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Reproducible quantification of metabolites in tissue samples is of high importance for characterization of animal models and identification of metabolic changes that occur in different tissue types in specific diseases. However, the extraction of metabolites from tissue is often the most labor-intensive and error-prone step in metabolomics studies. Here, we report the development of a standardized high-throughput method for rapid and reproducible extraction of metabolites from multiple tissue samples from different organs of several species. The method involves a bead-based homogenizer in combination with a simple extraction protocol and is compatible with state-of-the-art metabolomics kit technology for quantitative and targeted flow injection tandem mass spectrometry. We analyzed different extraction solvents for both reproducibility as well as suppression effects for a range of different animal tissue types including liver, kidney, muscle, brain, and fat tissue from mouse and bovine. In this study, we show that for most metabolites a simple methanolic extraction is best suited for reliable results. An additional extraction step with phosphate buffer can be used to improve the extraction yields for a few more polar metabolites. We provide a verified tissue extraction setup to be used with different indications. Our results demonstrate that this high-throughput procedure provides a basis for metabolomic assays with a wide spectrum of metabolites. The developed method can be used for tissue extraction setup for different indications like studies of metabolic syndrome, obesity, diabetes or cardiovascular disorders and nutrient transformation in livestock.

Francisella tularensis, the causative agent of tularaemia, is a potential agent of bioterrorism. The phenotypic discrimination of the closely related F. tularensis subspecies and individual strains with traditional methods is difficult and time consuming, often producing ambiguous results. Surface-enhanced laser desorption/ionization time-of-flight MS (SELDI-TOF MS) was used in this study to discriminate the different species and subspecies of the genus Francisella. We tested 18 Francisella strains including at least one representative of each species/subspecies on four different types of chromatographic chip surfaces. Multivariate analysis (hierarchical clustering and principal component analysis) allowed grouping of the strains according to their designated subspecies. Furthermore, single strains within F. tularensis subspecies could be discriminated.

A feasibility study to couple high throughput screening of packed bed chromatography with mass spectrometric detection by SELDI-TOF MS is presented. As model system monoclonal antibodies (mAb) versus host cell protein (HCP) from an industrial cultivation was chosen. Packed bed chromatography was screened on a TECAN Evo Freedom 200 station using miniaturized chromatographic columns placed on a specially designed array carrier linked to a commercially available T-Stack module. Gradient elution of the bound proteins was performed by applying a multiple step strategy. When analyzing selected HCP peaks as well as the detected antibody peaks throughout the chromatographic runs a direct correlation between applied and detected components was established. The sensitivity of conventional protein A chromatography was found to be lower than SELDI-TOF MS analysis. During initial screening a shift in the elution pattern for one of the monoclonal antibodies detected with all four resins was identified to be a heterogeneity in the mAb glycosylation pattern. In addition, a detailed differentiation between various HCP fractions through out the chromatographic process using SELDI-TOF analysis let to the detection of HCP components possibly adhering to the mAbs during chromatographic separations. Biotechnol. Bioeng. 2007;98; 440–450. © 2007 Wiley Periodicals, Inc.

The accurate diagnosis of the different forms of chronic mature B-cell lymphocytic malignancies is of primary importance to determine an appropriate and efficient treatment. Usually, the diagnosis is achieved by morphology and immunophenotyping. Nevertheless, the diagnostic tools available are not able to discriminate pathologies with variable evolution, or to classify some of them. To discover new biomarkers, we used peptide and protein profiling SELDI-TOF-MS, to analyze 39 chronic B-cell malignancies and 20 control serum samples. Markers of interest were subsequently identified and characterized. In the obtained SELDI-MS profiles, most of the differences were observed in three mass ranges (m/z = 13 000; m/z = 9000; m/z2000). Identification of these biomarkers was achieved either by direct enrichment on the ProteinChip arrays followed by on-chip-MS/MS or by chromatographic fractionation, 1D-gel followed by nanoLC-MS/MS analysis. An increase of a sulfite form of transthyretin (13,841 Da) was observed in the patient group. A second set of markers at 8.6 and 8.9 kDa was identified as complement related fragment proteins, the C3a and C4a anaphylatoxins. In the low mass range, several peptides originating from N-terminal and C-terminal processing of the C3 alpha and C4 alpha chains were specifically observed in 38% of the patient sera, but in none of the control sera. This study emphasizes the usefulness of mass spectrometry studies in such malignancies.

