N-palmitoylation has been reported in a number of proteins and suggested to play an important role in protein localization and functions. However, it remains unclear whether N-palmitoylation is a direct enzyme-catalyzed process, or results from intramolecular S- to N-palmitoyl transfer. Here, using the S-palmitoyl peptide standard, GCpalmLGNAK, as the model system, we observed palmitoyl migration from the cysteine residue to either the peptide N-terminus or the lysine side chain during incubation in both neutral and slightly basic buffers commonly used in proteomic sample preparation. Palmitoyl transfer can take place either intra- or inter-molecularly, with the peptide N-terminus being the preferred migration site, presumably because of its lower basicity. The extent of intramolecular palmitoyl migration was low in the system studied, as it required the formation of an entropically unfavored macrocycle intermediate. Intermolecular palmitoyl transfer, however, remained a tangible problem, and may lead to erroneous reporting of in vivo N-palmitoylation. It was found that addition of the MS-compatible detergent RapiGest could significantly inhibit intermolecular palmitoyl transfer, as well as thioester hydrolysis and DTT-induced thioester cleavage. Finally, palmitoyl transfer from the cysteine residue to the peptide N-terminus can also occur in the gas phase, during collision-induced dissociation, and result in false identification of N-palmitoylation. Therefore, one must be careful with both sample preparation and interpretation of tandem mass spectra in the study of N-palmitoylation.
Species of genus Burkholderia display different interaction profiles in the environment, causing either several diseases in plants and animals or being beneficial to some plants, promoting their growth, and suppressing phytopathogens. Burkholderia spp. also produce many types of biomolecules with antimicrobial activity, which may be commercially used to protect crops of economic interest, mainly against fungal diseases. Herein we have applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to investigate secondary metabolites produced by B. seminalis TC3.4.2R3 in monoculture and coculture with plant pathogen Fusarium oxysporum. The siderophore pyochelin and the rhamnolipid Rha-Rha-C15-C14 were detected in wild-type B. seminalis strain, and their productions were found to vary in mutant strains carrying disruptions in gene clusters associated with antimicrobial compounds. Two mycotoxins were detected in F. oxysporum. During coculture with B. seminalis, metabolites probably related to defense mechanisms of these microorganisms were observed in the interspecies interaction zone. Our findings demonstrate the effective application of MALDI-MSI in the detection of bioactive molecules involved in the defense mechanism of B. seminalis, and these findings suggest the potential use of this bacterium in the biocontrol of plant diseases caused by F. oxysporum.
Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue.
Phospholipids generally dominate in bacterial lipids. The negatively charged nature of phospholipids renders bacteria susceptible to cationic antibiotic peptides. In comparison with Gram-negative bacteria, Gram-positive bacteria in general have much less zwitterionic phosphatidylethanolamine. However, they are known for producing aminoacylated phosphatidylglycerol (PG), especially positively charged l-lysyl-PG, which is catalyzed by lysyl-PG synthase MprF, which appears to have a broad range of specificity for l-aminoacyl transfer RNAs. In addition, many Gram-positive bacteria also have a dlt-gene-coded d-alanylation pathway for lipoteichoic acids and wall teichoic acids covalently attached to a glycolipid or peptidoglycan. d-Alanylation also masks the dominant negative charge of the phosphate-rich polymers of teichoic acids. Using mass spectrometry, we have recently observed that precursor scans in negative mode for deprotonated amino acid fragments were most sensitive for ester-linked amino acids. Such a scan for precursors generating an m/z 145 lysyl anion revealed lysyl-PG as well as an additional species 100?m/z units greater than lysyl-PG. This unexpected species corresponded precisely to the expected mass of N-succinylated lysyl-PG. Tandem mass spectrometry revealed a precise match to the fragmentation pattern of this putative new species. PG, lysyl-PG, and N-succinyl-lysyl-PG may form a complete loop of charge reversal from -1 to +1 and then back to -1. Analogous charge reversal by N-succinylation of lysine residues in the bacterial as well as eukaryotic proteomes has been recently discovered as a major posttranslational modification. Such modification in bacterial lipids is possibly catalyzed by an enzyme homologous to the enzymes that modify lysine residues in proteins.
