Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.

Inactivating mutations in single genes can trigger, prevent, promote, or alleviate diseases. Identifying such disease-related genes is a main pillar of medical research. Since proteins play a crucial role in mediating these effects, their impact on the diseased cells' proteome including posttranslational modifications has to be elucidated for a detailed understanding of the role of these genes in the disease process. In complex disorders, like cancer, several genes contribute to the disease process, thereby hampering the assignment of a proteomic change to the corresponding causative gene. To enable comprehensive screening for the impact of inactivation of a gene, e.g., loss of a tumor suppressor in cancer, on the cellular proteome, we present a strategy based on combination of three technologies that is recombinase-mediated cassette exchange, click chemistry, and mass spectrometry. The methodology is exemplified by the analysis of the proteomic changes induced by the loss of a tumor suppressor gene in colorectal cancer cells. To demonstrate the applicability to screen for posttranslational modification changes, we also describe the analysis of protein glycosylation changes caused by the tumor suppressor inactivation. In principle, this strategy can be applied to analyze the effects of any gene of interest on protein expression as well as posttranslational modification by glycosylation. Moreover adaptation of the strategy to an appropriate cell culture model has the potential for application on a broad range of diseases where the disease-promoting mutations have been identified.

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

PAMAM structure-based multifunctional fluorescent conjugates for improved fluorescent labelling of biomacromolecules.

Fluorescent probes are of increasing interest in medicinal and biological applications for the elucidation of the structures and functions of healthy as well as tumour cells. The quality of these investigations is determined by the intensity of the fluorescence signal. High dye/carrier ratios give strong signals. However, these are achieved by the occupation of a high number of derivatisation sites and therefore are accompanied by strong structural alterations of the carrier. Hence, polyvalent substances containing a high number of fluorescent dyes would be favourable because they would allow the introduction of many dyes at one position of the compound to be labelled.A large number of different dyes have been investigated to determine the efficiency of coupling to a dendrimer scaffold and the fluorescence properties of the oligomeric dyes, but compounds that fulfil the requirements of both strong fluorescence signals and reactivities are rare. Herein we describe the synthesis and characterisation of dye oligomers containing dansyl-, 7-nitro-2,1,3-benzoxadiazol-4-yl- (NBD), coumarin-343, 5(6)-carboxyfluorescein and sulforhodamine B2 moieties based on polyamidoamine (PAMAM) dendrimers. The PAMAM dendrimers were synthesised by an improved protocol that yielded highly homogeneous scaffolds with up to 128 conjugation sites. When comparing the fluorescent properties of the dye oligomers it was found that only the dansylated dendrimers met the requirements of enhanced fluorescence signals. The dendrimer containing 16 fluorescent dyes was conjugated to the anti-epidermal-growth-factor receptor (EGFR) antibody hMAb425 as a model compound to show the applicability of the dye multimer compounds. This conjugate revealed a preserved immunoreactivity of 54%.We demonstrate the applicability of the dye oligomers to the efficient and applicable labelling of proteins and other large molecules that enables high dye concentrations and therefore high contrasts in fluorescence applications.

Calcium-signaling networks in olfactory receptor neurons.

The olfactory neuroepithelium represents a unique interface between the brain and the external environment. Olfactory function comprises a distinct set of molecular tasks: sensory signal transduction, cytoprotection and adult neurogenesis. A multitude of biochemical studies has revealed the central role of Ca(2+) signaling in the function of olfactory receptor neurons (ORNs). We set out to establish Ca(2+)-dependent signaling networks in ORN cilia by proteomic analysis. We subjected a ciliary membrane preparation to Ca(2+)/calmodulin-affinity chromatography using mild detergent conditions in order to maintain functional protein complexes involved in olfactory Ca(2+) signaling. Thus, calmodulin serves as a valuable tool to gain access to novel Ca(2+)-regulated protein complexes. Tandem mass spectrometry (nanoscale liquid-chromatography-electrospray injection) identified 123 distinct proteins. Ninety-seven proteins (79%) could be assigned to specific olfactory functions, including 32 to sensory signal transduction and 40 to cytoprotection. We point out novel perspectives for research on the Ca(2+)-signaling networks in the olfactory system of the rat.

Proteomic analysis reveals successive aberrations in protein expression from healthy mucosa to invasive head and neck cancer.

