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Biomedical subjects

J C Sanchez

Publications and source records attributed to J C Sanchez.

At least 37 records · Page 2Linked to original sources
PubMed2000

Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14 (S100A9), two calcium-binding proteins of the S100 family.

Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (SI00A8), a member of the S100 family of calcium-binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases.

Adult↗
PubMed1999

Toward a clinical molecular scanner for proteome research: parallel protein chemical processing before and during western blot.

To increase the throughput of protein identification and characterization in proteome studies, we investigated three methods of performing protein digestion in parallel. The first, which we term "one-step digestion-transfer" (OSDT), is based on protein digestion during the transblotting process. It involves the use of membranes containing immobilized trypsin which are intercalated between the gel and a PVDF collecting membrane. During electrotransfer, some digestion of the transferred proteins occurs, although poorly for basic and/or high molecular weight proteins. The second method is based on "in-gel" digestion of all proteins in parallel and termed "parallel in-gel digestion" (PIGD) to denote this fact. The PIGD led to more efficient digestion of basic and high molecular weight proteins (> 40,000) but suffered from a major drawback: loss of resolution for low molecular weight polypeptides (< 60,000) through diffusion during the digestion process. The third method examined was the combination of PIGD and OSDT procedures. This combination, called "double parallel digestion" (DPD), led to greatly improved digestion of high molecular weight and basic proteins without losses of low molecular weight polypeptides. Peptides liberated during transblotting of proteins through the immobilized trypsin membrane were trapped on a PVDF membrane and identified by mass spectrometry in scanning mode.

Bacterial Proteins↗
PubMed1999

A molecular scanner to automate proteomic research and to display proteome images.

Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today's commonly used high-throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification and annotation in 2-DE databases: the sequential excision process of pieces of gel containing protein; the enzymatic digestion step; the interpretation of mass spectra (reliability of identifications); and the manual updating of 2-DE databases. We present a highly automated method that generates a fully annoated 2-DE map. Using a parallel process, all proteins of a 2-DE are first simultaneously digested proteolytically and electro-transferred onto a poly(vinylidene difluoride) membrane. The membrane is then directly scanned by MALDI-TOF MS. After automated protein identification from the obtained peptide mass fingerprints using PeptIdent software (http://www.expasy.ch/tools/peptident.html + ++), a fully annotated 2-D map is created on-line. It is a multidimensional representation of a proteome that contains interpreted PMF data in addition to protein identification results. This "MS-imaging" method represents a major step toward the development of a clinical molecular scanner.

Automation↗
PubMed1999

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Acetylation↗
PubMed1999

The SWISS-2DPAGE database: what has changed during the last year.

SWISS-2DPAGE (http://www.expasy.ch/ch2d/) is an annotated two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) database established in 1993. The current release contains 21 reference maps from human and mouse biological samples, as well as from Saccharomyces cerevisiae, Escherichia coli and Dictyostelium discoideum origin. These reference maps now have 2480 identified spots, corresponding to 528 separate protein entries in the database, in addition to virtual entries for each SWISS-PROT sequence. During the last year, the SWISS-2DPAGE has undergone major changes. Six new maps have been added, and new functions to access the data have been provided through the ExPASy server. Finally, an important change concerns the database funding source.

Animals↗
PubMed1999

Modified expression of plasma glutathione peroxidase and manganese superoxide dismutase in human renal cell carcinoma.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool to separate thousands of polypeptides and to highlight the modification of protein expression in malignant diseases. By applying 2-D PAGE to ten normal human kidney and ten homologous renal cell carcinoma (RCC) tissues, we found two peptides in all ten normal tissues but not in RCCs and, conversely, two peptides were detected in all RCCs but not in normal tissues. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and internal sequence analysis, the two first peptides were identified as two isoforms of plasma glutathione peroxidase (GPxP). The two other peptides isolated in all RCCs but not in normal tissues were identified by N-terminal sequence analysis as multimeric forms of manganese superoxide dismutase (Mn-SOD). No multimeric Mn-SODs and only two monomeric forms were detected in normal tissues. GPxP and Mn-SOD are metallo-enzymes encoded on chromosome 5q32 and on chromosome 6p25, respectively. Their regions are within the locus 5q21-->qter and 6q21-6q27 on which deletions and translocations are described in some cytogenetic studies of RCC transformation. Therefore, our results might suggest a correlation between the modified expression of GPxP and Mn-SOD in tumor tissues and chromosomal modifications, and that the two proteins may be putative markers for diagnosis of RCC.

Carcinoma, Renal Cell↗
PubMed1999

Information transfer between large and small two-dimensional polyacrylamide gel electrophoresis.

To determine the feasibility of data transfer, an interlaboratory comparison was conducted on colon carcinoma cell line (DLD-1) proteins resolved by two-dimensional polyacrylamide gel electrophoresis either on small (6 x 7 cm) or large (16x18 cm) gels. The gels were silver-stained and scanned by laser densitometry, and the image obtained was analyzed using Melanie software. The number of spots detected was 1337+/-161 vs. 2382+/-176 for small vs. large format gels, respectively. After gel calibration using landmarks determined using pl and Mr markers, large- and small-format gels were matched and 712+/-36 proteins were found on both types of gels. Having performed accurate gel matching it was possible to acquire additional information after accessing a 2-D PAGE reference database (http://www.expasy.ch/ cgibin/map2/def?DLD1_HUMAN). Thus, the difference in gel size is not an obstacle for data transfer. This will facilitate exchanges between laboratories or consultation concerning existing databases.