Although colorectal cancer is one of the best-characterized tumors with regard to the multistep progression, it remains one of the most frequent and deadly neoplasms. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, changes in protein expression between microdissected normal and tumorous colonic epithelium were analyzed. Cryostat sections from colorectal tumors, adenoma tissue, and adjacent normal mucosa were laser-microdissected and analyzed using ProteinChip Arrays. The derived MS profiles exhibited numerous statistical differences. One peak showing significantly high expression in the tumor was purified by reverse-phase chromatography and SDS-PAGE. The protein band of interest was passively eluted from the gel and identified as heat shock protein 10 (HSP 10) by tryptic digestion, peptide mapping, and MS/MS analysis. This tumor marker was further characterized by immunohistochemistry. Analysis of HSP 10-positive tissue by ProteinChip technology confirmed the identity of this protein. This work demonstrates that biomarker in colorectal cancer can be detected, identified, and assessed by a proteomic approach comprising tissue microdissection, protein profiling, and immunological techniques. In our experience, histological defined microdissected tissue areas should be used to identify proteins that might be responsible for tumorigenesis.

In this study, we describe a differential mass spectrometric technique for the immuno-proteomic analysis of the major histocompatibility complex (MHC) peptides of a renal cell carcinoma (RCC) biopsy compared with the healthy kidney tissue of the same patient after nephrectomy. Using a stable isotope labeling approach, we could directly compare and relatively quantify 43 MHC-peptide pairs, most of which were present in similar proportions on both normal kidney and tumor. Significantly, two dominant peptides of monoisotopic masses ([M+H](+)) 973.43 u and 967.59 u, respectively, were found exclusively in the tumor sample. One of these was identified as originating from heme oxygenase-1 (HO-1), a protein involved in induction of apoptosis resistance, immuno-suppression and neoangiogenesis and reported to be up-regulated in various cancer types. Moreover, the corresponding synthetic HO-1-derived peptide was shown to be immunogenic in vitro by generation of CD8+ T cell lines with peptide-specific cytolytic activity. Thus, this peptide is an example of a differentially identified T cell epitope that could be considered as a target for immunotherapy.

The calcium-binding proteins of the S100 and the annexin protein families have been implicated in a variety of important physiological functions including membrane remodeling, calcium-related intracellular signaling, cytoskeleton dynamics, tissue homeostasis, and formation of the cornified envelope in differentiating keratinocytes. Deregulated expression of members of these families has been reported in different types of neoplasia and other diseases, but the results were not consistent. Here we have applied a combination of cDNA microarrays, quantitative reverse transcriptase-PCR (qRT-PCR) and surface enhanced laser desorption ionisation-time of flight mass spectrometry (SELDI-TOF MS) to study differential expression of these genes in head and neck squamous cell carcinoma (HNSCC). The calgranulins A and B and annexins 1 and 2 were found to be down-regulated in HNSCC, compared with normal mucosa, at both the mRNA and protein level. Upon validation of the differential gene expression by tissue microarray immunohistochemistry, we detected novel expression patterns of calgranulins A and B both in normal mucosa as well as in HNSCC. In contrast to squamous cancer of skin and other cancers in which the calgranulins were found to be up-regulated, most HNSCC showed reduced and widely deranged staining patterns including heterogeneous nuclear, cytoplasmic and membranous staining, and even enhanced staining in the tumor stroma. These observations suggest that the normal function of the calgranulins A and B in mucosa might be different from that in skin.

The molecular analysis of serum is an important field for the definition of potential diagnostic markers or disease-related protein alterations. Novel proteomic technologies such as the mass spectrometric-based surface-enhanced laser desorption/ionization (SELDI) ProteinChip technique facilitate a rapid and reproducible analysis of such protein mixtures and affords the researcher a new dimension in the search for biomarkers of disease. Here, we have applied this technology to the study of a cohort of serum samples from well-characterized renal cell carcinoma patients for the identification of such proteins by comparison to healthy controls. We detected and characterized haptoglobin 1 alpha and serum amyloid alpha-1 (SAA-1) as disease related, in addition to an as-yet-unidentified marker of 10.84 kDa. Of particular note is the detection of multiple variants of SAA-1 in multiplex that have not been described in the sera of cancer patients. SAA-1 is detected as full-length protein, des-Arginine and des-Arginine/des-Serine variants at the N terminus by SELDI. In addition, we could also detect a low-abundant variant minus the first five N-terminal amino acids. Such variants may impact the function of the protein. We conclude the technique to be a reproducible, fast and simple mode for the discovery and analysis of marker proteins of disease in serum.

Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 × 10−5 and 4.6 × 10−5) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.

Recently, a novel antimicrobial peptide DCD-1, derived from the Dermcidin (DCD) gene and secreted by sweat glands, has been described by Schittek et al. [Nat. Immunol. 2 (2001) 1133.]. Here we describe the application of the surface-enhanced laser desorption/ionisation (SELDI) technology for the detection of DCD-1 and other dermcidin-derived peptides directly from microlitre amounts of human sweat. The advantages of the technique are as follows: (a) it can be carried out with ease and rapidity; (b) multiple samples can be processed simultaneously; (c) prior purification is not required; and (d) only a limited sample volume is necessary for both protein profiling and semiquantitation. Profiling of human sweat from various donors revealed that in addition to DCD-1, other DCD-derived peptide species were also present in significant quantities. Four of five identified peptides were DCD-1 related, while the fifth corresponded to a portion of the DCD protein outside the DCD-1 core. This provides clues as to how the novel protein is processed to its active form, though further work remains to elucidate this fully. Thus, we have demonstrated the applicability of such technology to the detection of DCD-1 and for the protein profiling of sweat in general. Such studies could reveal valuable new biomarkers for diagnosis and treatment of skin and sweat gland disorders.

Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here, we screened mouse serum for metabolic alterations following an acute exposure to γ radiation using a multiplatform mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and low-abundance, oxygenated, polyunsaturated fatty acids (PUFAs) in the two groups of animals. Exposure to γ radiation induced a significant increase in the serum levels of ether phosphatidylcholines (PCs) while decreasing the levels of diacyl PCs carrying PUFAs. In exposed mice, levels of pro-inflammatory, oxygenated metabolites of arachidonic acid increased, whereas levels of anti-inflammatory metabolites of omega-3 PUFAs decreased. Our results indicate a specific serum lipidomic biosignature that could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine.

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe-Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3-8 microM and the inactivation was reversed by 2, 3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.

Deficiency of neuronal growth inhibitory factor (GIF) and abnormalities in zinc homeostasis have been suggested to play a role in the neuropathogenesis of Alzheimer's disease. We report here that embryonic chick cerebral cell cultures zinc and copper containing GIF in the presence of marmoset hippocampal extract reduces significantly and concentration dependently mitochondrial succinate dehydrogenase activity (MTT) and cell mass. In contrast, no indications could be found that GIF affected neural retina cell cultures. Our results suggest that the observed effects of GIF are not elicited by zinc.

Neuronal-growth-inhibitory factor (GIF) is a metalloprotein specific to the central nervous system which has been linked to Alzheimer's disease. The high metal content, approximately seven metal atoms/protein molecule, and 70% sequence identity to mammalian metallothioneins (MT), including a preserved array of 20 cysteinyl residues, place GIF in the family of MT. In contrast to MT, native GIF isolated from human or bovine brain contains an unusual metal composition, viz. four Cu(I) and three Zn(II) per polypeptide chain. Cu and/or Zn K-edge X-ray absorption spectra have been recorded for native Cu, Zn-GIF, Zn-substituted GIF, and these metals bound to the 32-residue N-terminal domain, Cu4-, Cu6- or Zn3-GIF-(1-32) at 77 K. The results are consistent with the metals being bound to the protein by cysteinyl residues in every case. The Cu-S distance is approximately 2.25 A and the EXAFS is considered to be consistent with primarily trigonal coordination of the Cu(I); Cu...Cu backscattering is observed at approximately 2.67 A, indicative of the formation of Cu(x)(Scys)y clusters. Thus, the Cu(I) environment is similar to that observed in MT. This is also the case for Zn(II), with 4 S at approximately 2.34 A. However, in contrast to Zn-MT for Zn-substituted GIF and Zn3-GIF-(1-32), Zn...Zn backscattering is observed at approximately 3.28 A. The significance of these results are discussed with respect to the specific biological activity of GIF.