Development of head and neck squamous cell carcinoma (HNSCC) is a multistep process and in many cases involves a phenomenon coined 'field cancerization'. In order to identify changes in protein expression occurring at different stages of tumorigenesis and field cancerization, we analysed 113 HNSCCs and 73 healthy, 99 tumor-distant and 18 tumor-adjacent squamous mucosae by SELDI-TOF-MS on IMAC30 ProteinChip Arrays. Forty-eight protein peaks were differentially expressed between healthy mucosa and HNSCC. Calgizarrin (S100A11), the Cystein proteinase inhibitor Cystatin A, Acyl-CoA-binding protein, Stratifin (14-3-3 sigma), Histone H4, alpha- and beta-Hemoglobin, a C-terminal fragment of beta-hemoglobin and the alpha-defensins 1-3 were identified by mass spectrometry. The alpha-defensins showed various alterations in expression as validated by immunohistochemistry (IHC). Supervised prediction analysis revealed excellent classification of healthy mucosa (94.5% correctly classified) and tumor samples (92.9% correctly classified). Application of this classifier to the tumor-adjacent and tumor-distant mucosa samples disclosed dramatic changes: only 59.6% of the tumor-distant biopsies were classified as normal, 27.3% were predicted as aberrant or HNSCC. Strikingly, 72% of the tumor-adjacent mucosae were predicted as aberrant. These data provide evidence for the existence of genetically altered fields with inconspicuous histology. Comparison of the protein profiles in the tumor-distant-samples with clinical outcome of 32 patients revealed a significant association between aberrant profiles with tumor relapse events (P=0.018; Fisher's exact test, two-tailed). We conclude that proteomic profiling in conjunction with protein identification greatly outperforms histopathological diagnosis and may have significant predictive power for clinical outcome and personalized risk assessment.

Differential protein expression in the epidermis of wild-type and COX-2 transgenic mice.

Cyclooxygenases (COX) 1 and 2 are the key enzymes of prostaglandin biosynthesis. Like in many tissues, in adult skin COX-1 is a constitutive 'housekeeping' enzyme, while COX-2 is induced transiently in stress situations such as tissue damage and regeneration. In human skin carcinomas and corresponding early-stage cancer lesions, permanent COX-2 expression and activation is a consistent feature. Knockout and various transgenic approaches and pharmacologic studies show strong evidence for a cause-and-effect relationship between the aberrant COX-2 activation and tumor formation. In skin epidermis, keratin 5 promoter-driven overexpression of COX-2 caused hyperplasia and dysplasia, and sensitized skin for carcinogenesis. Therefore, this model offers the unique possibility of identifying COX-2-dependent and prostaglandin-mediated molecular pathways leading to the formation and malignant progression of early-stage cancer lesions.

Proteome analysis of isolated myenteric plexus reveals significant changes in protein expression during postnatal development.

The enteric nervous system in vertebrates is the most complex part of the peripheral nervous system. Concerning chemical coding, ultrastructure and neuronal circuits, it is more similar to the central than to the peripheral nervous system. Its networks, the myenteric and submucous plexus are integrated in the gut wall. The enteric nervous system is a system of high plasticity, which not only changes during pre- and postnatal development, but also with disease or changing dietary habits. The Aim of this study was to elucidate changes in protein expression during the first two postnatal weeks in the rat myenteric plexus. Colonic and duodenal myenteric plexus from newborn (P1) and fourteen-day old (P14) Sprague-Dawley rats was isolated following a procedure that combines enzymatic digestion and mechanical agitation. The neuronal tissue was collected and processed for two-dimensional gel electrophoresis (2-DE). The obtained 2-D gels were stained with silver for image analysis or with colloidal Coomassie for subsequent protein identification. Gels from the various samples showed a high degree of consistence concerning protein-spots found in all preparations. Nevertheless, there was a number of proteins that were clearly detected in one sample but not, or only in significantly smaller amounts in the other. Several differentially expressed proteins in the postnatal myenteric plexus were identified with MALDI-TOF mass spectrometry. Especially stathmin, polyubiquitin and heterogeneous nuclear ribonucleoprotein seem to play an important role in pre- and postnatal development. 2-DE combined with mass spectrometry can help to identify pathological relevant proteins in the enteric nervous system, and so deliver a valuable tool for the early diagnosis of also central nervous system diseases by using biopsies from the gut.

Distinct expression of calnexin in major human salivary glands.

