Predominant identification of RNA-binding proteins in Fas-induced apoptosis by proteome analysis.

Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54(nrb), SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs.

A two dimensional electrophoresis database of a human Jurkat T-cell line.

About 2000 protein spots of human Jurkat T-cells were detected by high resolution two-dimensional gel electrophoresis (2-DE) and were characterized in terms of their isoelectric point and molecular mass. A 2-DE database was constructed and is available at http://www.mpiib-berlin.mpg.de/2D-PAGE/. At present the database contains 67 identified protein spots. These proteins were identified after tryptic digestion by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). Proteins with a sequence coverage of at least 30% were introduced in the database. This sequence coverage could not always be obtained by using only the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) for the mass analysis. Therefore, an additional mass spectrum was recorded by using 2,5-dihydroxybenzoic acid (DHB). Usually, additional mass peaks were detected and together with the mass spectrum of CHCA this resulted in the desired sequence coverage.

Comparative proteome analysis of Helicobacter pylori.

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.

Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion.

Peptide mass fingerprinting is a powerful tool for the identification of proteins. Trypsin is the most widely used enzyme for this purpose. Therefore, 104 protein digests from human Jurkat T cells and Mycobacterium were analyzed considering missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation. About 90% of the matched peptides with missed cleavage sites could be classified into three groups: (i) lysine and arginine with a neighbouring proline on the carboxy-terminal side, (ii) neighboring lysines/arginines, and (iii) lysines and arginines with an aspartic acid or glutamic acid residue on either the amino- or carboxy-terminal side. The first group is already accounted for by search programs. The number of missed cleavage sites can be increased without reducing the precision of the database search by taking the other two groups into consideration. Peptides with tryptophan were observed in non, singly (+16 Da) and doubly (+32 Da) oxidized forms. The higher oxidized form was only observed with lower intensity in the presence of the lower oxidized form. Peptides with N-terminal glutamine were found always as pyroglutamate (-17 Da), and in the majority of cases in pairs with unmodified glutamine. These data can be used for the refinement of protein searches by peptide mass fingerprinting.