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P Chaurand

Publications and source records attributed to P Chaurand.

14 recordsLinked to original sources
PubMed2001

Strain-based sequence variations and structure analysis of murine prostate specific spermine binding protein using mass spectrometry.

Mouse spermine binding protein (SBP) has been characterized using mass spectrometry, including its localization within the prostate, sequence verification, and its posttranslational modifications. MALDI (matrix-assisted laser desorption/ionization) mass spectrometry was employed for localization of proteins expressed by different lobes of the mouse prostate obtained after tissue blotting on a polyethylene membrane. The mass spectra showed complex protein profiles that were different for each lobe of the prostate. The prostate-specific spermine binding protein (SBP), primarily identified by its in-source decay fragment ion signals, was found predominantly expressed by the ventral lobe of the prostate. The MALDI in-source decay measurements combined with nanoESI (nanoelectrospay ionization) MS/MS measurements obtained after specific proteolysis of SBP, allowed the exact positioning of a single N-linked carbohydrate group, and the identification of a pyroglutamate residue at the sequence N-terminus. The N-linked carbohydrate component was further investigated and the general pattern of the N-linked carbohydrate identified. The presence of a disulfide bridge between cysteine78 and cysteine124 was also established. The full sequence characterization of SBP showed several strain-based sequence differences when compared to the published gene sequence.

Amino Acid Sequence↗
PubMed2001

Characterization of the lysyl adducts of prostaglandin H-synthases that are derived from oxygenation of arachidonic acid.

These investigations characterize the covalent binding of reactive products of prostaglandin H-synthases (PGHSs) to the enzyme and to other molecules. The intermediate product of oxygenation of arachidonic acid by the PGHSs, prostaglandin (PG) H2, undergoes rearrangement to the highly reactive gamma-keto aldehydes, levuglandin (LG) E2 and D2. We previously have demonstrated that LGE2 reacts with the epsilon-amine of lysine to form both the lysyl-levuglandin Shiff base and the pyrrole-derived lysyl-levuglandin lactam adducts. We now demonstrate that these lysyl-levuglandin adducts are formed on the PGHSs following the oxygenation of arachidonic acid; after reduction of the putative Schiff base, proteolytic digestion of the enzyme, and isolation of the adducted amino acid residues, these adducts were identified by liquid chromatography-tandem mass spectrometry. The reactivity of the LGs is reflected by the finding that virtually all of the LG predicted to be formed from PGH2 can be accounted for as adducts of the PGH-synthase and that oxygenation of arachidonic acid by PGH-synthases also leads to the formation of adducts of other proteins present in the reaction solution. The reactivity of the PGH-synthase adducts themselves is demonstrated by the formation of intermolecular cross-links.

Animals↗
PubMed2001

A probasin-large T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential.

Neuroendocrine (NE) cells may be involved not only in growth and differentiation of the normal prostate but also in carcinogenesis and progression of prostate adenocarcinoma (Pca), including development of androgen resistance. However, the exact pathophysiology of NE cells in Pca remains poorly understood. Here we describe a transgenic model of Pca with progressive NE differentiation. Seven lines of transgenic mice with the rat prostate-specific large probasin promoter linked to the SV40-large T antigen (Tag) that develop prostatic neoplasia have been established. In this study, one of the seven lines (12T-10) was characterized by examination of 52 mice aged from 2-12 months. With advancing age, low-grade prostatic intraepithelial neoplasia, high-grade prostatic intraepithelial neoplasia, microinvasion, invasive carcinoma, and poorly or undifferentiated carcinoma with NE differentiation appeared in the prostates in sequential order. Whereas Tag is expressed uniformly in prostate epithelium, only an increasing subset of cells in prostatic intraepithelial neoplasia showed NE differentiation by chromogranin immunostaining. Frankly invasive carcinoma developing subsequently showed occasional definitive glandular differentiation (adenocarcinoma) and particularly undifferentiated carcinoma with NE histological features similar to those observed in NE carcinomas in humans. The NE carcinomas occurred in the dorsolateral and ventral lobes and were generally androgen receptor negative. Twenty-one of 32 (66%) mice aged > or = 6 months and 15 of 17 (88%) mice aged > or = 9 months developed metastatic tumors, as confirmed by histology and/or Tag immunohistochemistry. Metastases occurred at the later time points, with metastasis to regional lymph nodes, liver, and lung being particularly common. Metastases showed histological features of NE differentiation, as confirmed by chromogranin immunostaining and electron microscopy. An athymic nude mouse that received a s.c. implant of a primary NE tumor developed Tag-positive metastatic tumors with similar NE differentiation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified identical protein profiles between the primary NE tumor and lesions in the extraprostatic organs. Hence, in the 12T-10 large probasin promoter-Tag mouse, high-grade prostatic intraepithelial neoplasia develops progressively greater NE differentiation and progresses to invasive adenocarcinoma and NE carcinoma, with a high percentage of metastases. The predictable progression through these stages will allow testing of therapeutic interventions as well as possible further delineation of the role of NE cells in Pca progression.

Adenocarcinoma↗
PubMed2001

Profiling proteins from azoxymethane-induced colon tumors at the molecular level by matrix-assisted laser desorption/ionization mass spectrometry.