Calnexin (Cnx) has been characterized as a membrane-bound protein that transiently interacts in a unique chaperone system with newly synthesized glycoproteins in order to allow the establishment of their proper tertiary and, in most cases, quarternary structures. The aim of the study was to identify and to locate the expression of Cnx in the three major salivary glands of humans by different methods. Strong expression of Cnx protein and mRNA were generally found in serous salivary secretory units. With regard to mucous secretory units, expression of Cnx was only detectable at a low level in mucous acinar cells of sublingual glands, but not of submandibular glands. Expression of Cnx was always preserved in the surface epithelium of intralobar and interlobular duct segments. In addition, expression of Cnx was detected in sebaceous glands of parotid tissues, with a distribution pattern resembling that seen in sebaceous glands of the normal skin. In conclusion, production of saliva is associated with the expression of Cnx. Synthesis of molecules in mucous secretory units is not necessarily associated with a strong Cnx expression, whereas synthesis in serous secretory units apparently is. The tissue-specific Cnx expression is also paralleled by the observation that the secretions produced by the major salivary glands differ in their composition and amount.

Proteome analysis of lipofuscin in human retinal pigment epithelial cells.

Excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial cells is a common pathogenetic pathway in various blinding retinal diseases including age-related macular degeneration, which is now the most common cause of registerable blindness in the industrialized nations. To better understand the role of lipofuscin accumulation and to manipulate the pathogenetic mechanisms on both experimental and therapeutic levels we analyzed the proteome of isolated human ocular lipofuscin granules from human RPE cells. After homogenization and fractionation by gradient ultracentrifugation of the RPE/choroid complex from 10 pairs of human donors, protein compounds were separated by 2D gel electrophoresis and analyzed using matrix-assisted laser desorption/ionization mass spectrometry and HPLC-coupled electrospray tandem mass spectrometry. Besides a better understanding of downstream pathways, this approach may provide new targets for therapeutic interventions in a currently untreatable disease.

A novel karyoskeletal protein: characterization of protein NO145, the major component of nucleolar cortical skeleton in Xenopus oocytes.

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to M(r) 145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from different Xenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli of Xenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.

Characterization of the differential protein expression associated with thermoresistance in human gastric carcinoma cell lines.

Resistance to chemotherapeutic agents is one of the major problems faced during palliative therapy of tumor cells. Thus, chemotherapy is frequently combined with other modes of therapy such as radiation therapy and/or hyperthermia. Tumor cells respond to heat stress with development of thermotolerance and the interactions between chemo- and thermoresistance phenomena are not clearly understood. In this paper, we analyze the differential protein expression in vitro in human stomach cancer cells, their chemoresistant and thermoresistant counterparts using proteomics. The immediate aim was to identify sets of proteins that may lead to the development of thermoresistance. Based on these results, we aim to develop functional tests and methods for the modulation of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers.

A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis.

We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.

Nuclear particles containing RNA polymerase III complexes associated with the junctional plaque protein plakophilin 2.

Plakophilin 2, a member of the arm-repeat protein family, is a dual location protein that occurs both in the cytoplasmic plaques of desmosomes as an architectural component and in an extractable form in the nucleoplasm. Here we report the existence of two nuclear particles containing plakophilin 2 and the largest subunit of RNA polymerase (pol) III (RPC155), both of which colocalize and are coimmunoselected with other pol III subunits and with the transcription factor TFIIIB. We also show that plakophilin 2 is present in the pol III holoenzyme, but not the core complex, and that it binds specifically to RPC155 in vitro. We propose the existence of diverse nuclear particles in which proteins known as plaque proteins of intercellular junctions are complexed with specific nuclear proteins.

Identification of a novel structural variant of the alpha 6 integrin.

The alpha(6) integrin is a 140-kDa (nonreduced) laminin receptor. We have identified a novel 70-kDa (nonreduced) form of the alpha(6) integrin called alpha(6)p for the latin word parvus, meaning small. The variant was immunoprecipitated from human cells using four different alpha(6)-specific monoclonal antibodies but not with alpha(3) or alpha(5) antibodies. The alpha(6)p integrin contained identical amino acid sequences within exons 13--25, corresponding to the extracellular "stalk region" and the cytoplasmic tail of the alpha(6) integrin. The light chains of alpha(6) and alpha(6)p were identical as judged by alpha(6)A-specific antibodies and electrophoretic properties. The alpha(6)p variant paired with either beta(1) or beta(4) subunits and was retained on the cell surface three times longer than alpha(6). Reverse transcription/polymerase chain reaction analysis revealed a single polymerase chain reaction product. The alpha(6)p variant was found in human prostate (DU145H, LnCaP, PC3) and colon (SW480) cancer cell lines but not in normal prostate (PrEC), breast cancer (MCF-7), or lung cancer (H69) cell lines or a variant of a prostate carcinoma cell line (PC3-N). Protein levels of alpha(6)p increased 3-fold during calcium-induced terminal differentiation in a normal mouse keratinocyte model system. A novel form of the alpha(6) integrin exists on cell surfaces that contains a dramatically altered extracellular domain.

