mass spectrometry analyses of virus lipid extracts. Novel details on the membrane lipid composition of selected viruses have also been obtained. In addition we show that this technique allows the study of lipid distribution easily in subviral particles during virus fractionation. The possibility to reliably analyse minute amounts of intact viruses by mass spectrometry opens new perspectives in analytical and functional lipid studies on a wider range of viruses including pathogenic human ones, which are difficult to purify in large amounts.  相似文献   

18.
The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an ∼ 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove ∼ 86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to ∼ 800-fold.  相似文献   

19.
Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38α MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38α are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38α alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38α and X-ray structures of phosphorylated p38γ. Conformational differences account for altered HX within the activation lip, the P + 1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (αF-αG, β7-β8), the C-terminal core (αE), and the N-terminal core region (β4-β5, αL16, αC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38α. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38α because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38α are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38α and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.  相似文献   

20.
The isoforms Iso-2, Iso-3, and Iso-4 of Escherichia coli-derived recombinant human interferon alpha-2b (rhIFN α-2b), generated by posttranslational modifications of the protein during fermentation, present a major problem in terms of purification and the yield of the drug substance. We report here the structural characterization of these isoforms by mass spectrometry (MS) methods. An extensive MS study was conducted on Iso-4, which is composed of up to 75% of the in-process IFN, and on the native rhIFN α-2b. The trypsin-digested peptide mixtures generated from the two samples were analyzed by liquid chromatography (LC)–MS, and targeted peptides were further studied by LC–tandem MS (triple quadrupole mass spectrometer), high-resolution MSn (LTQ Orbitrap), and matrix-assisted laser desorption/ionization MS (MALDI–MS). The structure of Iso-4 was elucidated as a novel pyruvic acid ketimine derivative of the N-terminal cysteine (Cys1) of IFN α-2b, where the disulfide bond between Cys1 and Cys98 was fully reduced and the other disulfide bond pair, Cys29-ss-Cys138, was partially reduced. Similarly, Iso-2 was identified as a correctly disulfide-folded rhIFN α-2b with acetylation on Cys1, and Iso-3 was identified as an S-glutathionylated form (Cys98) of partially reduced rhIFN α-2b that was pyruvated on Cys1. Based on the characterization work, a reproducible conversion procedure was successfully implemented to convert Iso-4 to rhIFN α-2b.  相似文献   

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