1,721,132 research outputs found
Primary structure and glycan moiety characterization of PD-Ss, type 1 ribosome-inactivating proteins from Phytolacca dioica L. seeds, by precursor ion discovery on a Q-TOF mass spectrometer
No full textSeeds from Phytolacca dioica L. contain at least three N-glycosylated PD-Ss, type 1 ribosome-inactivating proteins (RIPs), which were separated and purified to homogeneity by conventional chromatographic techniques. ESI-Q-TOF mass spectrometry provided the accurate Mr of native PD-S1 and PD-S3 (30957.1 and 29785.1, respectively) and the major form PD-S2 (30753.8). As the amino acid sequence of PD-S2 was already known, its disulfide pairing was determined and found to be Cys34-Cys262 and Cys88-Cys110. Further structural characterization of PD-S1 and PD-S3 (N-terminal sequence determination up to residue 30, amino acid analysis and tryptic peptide mapping) showed that the three PD-Ss shared the entire protein sequence. To explain the different chromatographic behaviour, their glycosylation patterns were characterized by a fast and sensitive mass spectrometry-based approach, applying a precursor ion discovery mode on a Q-TOF mass spectrometer. A standard plant paucidomannosidic N-glycosylation pattern [Hex3, HexNAc2, deoxyhexose1, pentose1] was found for PD-S1 and PD-S2 on Asn120. Furthermore, a glycosylation site carrying only a HexNAc residue was identified on Asn112 in PD-S1 and PD-S3. Finally, considering the two disulfide bridges and the glycan moieties, the experimental Mr values were in agreement with the mass values calculated from the primary structure. The complete characterization of PD-Ss shows the high potential of mass spectrometry to rapidly characterize proteins, widespread in eukaryotes, differing only in their glycosylation motifs. © 2008 Elsevier Ltd. All rights reserved
A Novel Precursor Ion Discovery Method on a Q-TOF Mass Spectrometer for GnRH Detection in Complex Biological Mixtures
No full textIdentification of Lactic Acid Bacteria in Food Matrices by High-Resolution Nano-LC-ESI MS/MS
No full textPrecursor ion discovery on a hybrid quadrupole-time-of-flight mass spectrometer for gonadotropin-releasing hormone detection in complex biological mixtures
No full textThe gonadotropin-releasing hormone (GnRH) family includes several hypophysiotropic peptides occupying a central position in the regulatory loop controlling reproduction. Studies are still under way to clarify its biological role and evolutionary implication. Although sequencing of multiple genomes is bringing further advances in the understanding of the evolution of GnRH, there is still a need for biochemical studies aiming to identify GnRH from different species. Using a hybrid quadrupole-time-of-flight (Q-TOF) instrument, a new method for selective and sensitive GnRH detection and characterization from tissue extracts has been developed. The method uses the "precursor ion discovery" mode based on the capability of the Q-TOF analyzer to quickly record alternate mass spectra at low and high collision energy of precursor and product ion spectra, respectively, following liquid chromatographic separation of complex biological mixtures. The method exploits the selective detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine, highly conserved among nearly all species (22 of 24), and deriving from the preferential fragmentation of GnRHs carrying the dipeptide. Importantly, the method also includes acquisition of the product ion spectra from any candidate precursor ion, thereby allowing the determination of sequence information to confirm the GnRH identity or to isolate new ones. © 2007 Elsevier Inc. All rights reserved
Quantitative Neuroproteomics: Classical and Novel Tools for Studying Neural Differentiation and Function
No full textMechanisms underlying neural stem cell proliferation, differentiation and maturation play a critical role in the formation and wiring of neuronal connections. This process involves the activation of multiple serial events, which guide the undifferentiated cells to different lineages via distinctive developmental programs, forming neuronal circuits and thus shaping the adult nervous system. Furthermore, alterations within these strictly regulated pathways can lead to severe neurological and psychiatric diseases. In this framework, the investigation of the high dynamic protein expression changes and other factors affecting protein functions, for example post-translational modifications, the alterations of protein interaction networks, is of pivotal importance for the understanding of the molecular mechanisms responsible for cell differentiation. More recently, proteomic studies in neuroscience ("neuroproteomics") are receiving increased interest for the primary understanding of the regulatory networks underlying neuronal differentiation processes. Besides the classical two-dimensional-based proteomic strategies, the emerging platforms for LC-MS shotgun proteomic analysis hold great promise in unraveling the molecular basis of neural stem cell differentiation. In this review, recent advancements in label-free LC-MS quantitative neuroproteomics are highlighted as a new tool for the study of neural differentiation and functions, in comparison to mass spectrometry-based labeling approaches. The more commonly used protein profiling strategies and model systems for the analysis of neural differentiation are also discussed, along with the challenging proteomic approaches aimed to analyze the nervous system-specific organelles, the neural cells secretome and the specific protein interaction networks. © 2010 Springer Science+Business Media, LLC