Profiling cellular protein glycosylation is challenging due to the presence of highly similar glycan structures that play diverse roles in cellular physiology. As the anomericity and the exact linkage type of a single glycosidic bond can influence glycan function, there is a demand for improved and automated methods to confirm detailed structural features and to discriminate between structurally similar isomers, overcoming a significant bottleneck in the analysis of data generated by glycomics experiments. We used porous graphitized carbon-LC-ESI-MS/MS to separate and detect released N- and O-glycan isomers from mammalian model glycoproteins using negative mode resonance activation CID-MS/MS. By interrogating similar fragment spectra from closely related glycan isomers that differ only in arm position and sialyl linkage, product fragment ions for discrimination between these features were discovered. Using the Skyline software, at least two diagnostic fragment ions of high specificity were validated for automated discrimination of sialylation and arm position in N-glycan structures, and sialylation in O-glycan structures, complementing existing structural diagnostic ions. These diagnostic ions were shown to be useful for isomer discrimination using both linear and 3D ion trap mass spectrometers when analyzing complex glycan mixtures from cell lysates. Skyline was found to serve as a useful tool for automated assessment of glycan isomer discrimination. This platform-independent workflow can potentially be extended to automate the characterization and quantitation of other challenging glycan isomers.
Bacteriochlorophyll a (BChl a), a photosynthetic pigment performing the same functions of chlorophylls in plants, features a bacteriochlorin macrocycle ring (18 π electrons) with two reduced pyrrole rings along with a hydrophobic terpenoid side chain (i.e., the phytol residue). Chlorophylls analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is not so straightforward since pheophytinization (i.e., release of the central metal ion) and cleavage of the phytol–ester linkage are invariably observed by employing protonating matrices such as 2,5-dihydroxybenzoic acid, sinapinic acid, and α-cyano-4-hydroxycinnamic acid. Using BChl a from Rhodobacter sphaeroides R26 strain as a model system, different electron-transfer (ET) secondary reaction matrices, leading to the formation of almost stable radical ions in both positive ([M]+?) and negative ([M]??) ionization modes at m/z 910.55, were evaluated. Compared with ET matrices such as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 2,2':5',2''-terthiophene (TER), anthracene (ANT), and 9,10-diphenylanthracene (DP-ANT), 1,5-diaminonaphthalene (DAN) was found to provide the highest ionization yield with a negligible fragmentation. DAN also displayed excellent ionization properties for two metal ion-substituted bacteriochlorophylls, (i.e., Zn- and Cu-BChl a at m/z 950.49 and 949.49), respectively. MALDI MS/MS of both radical charged molecular species provide complementary information, thus making analyte identification more straightforward.
Food intake is regulated by various neuromodulators, including numerous neuropeptides. However, it remains elusive at the molecular and cellular level as to how these important chemicals regulate internal processes and which regions of the neuronal organs are responsible for regulating the behavior. Here we report a comparative neuropeptidomic analysis of the brain and pericardial organ (PO) in response to feeding in two well-studied crustacean physiology model organisms, Callinectes sapidus and Carcinus maenas, using mass spectrometry (MS) techniques. A multifaceted MS-based approach has been developed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and PO. The method employs stable isotope labeling of brain and PO extracts for relative MS quantitation, capillary electrophoresis (CE)-MS for fractionation and high-specificity analysis, and mass spectrometric imaging (MSI) for in-situ molecular mapping of peptides. A number of neuropeptides, including RFamides, B-type allatostatins (AST-B), RYamides, and orcokinins exhibit significant changes in abundance after feeding in this investigation. Peptides from the AST-B family found in PO tissue were shown to have both altered expression and localization changes after feeding, indicating that they may be a class of vital neuropeptide regulators involved in feeding behavior.
Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.
Glycoconjugates are directly or indirectly involved in many biological processes. Due to their complex structures, the structural elucidation of glycans and the exploration of their role in biological systems have been challenging. Glycan pools generated through release from glycoprotein or glycolipid mixtures can often be very complex. For the sake of procedural simplicity, many glycan profiling studies choose to concentrate on a single class of glycoconjugates. In this paper, we demonstrate it feasible to cover glycosphingolipids, N-glycans, and O-glycans isolated from the same sample. Small volumes of human blood serum and ascites fluid as well as small mouse brain tissue samples are sufficient to profile sequentially glycans from all three classes of glycoconjugates and even positively identify some mixture components through MALDI-MS and LC-ESI-MS. The results show that comprehensive glycan profiles can be obtained from the equivalent of 500-μg protein starting material or possibly less. These methodological improvements can help accelerating future glycomic comprehensive studies, especially for precious clinical samples.