Activation of pro-astacin. Immunological and model peptide studies on the processing of immature astacin, a zinc-endopeptidase from the crayfish Astacus astacus.

To contribute knowledge of the processing and activation of invertebrate proteolytic enzymes, we studied the metalloprotease astacin, a digestive enzyme from the freshwater crayfish Astacus astacus (decapod crustacean). It is the prototype of the protein family of astacins, members of which occur in organisms from bacteria to man and are involved in a variety of physiological reactions. According to its genomic structure, astacin is produced as a zymogen [Geier, G., Jacob, E., Stöcker, W. & Zwilling, R. (1997) Arch. Biochem. Biophys. 337, 300-307]. To localize and follow the processing of pro-astacin in different parts of the digestive tract, we synthesized two peptides covering the pro part of pro-astacin and raised antibodies against them. In addition, antiserum against the whole active astacin was produced. Using immunohistochemical investigation, we detected pro-astacin in the F cells of the hepatopancreas and all the way into the tubular lumen and the collecting ducts of this gland. Immunoblot assays revealed only active astacin, and never pro-astacin, present in the cardiac stomach. We conclude from these studies that astacin is secreted into the lumen of the hepatopancreatic tubules in its pro form and is activated on its way to the stomach. To investigate which of the two endopeptidases found in the digestive tract of crayfish, astacin or trypsin, is responsible for cleaving the propeptide from pro-astacin, we synthesized different peptides that mimick the activation site. MS analysis of the cleavage products of astacin and trypsin showed that astacin is capable of catalyzing its own activation. Any contribution of trypsin would require the successive action of an aminopeptidase. Substituting glycine for arginine at position -1 of the activation site does not prevent astacin activity. As most members of the astacin protein family have basic amino-acid residues in this position, in these cases also astacin-specific cleavage would be possible.

The AN2 protein is a novel marker for the Schwann cell lineage expressed by immature and nonmyelinating Schwann cells.

The expression of the 330 kDa AN2 glycoprotein was studied in the rodent peripheral nervous system. AN2 is expressed by immature Schwann cells in vitro and in vivo and downregulated as the cells upregulate myelin genes. A subpopulation of nonmyelinating Schwann cells in the adult sciatic nerve retains expression of AN2. In rat sciatic nerve crushes, where Schwann cell numbers increase after initial axonal loss and markers of immature Schwann cells show an upregulation, no increased expression of AN2 was observed. In contrast, AN2 expression was upregulated in nerves from peripheral myelin protein-22-transgenic rats, where immature Schwann cells expand without axonal loss. Furthermore, coculture with neurons upregulated AN2 expression on Schwann cells in vitro. Polyclonal antibodies against AN2 inhibited the migration of an immortalized Schwann cell clone in an in vitro migration assay, and the purified AN2 protein was shown to be neither inhibitory nor permissive for outgrowing dorsal root ganglion neurites. AN2 is thus a novel marker for the Schwann cell lineage. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of purified AN2 from early postnatal mouse brain demonstrated that AN2 is the murine homolog of the rat NG2 proteoglycan.

Characterization of peptide substrates and viral enzyme that affect the cleavage site specificity of the human spumaretrovirus proteinase.

