Biomedical subjects

Jean-Charles Sanchez

Publications and source records attributed to Jean-Charles Sanchez.

45 records · Page 3Linked to original sources

PubMed2002

IgM are associated to Sp alpha (CD5 antigen-like).

In 1993, we reported the presence of an IgM-associated peptide (M(r) 44 kDa; pI 5.45) in all immunoglobulin M (IgM) fractions purified from plasma/serum by various methods. This peptide was absent in Ig fractions of non-IgM isotypes. The N-terminal sequence was determined as being APPSGVRLVGGLH. To gain insight into the nature of this peptide, we further analyzed, using modern proteomic tools, the IgM-associated peptide isolated from cryoglobulins. Mass spectrometry revealed three peptides of different masses: 2203.13 (ELGCGAASGTPSGILYEPPAEK), 1564.83 (KPIWLSQMSCSGR), and 1544.77 (EATLQDCPSGPWGK). Theses sequences together with the already known N-terminal sequence allowed us to identity the IgM-associated peptide as Sp alpha (O43866 in TrEMBL database; CD5 antigen-like). Sp alpha is a member of the scavenger receptor cysteine-rich superfamily of proteins. This family includes the T-and B-cell antigens CD5 and CD6, and several of its members influence immune cell fate. Our finding may have important implications in the understanding of the homeostasis of IgM antibodies.

Amino Acid Sequence↗

PubMed2002

Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study.

We used two-dimensional (2-D) electrophoresis to analyze the protein composition of fluid recovered by bronchoalveolar lavage (BALF) from patients with sarcoidosis and idiopathic pulmonary fibrosis, two forms of interstitial lung disease with different cellular composition and cytokine profile in BALF. They are also characterized by different pathogenesis and clinical evolution, idiopathic pulmonary fibrosis being less favorable than sarcoidosis due to rapidly progressive pulmonary fibrosis. Thirty-eight proteins or protein fragments, never previously assigned in BALF samples, were identified by various methods including mass fingerprinting of tryptic digests. Comparison of the BALF protein maps of the two groups of patients showed 32 spots with statistically significant disease-related variations in relative abundance. In sarcoidosis we found an increase in the amount of several plasma proteins, while in idiopathic pulmonary fibrosis we observed a statistically significant increase in low-molecular-weight proteins, many of which are involved in inflammatory processes (such as MIF and calgranulin) or antioxidant response (such as antioxidant peroxysomal enzyme and thioredoxin peroxidase 2). 2-D electrophoresis allowed us to identify new BALF proteins and to characterize protein composition in patients with sarcoidosis and idiophatic pulmonary fibrosis. Comparison of the gels of the two diseases showed that they differ in BALF protein profiles as they do in type of immune response.

Aged↗

PubMed2002

Proteomics and its trends facing nature's complexity.

The complexity of nature is tremendous, particularly at the epigenetic level. Proteomic studies must therefore complement genomic discoveries to better understand biological processes. Because of the very large number of modified proteins and their great variability in physico-chemical properties, no single method can be used to analyze all of them. Mass spectrometry has demonstrated its superior ability to rapidly identify and partially characterize numerous proteins in low abundance and has become a central element in most proteomic projects. Studies of protein function are necessary to understand biological pathways and this is being tackled using several approaches such as two hybrid systems, phage technology or affinity methods. Finally, mathematical and bioinformatic developments will be essential to study nature's complex systems.

Computational Biology↗

PubMed2002

Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins.

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.

Animals↗

PubMed2002

Hydrogen/deuterium exchange for higher specificity of protein identification by peptide mass fingerprinting.

Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments.

Amino Acid Sequence↗

PubMed2002

Effect of rosiglitazone on the differential expression of diabetes-associated proteins in pancreatic islets of C57Bl/6 lep/lep mice.