Among dissociation methods, negative electron transfer dissociation (NETD) has been proven the most useful for glycosaminoglycan (GAG) sequencing because it produces informative fragmentation, a low degree of sulfate losses, high sensitivity, and translatability to multiple instrument types. The challenge, however, is to distinguish positional sulfation. In particular, NETD has been reported to fail to differentiate 4-O- versus 6-O-sulfation in chondroitin sulfate decasaccharide. This raised the concern of whether NETD is able to differentiate the rare 3-O-sulfation from predominant 6-O-sulfation in heparan sulfate (HS) oligosaccharides. Here, we report that NETD generates highly informative spectra that differentiate sites of O-sulfation on glucosamine residues, enabling structural characterizations of synthetic HS isomers containing 3-O-sulfation. Further, lyase-resistant 3-O-sulfated tetrasaccharides from natural sources were successfully sequenced. Notably, for all of the oligosaccharides in this study, the successful sequencing is based on NETD tandem mass spectra of commonly observed deprotonated precursor ions without derivatization or metal cation adduction, simplifying the experimental workflow and data interpretation. These results demonstrate the potential of NETD as a sensitive analytical tool for detailed, high-throughput structural analysis of highly sulfated GAGs.
A strategy for rapid identification of the chemical constituents from crude extracts of Tribulus terrestris was proposed using an informatics platform for the UHPLC/Q-TOF MSE data analyses. This strategy mainly utilizes neutral losses, characteristic fragments, and in-house library to rapidly identify the structure of the compounds. With this strategy, rapid characterization of the chemical components of T. terrestris from Beijing, China was successfully achieved. A total of 82 steroidal saponins and nine flavonoids were identified or tentatively identified from T. terrestris. Among them, 15 new components were deduced based on retention times and characteristic MS fragmentation patterns. Furthermore, the chemical components of T. terrestris, including the other two samples from Xinjiang Uygur Autonomous region, China, and Rome, Italy, were also identified with this strategy. Altogether, 141 chemical components were identified from these three samples, of which 39 components were identified or tentatively identified as new compounds, including 35 groups of isomers. It demonstrated that this strategy provided an efficient protocol for the rapid identification of chemical constituents in complex samples such as traditional Chinese medicines (TCMs) by UHPLC/Q-TOF MSE with informatics platform.
The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.
An ion of m/z 110.06036 (ion formula [C6H8NO]+; error: 0.32 mDa) was observed in the collision induced dissociation tandem mass spectrometry experiments of protonated N-(3-aminophenyl)benzamide, which is a rearrangement product ion purportedly through nitrogen-oxygen (N–O) exchange. The N–O exchange rearrangement was confirmed by the MS/MS spectrum of protonated N-(3-aminophenyl)-O18-benzamide, where the rearranged ion, [C6H8NO18]+ of m/z 112 was available because of the presence of O18. Theoretical calculations using Density Functional Theory (DFT) at B3LYP/6-31 g(d) level suggest that an ion-neutral complex containing a water molecule and a nitrilium ion was formed via a transition state (TS-1), followed by the water molecule migrating to the anilide ring, eventually leading to the formation of the rearranged ion of m/z 110. The rearrangement can be generalized to other protonated amide compounds with electron-donating groups at the meta position, such as, –OH, –CH3, –OCH3, –NH(CH3)2, –NH-Ph, and –NHCOCH3, all of which show the corresponding rearranged ions in MS/MS spectra. However, the protonated amide compounds containing electron-withdrawing groups, including –Cl, –Br, –CN, –NO2, and –CF3, at the meta position did not display this type of rearrangement during dissociation. Additionally, effects of various acyl groups on the rearrangement were investigated. It was found that the rearrangement can be enhanced by substitution on the ring of the benzoyl with electron-withdrawing groups.
Boiling in toluene of 5-(arylmethylidene)pyrimidine-2,4,6(1H,3H,5H)-triones with L-proline and aldehyde led to the formation of previously unknown substituted 1′-arylhexahydro-1H-spiro[pyrimidine-5,2′-pyrrolysine]-2,4,6(1H,3Н,5Н)-triones. 相似文献
Neuropeptides are important signaling molecules used by nervous systems to mediate and fine-tune neuronal communication. They can function as neurotransmitters or neuromodulators in neural circuits, or they can be released as neurohormones to target distant cells and tissues. Neuropeptides are typically cleaved from larger precursor proteins by the action of proteases and can be the subject of post-translational modifications. The short, mature neuropeptide sequences often entail the only evolutionarily reasonably conserved regions in these precursor proteins. Therefore, it is particularly challenging to predict all putative bioactive peptides through in silico mining of neuropeptide precursor sequences. Peptidomics is an approach that allows de novo characterization of peptides extracted from body fluids, cells, tissues, organs, or whole-body preparations. Mass spectrometry, often combined with on-line liquid chromatography, is a hallmark technique used in peptidomics research. Here, we used an acidified methanol extraction procedure and a quadrupole-Orbitrap LC-MS/MS pipeline to analyze the neuropeptidome of Caenorhabditis elegans. We identified an unprecedented number of 203 mature neuropeptides from C. elegans whole-body extracts, including 35 peptides from known, hypothetical, as well as from completely novel neuropeptide precursor proteins that have not been predicted in silico. This set of biochemically verified peptide sequences provides the most elaborate C. elegans reference neurpeptidome so far. To exploit this resource to the fullest, we make our in-house database of known and predicted neuropeptides available to the community as a valuable resource. We are providing these collective data to help the community progress, amongst others, by supporting future differential and/or functional studies.