Oligopeptides that correspond to proteolytic cleavage site junctions of the native Gag and Pol proteins are specifically cleaved by retroviral aspartate proteases (PRs). The role of the flap subdomain of the PR of the human spumaretrovirus (HSRV) and of substrate peptides in cleavage site specificity was analyzed by site-directed mutagenesis. Native and mutant peptides were subjected to proteolysis by the authentic and mutated recombinant viral enzyme. The results reveal that Glu residue 54 of the HSRV PR is an essential specificity determinant for proteolytic processing of the structural proteins. Peptides that represent in vivo cleavage sites were susceptible to proteolysis by the recombinant HSRV PR, but one peptide located at the junction between the PR and reverse transcriptase domains was completely resistant to cleavage. Thus the data indicate that a proteolytic cleavage between these domains does not occur in vivo. Naturally occurring and mutant forms of the cleavage-resistant peptide were therefore analyzed by circular dichroism to determine if differences existed in the secondary structures of the peptides that did or did not serve as substrates. The data show that differences in the secondary structure of the native and mutant peptides analyzed does not seem to play a crucial role for cleavage site specificity in HSRV PR. Instead highly conserved hydrophobic residues at distinct positions of the HSRV cleavage site junctions contribute to the specificity observed as reported for HIV-1 PR.

First isolation of human UDP-D-xylose: proteoglycan core protein beta-D-xylosyltransferase secreted from cultured JAR choriocarcinoma cells.

Human UDP-d-xylose:proteoglycan core protein beta-d-xylosyltransferase (EC, XT) initiates the biosynthesis of glycosaminoglycan lateral chains in proteoglycans by transfer of xylose from UDP-xylose to specific serine residues of the core protein. In this study, we report the first isolation of the XT and present the first partial amino acid sequence of this enzyme. We purified XT 4,700-fold with 1% yield from serum-free JAR choriocarcinoma cell culture supernatant. The isolation procedure included a combination of ammonium sulfate precipitation, heparin affinity chromatography, ion exchange chromatography, and protamine affinity chromatography. Among other proteins an unknown protein was detected by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis in the purified sample. The molecular mass of this protein was determined as 120 kDa by SDS-polyacrylamide gel electrophoresis. The isolated protein was enzymatically cleaved by trypsin and endoproteinase Lys-C. Eleven peptide fragments were sequenced by Edman degradation. Searches with the amino acid sequences in protein and EST data bases showed no homology to known sequences. XT was enriched by immunoaffinity chromatography with an immobilized antibody against a synthetic peptide deduced from the sequenced peptide fragments and was specifically eluted with the antigen. In addition, XT was purified alternatively with an aprotinin affinity chromatography and was detected by Western blot analysis in the enzyme-containing fraction.

Meiotic lamin C2: the unique amino-terminal hexapeptide GNAEGR is essential for nuclear envelope association.

Meiotic lamin C2 is the only A-type lamin expressed during mammalian spermatogenesis. Typical for this short lamin is the unique hexapeptide GNAEGR, which substitutes the nonhelical amino terminus and part of the alpha-helical rod domain present in somatic lamins. Meiotic lamin C2 also lacks a carboxyl-terminal CaaX box, which is modified by isoprenylation and involved in nuclear envelope (NE) association of somatic isoforms. The mechanism by which lamin C2 becomes localized in the NE is totally unknown. Here we demonstrate that the hexapeptide GNAEGR is essential for this process: (i) Its deletion resulted in a diffuse distribution of lamin C2 within nuclei of transfected COS-7 cells; (ii) Mutated somatic lamin C, containing the sequence GNAEGR at its amino terminus, was located at the NE. The mass spectrometric analysis of the amino terminus of lamin C2 revealed that it is modified by myristoylation. Correspondingly, the substitution of the first glycine residue abolishes the NE association of lamin C2. We conclude that NE association of lamin C2 is achieved by a mechanism different from that of somatic lamins.

Identification of novel proteins associated with the development of chemoresistance in malignant melanoma using two-dimensional electrophoresis.

A model system for studying chemoresistance in human melanoma cells (MeWo) has been established utilizing the four commonly used cytotoxic drugs vindesine, cisplatin, fotemustine and etoposide to yield stable drug-resistant sublines. We analyzed phenotypical differences between MeWo cells and their chemoresistant counterparts using two-dimensional electrophoresis. Proteins that were overexpressed in chemoresistant cell lines were purified and identified using matrix assisted laser desorption/ionization-time of flight - mass spectrometry (MALDI-TOF-MS) and microsequencing. Here we show that four proteins, namely the translationally controlled tumor protein, the human elongation factor 1-delta, tetratricopeptide repeat protein and the isoform 14-3-3-gamma of the 14-3-3-family are overexpressed in chemoresistant melanoma cell lines. The significance of these findings is now being verified using transfection experiments with the aim of developing more effective chemotherapy protocols.