Proteomic profiling of rat liver regeneration after partial hepatectomy and triiodothyronine stimulation
No full textA novel polygalacturonase-inhibiting protein (PGIP) from Lathyrus sativus L. seeds
No full textPolygalacturonase-inhibiting proteins (PGIPs) are extracellular plant proteins bound to the plant cell wall containing leucine-rich repeats (LRR). They play an important role in plant defence being able to inhibit fungal endopolygalacturonases (EPGs), the first enzymes secreted by phytopathogenic fungi during plant infection. In the present work, a novel PGIP (LsPGIP) has been isolated from Lathyrus sativus seeds. LsPGIP exhibited an inhibitory activity towards EPGs from Aspergillus niger and Rhizopus spp. A pI value of 8.3 and a molecular mass of 40 kDa were determined for the purified inhibitor. Furthermore, N-terminal sequence up to residue 20 revealed that LsPGIP exhibit a high percentage of identity with PGIP from Actinidia deliciosa. A secondary structure similar to those of other polygalacturonase inhibitors was also inferred form circular dichroism data. © 2012 Bentham Science Publishers
Influence of components of tumour microenvironment on the response of HCT-116 colorectal cancer to the ruthenium-based drug NAMI-A
Full text linkSolid tumours are constituted of tumour cells, healthy cells recruited from the host tissues and soluble factors released by both these cell types. The present investigation examines the capacity of co-cultures between the HCEC colon epithelial cells and the HCT-116 colorectal cancer cells (mimicking the primary site of tumour growth) and between IHH hepatocytes and the HCT-116 colorectal cancer cells (metastatic site) to influence the effects of NAMI-A (imidazolium trans-imidazoledimethylsulphoxidetetrachloro ruthenate) on the tumour cells themselves. The growth of HCT-116 cells is significantly influenced when the cancer cells are sown on a monolayer of HCEC. The release of soluble factors by the healthy cells promotes, in HCT-116 colorectal cancer cells, the transcription of genes involved in growth, invasion and migration. NAMI-A is not cytotoxic to HCT-116 cells grown on plastics or co-cultured with HCEC or IHH cells, and maintains its ability to control the cell pseudo-metastatic ability, mimicked by the migration in the scratch test. The effects of NAMI-A on HCT-116 migration are supported by its inhibition of the transcription of the ABL-2, ATF-3 and RND-1 genes. In conclusion the study highlights the need of test systems more complex than a single cancer cell culture to study an anticancer drug in vitro and reinforces the hypothesis that NAMI-A targets the ability of the cancer cell to interact with the tumour microenvironment and with the signals that support its metastatic ability
Studies toward the synthesis of inhibitors of mycobacterium tuberculosis cell-wall biosynthesis: The assembly of triazole-linked 1,6-a-D-oligomannosides via click CuAAC
No full textA versatile synthesis of 1,6 - d-oligomannosides featuring the 1,4-disubstituted triazole ring as interglycosidic tether is presented via iterative copper(I)-catalyzed azide-alkyne cycloaddition. Free hydroxy hexamannoside and decamannoside featuring a capping 6-deoxymannose fragment have been prepared and characterized
Peptide fingerprint of high quality Campania white wines by MALDI-TOF mass spectrometry
No full textFood traceability is essential to preserve the identity of unique quality traits against frauds or commercial disputes. Therefore, there is a growing demand of new traceability systems for the collection of information related to units/batches of food ingredients and products. A rapid method based on peptide profiles obtained from tryptic digests of whole wine proteins by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry is described. Reliable peptide fingerprints were obtained for high quality Campania white wines, providing a signature of the finished products. The MALDI spectra revealed the presence of common diagnostic ions, but also evidenced differences between wines. Furthermore, the MALDI-TOF spectral traces were converted into simulated images to obtain a graphical representation of spectra. The resulting "mass codes" constitute a simple tool to display differences between samples, suggesting their potential use as "biological bar codes" for food authenticity and traceability, probably applicable to other classes of certified food products. © 2008 Elsevier Ltd. All rights reserved
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