The insulin sensitizer drug, rosiglitazone, has been shown to have a protective effect on pancreatic islet cell structure and function in animal models of type 2 diabetes. The identification of new molecular targets associated both with islet cell dysfunction and protection is a crucial research goal. In the present study, a proteomics approach has been used to identify such targets. Obese C57Bl/6J lep/lep mice and lean littermates were given the insulin sensitizer drug BRL49653, rosiglitazone. It normalized the impaired glucose tolerance in lep/lep mice but had no significant effect on glucose tolerance in the lean mice. Pancreatic islet polypeptides were arrayed by a two-dimensional gel electrophoresis system that separated more than 2500 individual spots. Three overexpressed and six underexpressed proteins were significant (p < 0.05) between lep/lep and lean mice, and four were modulated significantly (p < 0.05) by the rosiglitazone treatment of the obese mice. The identity of these differentially expressed proteins was made using mass spectrometric analysis and provided evidence that differential expression of actin-binding proteins may be an important aspect of defective islet function. Rosiglitazone increased carboxypeptidase B expression in both lep/lep and normal mice suggesting that this might be an independent effect of rosiglitazone that contributes to improved insulin processing.

Animals↗

PubMed2002

Functional proteomic analysis of human nucleolus.

The notion of a "plurifunctional" nucleolus is now well established. However, molecular mechanisms underlying the biological processes occurring within this nuclear domain remain only partially understood. As a first step in elucidating these mechanisms we have carried out a proteomic analysis to draw up a list of proteins present within nucleoli of HeLa cells. This analysis allowed the identification of 213 different nucleolar proteins. This catalog complements that of the 271 proteins obtained recently by others, giving a total of approximately 350 different nucleolar proteins. Functional classification of these proteins allowed outlining several biological processes taking place within nucleoli. Bioinformatic analyses permitted the assignment of hypothetical functions for 43 proteins for which no functional information is available. Notably, a role in ribosome biogenesis was proposed for 31 proteins. More generally, this functional classification reinforces the plurifunctional nature of nucleoli and provides convincing evidence that nucleoli may play a central role in the control of gene expression. Finally, this analysis supports the recent demonstration of a coupling of transcription and translation in higher eukaryotes.

Cell Nucleolus↗

PubMed2002

Identification of specific proteins in different lymphocyte populations by proteomic tools.

The solubilized proteins of purified CD19(+) (B), CD8(+) (T) as well as CD4(+) (T) lymphocytes were separated by high resolution two-dimensional polyacrylamide gel electrophoresis, and the gels were analyzed using Melanie 3.0. Nine gels were studied, three for each lymphocyte population. After image analysis, 1411 +/- 73 spots (mean + SD) were detected. The protein pattern of B lymphocytes segregated from the one of T lymphocytes by ascendant heuristic clustering analysis. In addition, computer analysis separated CD8(+) from CD4(+) lymphocytes. When a search was performed in order to detect subsets of specific spots (presence vs. absence), a group of three spots, detected in the area of the protein maps corresponding to isoelectric point (pI) of 5.2 to 5.4 and molecular weight (M(r)) of 50 to 51 kDa, were found in both CD8(+) and CD4(+) cells, but not in CD19(+) cells. Mass spectrometry analysis revealed that these spots were associated with several proteins such as vimentin, tubulin, desmin and cytokeratin. Two spots, located in the area of the gel corresponding to pI of about 5.0 and a M(r) of 30 kDa, appeared as CD8(+) cell associated. Mass spectrometry analysis showed that the two spots were related to the same non-identified protein. Moreover internal peptides sequences matched with two human expressed sequence tags: gi/9759776, gi/12798420. No spots were found as only B cell associated.

Blood Proteins↗

PubMedDate not supplied

Deciphering the human nucleolar proteome.

Nucleoli are plurifunctional nuclear domains involved in the regulation of several major cellular processes such as ribosome biogenesis, the biogenesis of non-ribosomal ribonucleoprotein complexes, cell cycle, and cellular aging. Until recently, the protein content of nucleoli was poorly described. Several proteomic analyses have been undertaken to discover the molecular bases of the biological roles fulfilled by nucleoli. These studies have led to the identification of more than 700 proteins. Extensive bibliographic and bioinformatic analyses allowed the classification of the identified proteins into functional groups and suggested potential functions of 150 human proteins previously uncharacterized. The combination of improvements in mass spectrometry technologies, the characterization of protein complexes, and data mining will assist in furthering our understanding of the role of nucleoli in different physiological and pathological cell states.

Cell Nucleolus↗