New developments in mass spectrometry allow for the profiling of the major proteomic content of fresh tissue sections. Briefly, fresh tissue sections are sampled and blotted onto a polyethylene membrane for protein transfer and then subsequently analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Using this technology, we have compared the protein expression of normal and cancerous mouse colon tissue obtained from the same animal. By difference, several protein signals specific to cancerous tissue were observed. A protein extract obtained from the tumors was fractionated by high-performance liquid chromatography and the individual fractions analyzed by MALDI-MS. The fractions containing the targeted proteins were subjected to trypsin digestion. The resulting tryptic peptides were sequenced by tandem mass spectrometry, and based on the recovered partial amino acid sequences, three of the tumor specific protein markers were identified as calgranulin A (S100A8), calgranulin B (S100A9) and calgizzarin (S100A11).

Animals↗
PubMed2001

Organic ion imaging of biological tissue with secondary ion mass spectrometry and matrix-assisted laser desorption/ionization.

Organic secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be used to produce molecular images of samples. This is achieved through ionization from a clearly identified point on a flat sample, and performing a raster of the sample by moving the point of ionization over the sample surface. The unique analytical capabilities of mass spectrometry for mapping a variety of biological samples at the tissue level are discussed. SIMS provides information on the spatial distribution of the elements and low molecular mass compounds as well as molecular structures on these compounds, while MALDI yields spatial information about higher molecular mass compounds, including their distributions in tissues at very low levels, as well as information on the molecular structures of these compounds. Application of these methods to analytical problems requires appropriate instrumentation, sample preparation methodology, and a data presentation usually in a three-coordinate plot where x and y are physical dimensions of the sample and z is the signal amplitude. The use of imaging mass spectrometry is illustrated with several biological systems.

Animals↗
PubMed2000

Sequencing of a branched peptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Chemical degradation methods combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and post-source decay (PSD)-MALDI reflex TOF mass spectrometry (MS) were used to determine the sequence of a peptide branched on to a known peptide backbone. This study was applied to a branched peptide model (derivative of substance P). The branched peptide mimics a digest of a membrane receptor on to which a derivative of substance P was photochemically linked. Chemical degradation based on N-terminal ladder sequencing in combination with MALDI-TOF-MS gave only partial sequence information. Although single PSD mass spectra still remain difficult to interpret unambiguously, PSD-MALDI-TOF-MS was combined with on-target acetylation and H -- D exchange to give a better and successful approach to the unambiguous determination of the complete amino acid side-chain sequence. This study shows the capability of MALDI-TOF-MS to help in characterizing ligand-receptor interactions.

Acetylation↗
PubMed1999

Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry.

The direct profiling of proteins present in tissue sections for several organs of the mouse has been accomplished using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Fresh tissue was sectioned and blotted on a conductive polyethylene membrane. The dried membrane blot was coated with matrix, typically sinapinic acid, and directly analyzed in the mass spectrometer. Generally, well over 100 peptide/protein signals in the 2000-30,000 Da range were observed, with 30-50 having relatively high signal intensities. Analysis of different areas of the same tissue gave remarkably similar mass spectra with greater than 90% homology. However, different parts of a segmented tissue, such as the proximal, intermediate, and distal colon, gave some unique protein signals. After treatment of the tissue blot with protease and subsequent MALDI MS analysis using postsource decay methods for peptide sequencing, some of the proteins were identified. The unique protein profiles measured from these tissue blots also showed differences from strain to strain of the mouse, with genetically similar strains having very similar patterns.

Animals↗
PubMed1999

Direct sequencing of neuropeptides in biological tissue by MALDI-PSD mass spectrometry.

Dissected tissue pieces of the pituitary pars intermedia from the amphibian Xenopus laevis was directly subjected to matrix-assisted laser desorption/ionization (MALDI) mass analysis. The obtained MALDI peptide profile revealed both previously known and unexpected processing products of the proopiomelanocortin gene. Mass spectrometric peptide sequencing of a few of these neuropeptides was performed by employing MALDI combined with postsource decay (PSD) fragment ion mass analysis. The potential of MALDI-PSD for sequence analysis of peptides directly from unfractionated tissue samples was examined for the first time for the known desacetyl-alpha-MSH-NH2 and the presumed vasotocin neuropeptide. In addition, the sequence of an unknown peptide which was present in the pars intermedia tissue sample at mass 1392.7 u was determined. The MALDI-PSD mass spectrum of precursor ion 1392.7 u contained sufficient structural information to uniquely identify the sequence by searching protein sequence databases. The determined amino acid sequence corresponds to the vasotocin peptide with a C-terminal extension of Gly-Lys-Arg ("vasotocinyl-GKR"), indicating incomplete processing of the vasotocin precursor protein in the pituitary pars intermediate of X. laevis. Both vasotocin and vasotocinyl-GKR are nonlinear peptides containing a disulfide (S-S) bridge between two cysteine residues. Interpretation of the spectra of these two peptides reveals three different forms of characteristic fragment ions of the cysteine side chain: peptide-CH2-SH (regular mass of Cys-containing fragment ions), peptide-CH2-S-SH (regular mass + 32 u) and peptide = CH2 (regular mass -34 u) due to cleavage on either side of the sulfur atoms.

