Bernd Thiede

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.

MALDI-MS for C-terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P

Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) has been used for C‐terminal amino acid sequence determination of peptides and proteins. The usefulness of MALDI‐MS was demonstrated by analyzing peptide mixtures (C‐terminal peptide ladder) which were generated by enzymatic digestion of substance P, glucagon, angiotensinogen, insulin B chain and myoglobin with the exopeptidases carboxypeptidase Y and P. The results clearly show that up to 11 amino acid residues can be determined in the pmol range by analyzing the molecular masses of the truncated peptides. For proteins it is possible to investigate enzymatic or chemical digests in the same manner.

Identification of interaction partners of the cytosolic polyproline region of CD95 ligand (CD178) 1

The CD95/Fas/Apo‐1 ligand (CD95L, CD178) induces apoptosis through the death receptor CD95. CD95L was also described as a co‐stimulatory receptor for T‐cell activation in mice in vivo. The molecular basis for the bidirectional signaling capacity and directed expression of CD95L is unknown. In the present study we identify proteins that precipitate from T‐cell lysates with constructs containing fragments of the CD95L cytosolic tail. The determined peptide mass fingerprints correspond to Grb2, actin, β‐tubulin, formin binding protein 17 (FBP17) and PACSIN2. Grb2 had been identified as a putative mediator of T‐cell receptor‐to‐CD95L signaling before. FBP17 and PACSIN2 may be associated with expression and trafficking of CD95L. When overexpressed, CD95L co‐precipitates with FBP17 and PACSIN. Protein–protein interactions are mediated via Src homology 3 (SH3) domain binding to the polyproline region of CD95L and can be abolished by mutation or deletion of the respective SH3 domain.

A Global Approach Combining Proteome Analysis and Phenotypic Screening with RNA Interference Yields Novel Apoptosis Regulators

Global approaches like proteome or transcriptome analyses have been performed extensively to identify candidate genes or proteins involved in biological and pathological processes. Here we describe the identification of proteins implicated in the regulation of apoptosis using proteome analysis and the functional validation of targets by RNA interference. A high-throughput platform for the validation of synthetic small interfering RNAs (siRNAs) by quantitative real-time PCR was established. Genes of the identified factors were silenced by automated siRNA transfection, and their role in apoptotic signaling was investigated. Using this strategy, nine new modulators of apoptosis were identified. A subsequent detailed study demonstrated that hepatoma-derived growth factor (HDGF) is required for TNFα-induced release of pro-apoptotic factors from mitochondria. The strategy described here may be used for hypothesis-free, global gene function analysis.

Prediction of translocation and cleavage of heterogeneous ribonuclear proteins and Rho guanine nucleotide dissociation inhibitor 2 during apoptosis by subcellular proteome analysis.

Jurkat T cells induced to undergo apoptosis by the CD95(Fas/Apo‐1) pathway were investigated by proteome analysis. The most prominent differing protein spots of apoptotic and nonapoptotic cells were identified as various heterogeneous ribonuclear proteins (hnRNPs) and Rho guanin nucleotide dissociation inhibitor (GDI) 2. In apoptotic cells, four spots slightly differing in molecular mass and/or isoelectric point were identified as Rho GDI 2 with the mass and pI as expected after caspase‐3 cleavage near the N‐terminus. Subcellular proteome analysis revealed that Rho GDI 2 was highly enriched in the cytosolic fraction, present in minor amounts in the nuclear fraction and absent from the mitochondrial fraction. In apoptotic cells however, the spots representing processed and modified Rho GDI 2 were found in the cytosol, in the nucleus and also the mitochondria at different spot positions. In addition, twelve different hnRNPs were identified to be altered after induction of cell death of which hnRNPs A/B, D, F, H, I and L were hitherto unknown to be modified during apoptosis. Most of the hnRNP spots were found in the nucleus of nonapoptotic cells, whereas these proteins, either modified or unmodified, relocated to the cytosol and/or the mitochondria in apoptotic cells. Our results demonstrate that modification of proteins during apoptosis is often accompanied by their relocalisation between cellular compartments.

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 α‐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.

Hydrogen peroxide produced by Aplysia ink toxin kills tumor cells independent of apoptosis via peroxiredoxin I sensitive pathways

AbstractMarine snails of the genus Aplysia possess numerous bioactive substances. We have purified a 60u2009kDa protein, APIT (Aplysia punctata ink toxin), from the defensive ink of A. punctata that triggers cell death with profound tumor specificity. Tumor cell death induced by APIT is independent of apoptosis but is characterized by the rapid loss of metabolic activity, membrane permeabilization, and shrinkage of nuclei. Proteome analysis of APIT-treated tumor cells indicated a modification of peroxiredoxin I, a cytoplasmic peroxidase involved in the detoxification of peroxides. Interestingly, knockdown of peroxiredoxin I expression by RNA interference sensitized cells for APIT-induced cell death. APIT induced the death of tumor cells via the enzymatic production of H2O2 and catalase completely blocked APITs activity. Our data suggest that H2O2 induced stress and the modulation of peroxiredoxins might be a promising approach for tumor therapy.