Adenocarcinoma↗
PubMed1999

Improving protein identification from peptide mass fingerprinting through a parameterized multi-level scoring algorithm and an optimized peak detection.

We have developed a new algorithm to identify proteins by means of peptide mass fingerprinting. Starting from the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectra and environmental data such as species, isoelectric point and molecular weight, as well as chemical modifications or number of missed cleavages of a protein, the program performs a fully automated identification of the protein. The first step is a peak detection algorithm, which allows precise and fast determination of peptide masses, even if the peaks are of low intensity or they overlap. In the second step the masses and environmental data are used by the identification algorithm to search in protein sequence databases (SWISS-PROT and/or TrEMBL) for protein entries that match the input data. Consequently, a list of candidate proteins is selected from the database, and a score calculation provides a ranking according to the quality of the match. To define the most discriminating scoring calculation we analyzed the respective role of each parameter in two directions. The first one is based on filtering and exploratory effects, while the second direction focuses on the levels where the parameters intervene in the identification process. Thus, according to our analysis, all input parameters contribute to the score, however with different weights. Since it is difficult to estimate the weights in advance, they have been computed with a generic algorithm, using a training set of 91 protein spectra with their environmental data. We tested the resulting scoring calculation on a test set of ten proteins and compared the identification results with those of other peptide mass fingerprinting programs.

Algorithms↗
PubMed1999

Two-dimensional electrophoresis resources available from ExPASy.

This paper describes the set of two-dimensional electrophoresis (2-DE) resources currently available from the ExPASy proteomics Web server. These resources include the SWISS-2DPAGE database, 2-DE software packages, 2-DE technical and educational services, as well as indexes and search engines for 2-DE related sites over the Internet.

Databases, Factual↗
PubMed1998

Protein identification with N and C-terminal sequence tags in proteome projects.

Genome sequences are available for increasing numbers of organisms. The proteomes (protein complement expressed by the genome) of many such organisms are being studied with two-dimensional (2D) gel electrophoresis. Here we have investigated the application of short N-terminal and C-terminal sequence tags to the identification of proteins separated on 2D gels. The theoretical N and C termini of 15, 519 proteins, representing all SWISS-PROT entries for the organisms Mycoplasma genitalium, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and human, were analysed. Sequence tags were found to be surprisingly specific, with N-terminal tags of four amino acid residues found to be unique for between 43% and 83% of proteins, and C-terminal tags of four amino acid residues unique for between 74% and 97% of proteins, depending on the species studied. Sequence tags of five amino acid residues were found to be even more specific. To utilise this specificity of sequence tags for protein identification, we created a world-wide web-accessible protein identification program, TagIdent (http://www.expasy.ch/www/tools.html), which matches sequence tags of up to six amino acid residues as well as estimated protein pI and mass against proteins in the SWISS-PROT database. We demonstrate the utility of this identification approach with sequence tags generated from 91 different E. coli proteins purified by 2D gel electrophoresis. Fifty-one proteins were unambiguously identified by virtue of their sequence tags and estimated pI and mass, and a further 11 proteins identified when sequence tags were combined with protein amino acid composition data. We conlcude that the TagIdent identification approach is best suited to the identification of proteins from prokaryotes whose complete genome sequences are available. The approach is less well suited to proteins from eukaryotes, as many eukaryotic proteins are not amenable to sequencing via Edman degradation, and tag protein identification cannot be unambiguous unless an organism's complete sequence is available.

Amino Acid Sequence↗
PubMed1998

Current status of the SWISS-2DPAGE database.

The SWISS-2DPAGE database (http: //www.expasy.ch/ch2d/ch2d-top.html ) consists of two-dimensional polyacrylamide gel electrophoresis images, as well as textual descriptions of the proteins that have been identified on them. The current release contains 15 reference maps from human biological samples, as well as from Saccharomyces cerevisiae , Escherichia coli and Dictyostelium discoideum origin. These reference maps have 2088 identified spots, corresponding to 410 separate protein entries in the database, in addition to virtual entries for each SWISS-PROT sequence.

Animals↗
PubMed1998

A two-dimensional electrophoretic study of serum amyloid A and C-reactive protein in infants and children.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to analyze C-reactive - (CRP) and serum amyloid A protein (SAA) in infants and children. Five SAA isotypes were identified. CRP showed vertical streaking, and its optical density values correlated with immunoturbidimetric measurements. As evaluated by densitometry, both proteins showed an age-dependent variation. In more than 50% of the neonates, SAA was present in equal or higher amounts than CRP, and only SAA1alpha could be detected. In children, CRP was expressed in higher amounts than SAA, and both SAA1alpha and SAA2alpha were present. N-terminally modified forms of both isotypes were present regardless of age, including in premature infants. These results suggest that the overall synthesis of the gene products SAA1alpha and SAA2alpha is developmentally regulated, but at the same time that their N-terminal processing occurs independently of developmental factors. The presented data suggest that SAA has an important function in neonates, and that the role of SAA as an infection marker in this population should be investigated further.

Adolescent↗
PubMed1998

Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis.

We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.

Bacterial Proteins↗