Amino Acid Sequence↗
PubMed1999

Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay time-of-flight mass spectrometry.

The potential of matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay (PSD) time-of-flight mass spectrometry for the characterization of peptides and proteins is discussed. Recent instrumental developments provide for levels of sensitivity and accuracy that make these techniques major analytical tools for proteome analysis. New software developments employing protein database searches have greatly enhanced the fields of application of MALDI-PSD. Peptides and proteins can be easily identified even if only a partial sequence information is determined. Derivatization procedures have been optimized for MALDI-PSD to increase the structural information and to obtain a complete peptide sequence even in critical cases. They are fast, simple and can be performed on target. MALDI-PSD is also a very powerful tool to characterize or elucidate post-translational or chemically induced modifications. In association with database searches, proteins issued from electrophoretic gels can be identified after specific enzymatic cleavages and peptide mapping.

Amino Acid Sequence↗
PubMed1998

Identification of a sequence-dependent reversible acylation of tosylarginine in a peptidyl-resin reacted with isonicotinyl p-nitrophenylcarbonate.

In this paper, we report on the identification of an unexpected acylation that occurred on the solid phase when a peptide containing an unprotected lysyl and a tosyl-protected arginyl residue was treated with a large excess of isonicotinyl p-nitrophenylcarbonate. NMR and matrix-assisted laser desorption/ionisation -post-source decay analysis of the purified peptide demonstrated the presence of one extra isonicotinyloxycarbonyl (iNoc) group located on the omega nitrogen atom of the arginine which was adjacent to the Lys(iNoc). The desired peptide was obtained by quantitative removal of the unwanted iNoc group during a brief treatment with diluted aqueous hydrazine.

Acylation↗
PubMed1996

Post-source decay and delayed extraction in matrix-assisted laser desorption/ionization-reflectron time-of-flight mass spectrometry. Are there trade-offs?

By the incorporation of delayed extraction (DE) into matrix-assisted laser desorption/ionization time-of-flight mass spectrometry a dramatic improvement of performance with respect to sensitivity, mass resolution and mass accuracy of precursor ions up to approximately 10 kDa has been achieved. Since DE reduces collisional in-source activation to a large extent, the rate of subsequent metastable decay is considerably reduced. Results are presented which demonstrate that under DE the loss of total post-source decay (PSD) fragment ion yield can be as large as one order of magnitude but that, in terms of sensitivity, part of this loss is balanced by a better S/N ratio which results from a significantly improved mass resolution of the PSD fragment ions (M/delta M up to 1800 compared with M/delta M = 200-500 under prompt extraction). While this compensatory effect is true for the middle to high mass range of PSD fragment ions, it gradually vanishes towards the low mass end of the PSD mass scale where, in the case of linear peptides some important information (immonium ions) is lost. It appears, however, that in the majority of practical PSD work, DE improves the qualty of the PSD spectra and that high energy collisional post-source activation can compensate for the occasional loss of analytical information.

Amino Acid Sequence↗
PubMed1993

Structural features of lipoarabinomannan from Mycobacterium bovis BCG. Determination of molecular mass by laser desorption mass spectrometry.

It was recently shown that mycobacterial lipoarabinomannan (LAM) can be classified into two types (Chatterjee, D., Lowell, K., Rivoire B., McNeil M. R., and Brennan, P. J. (1992) J. Biol. Chem. 267, 6234-6239) according to the presence or absence of mannosyl residues (Manp) located at the nonreducing end of the oligoarabinosyl side chains. These two types of LAM were found in a pathogenic Mycobacterium tuberculosis strain and in an avirulent M. tuberculosis strain, respectively, suggesting that LAM with Manp characterizes virulent and "disease-inducing strains." We now report the structure of the LAM from Mycobacterium bovis Bacille Calmette-Guérin (BCG) strain Pasteur, largely used throughout the world as vaccine against tuberculosis. Using an up-to-date analytical approach, we found that the LAM of M. bovis BCG belongs to the class of LAMs capped with Manp. By means of two-dimensional homonuclear and heteronuclear scalar coupling NMR analysis and methylation data, the sugar spin system assignments were partially established, revealing that the LAM contained two types of terminal Manp and 2-O-linked Manp. From the following four-step process: (i) partial hydrolysis of deacylated LAM (dLAM), (ii) oligosaccharide derivatization with aminobenzoic ethyl ester, (iii) HPLC purification, (iv) FAB/MS-MS analysis; it was shown that the dimannosyl unit alpha-D-Manp-(1-->2)-alpha-D-Manp is the major residue capping the termini of the arabinan of the LAM. In this report, LAM molecular mass determination was established using matrix-assisted UV-laser desorption/ionization mass spectrometry which reveals that the LAM molecular mass is around 17.4 kDa. The similarity of the LAM structures between M. bovis BCG and M. tuberculosis H37Rv is discussed in regard to their function in the immunopathology of mycobacterial infection.

Antigens, Bacterial↗