An efficient synthetic approach to 2E,4E and 2E,4Z isomers of ethyl 5-chloropenta-2,4-dienoate has been developed on the basis of one-pot oxidation–olefination of readily accessible (E)- and (Z)-3-chloroprop-2-en-1-ols by the action of barium manganate and ethyl (triphenyl-λ5-phosphanylidene)acetate. 相似文献
The plant Alisma plantago-aquatica Linnaeus, which is widely distributed in southwest of China, is the main material of traditional Chinese medicine “Zexie”. It was used as folk medicine for immune-modulation, anti-tumor, anti-inflammatory and antibacterial. Previous chemical studies on A. plantago-aquatica reported the identification of triterpenes, diterpenes, sesquiterpenes, steroids, alkaloids and phenolic acid. Terpenes and phenolic acid were regard as major secondary metabolites from this medicine plant.
Results
A new phenolic acid, plantain A (1), along with four known compounds (2–5) were isolated and identified from A. plantago-aquatica by extensive chromatographic and spectrometric methods. In the present study, the levels of TNF-α, IL-1β, COX-2, PEG2 and TGF-β1 were increased in model group rats, whereas on treatment with the isolated compound (1 and 4) at 50 mg/kg, there was a significant decrease in the cytokine levels. Therefore, the anti-CNP effect of 1 and 4 may be related to their anti-inflammatory properties.
Conclusions
A new phenolic acid and four known phenolic compounds were isolated from A. plantago-aquatica. Moreover, compounds 1 and 4 shows significant anti-chronic prostatitis activity in rats.
Native mass spectrometry (MS) is an emerging approach to study protein complexes in their near-native states and to elucidate their stoichiometry and topology. Here, we report a native MS study of the membrane-embedded reaction center (RC) protein complex from the purple photosynthetic bacterium Rhodobacter sphaeroides. The membrane-embedded RC protein complex is stabilized by detergent micelles in aqueous solution, directly introduced into a mass spectrometer by nano-electrospray (nESI), and freed of detergents and dissociated in the gas phase by collisional activation. As the collision energy is increased, the chlorophyll pigments are gradually released from the RC complex, suggesting that native MS introduces a near-native structure that continues to bind pigments. Two bacteriochlorophyll a pigments remain tightly bound to the RC protein at the highest collision energy. The order of pigment release and their resistance to release by gas-phase activation indicates the strength of pigment interaction in the RC complex. This investigation sets the stage for future native MS studies of membrane-embedded photosynthetic pigment–protein and related complexes.
Diisopropyl N-benzoyl-N-(trimethylsilyl)phosphoramidate reacts with ClCH2SiMe2Cl under mild conditions to form diisopropyl N-benzoyl-N-[(chlorodimethylsilyl)methyl]phosphoramidate (III). Diisopropyl N-methyl-N-(trimethylsilyl)phosphoramidate with ClCH2SiMe2Cl affords an N-transsilylation product which does not rearrange into diisopropyl N-[(chlorodimethylsilyl)methyl]-N-methylphosphoramidate (XV) even under severe conditions (4 h, 130°C). Compound XV was prepared by the reaction of diisopropyl phosphorochloridate with N-[(methoxydimethylsilyl)methyl]-N-methylamine followed by treatment of diisopropyl N-[(methoxydimethylsilyl)methyl]-N-methylphosphoramidate with boron trichloride. Analysis of experimental and calculated 29Si chemical shifts points to a five-coordinate silicon atom in compound III and a fourcoordinate silicon atom in compound XV. According to B3LYP calculations with due regard to solvent effects, compound III is an isomer with a C=O→Si bond. By variation of substituents at silicon, phosphorus, and carbonyl carbon atoms, chelate structures with either C=O→Si or P=O→Si dative bonds can be obtained. 相似文献