The ability of metallothionein (MT) to modulate DNA binding by a two-finger peptide of Tramtrack (TTK), a CCHH zinc transcription factor, was investigated using metal-bound and metal-deficient forms of rabbit MT-2 and the TTK peptide. Thionein inhibited DNA binding by zinc-bound TTK, and Zn-MT restored DNA-binding by zinc-deficient apo-TTK. "Free" zinc at low concentrations was as effective as Zn-MT in restoring DNA binding by apopeptide but was inhibitory at concentrations equal to zinc bound to 2 mol eq and higher of Zn-MT. Substitution of cadmium for zinc reduced the affinity of the peptide for its DNA binding site. This effect was reversed by incubation with Zn-MT. The circular dichroic spectra of the TTK peptide indicated that zinc removal resulted in loss of alpha-helical structures, which are sites of DNA contact points. Reconstitution with cadmium resulted in stoichiometric substitution of 2 mol of Cd/mol of peptide but not recovery of alpha-helical structures. Incubation of Cd-TTK with Zn-MT restored the secondary structure expected for zinc-bound TTK. The ability of Zn-MT and thionein to restore or inhibit DNA-binding by TTK was associated with effects on the metallation status of the peptide and related alterations in its secondary structure.

Dimethylargininase (EC 3.5.3.18) is involved in the regulation of the levels of the natural occurring free arginine derivatives L-Nomega,Nomega-dimethylarginine and L-Nomega-methylarginine, which are reversible inhibitors of nitric oxide synthase. A dimethylargininase has been isolated from bovine brain tissue and was characterized by using immunological, kinetic, and spectroscopic techniques. Western blot analysis using polyclonal antibodies revealed that the enzyme is widely distributed in bovine with the highest relative concentrations found in brain and kidney tissue. A similar tissue distribution has also been reported for the other so far isolated dimethylargininase from rat kidney [Ogawa, T., Kimoto, M., and Sasaoka, K. (1989) J. Biol. Chem. 264, 10205-10209]. The bovine enzyme is a monomeric, globular protein (molecular mass approximately 31.2 kDa) containing one tightly bound Zn2+ ion, which can be removed by dialysis against 1,10-phenanthroline. The determination of kinetic constants for both the native (holo-protein) and the zinc-depleted (apo-protein) enzyme at 37 degrees ¿C established that the dimethylargininase is not a zinc hydrolase. The specific activity was 0.66 unit/mg for the holo-protein and 0.19 unit/mg for the apo-protein. The secondary structure determination of the native enzyme by circular dichroism revealed 41% alpha-helix and 32% beta-sheet and beta-turn structure. In the apo-enzyme, a small, but significant decrease in the alpha-helical content (5%) was observed, consistent with a marked decrease in enzymatic activity to 30%. Upon preincubation of both enzyme forms at 50 degrees C, only the holo-enzyme showed a residual enzymatic activity. In thermostability studies, a 7 degrees C lower apparent Tm value was observed for the apo-enzyme compared to the 66 degrees C for the holo-enzyme, suggesting that the zinc ion has a structure-stabilizing role. Besides the tightly bound zinc, additional Zn2+ ions inhibit the enzyme competitively with a Ki value of 2.0 microM. A possible interrelationship between dimethylargininase and nitric oxide synthase is discussed.

The interactions of substrates and inhibitors with the Mn2+ ions in the binuclear active center of D-xylose isomerase (XylI) were investigated by EPR spectroscopy at X- and Q-band frequencies. The metal binding site 1 (A site) was specifically occupied with Mn2+ ions by blocking the high-affinity metal binding site 2 (B-site) either with Co2+ ions, resulting in a catalytically active enzyme, or with Cd2+ or Pb2+ ions yielding an inactive enzyme species. Incubation of both the Co2+/Mn2+- and the Cd2+/Mn2+-XylI with the acyclic inhibitor xylitol revealed EPR spectra with well-resolved hyperfine patterns, but with increased zero field splitting (zfs) parameter D compared to the spectra without inhibitor. D was estimated by spectral simulation of the central --1/2--1/2 fine structure transition. D values of 33 and 50 mT were obtained for the Co2+/Mn2+-XylI and the Cd2+/Mn2+-XylI samples, respectively. These results indicate direct interaction of the xylitol with the Mn2+ in the A-site. More drastic changes are observed with the substrates D-xylose and D-glucose and with the cyclic inhibitors 5-thio-alpha-D-glucose and 2-desoxy-D-glucose. For Cd2+/Mn2+-XylI, the EPR spectra with substrates and cyclic inhibitors are similar to each other but different from the spectra with the acylic inhibitor xylitol. They exhibit well-resolved line patterns with a relative large zero field splitting, which was estimated to be in the range of D = 65-85 mT in the various complexes. Binding of substrates or of cyclic inhibitors to the Co2+/ Mn2+-XylI yields EPR spectra without resolved hyperfine interactions, indicative of dipolar interaction between the two paramagnetic metal ions. This can be explained with a decrease in the metal-metal distance. Furthermore, the EPR data strongly suggest that the corresponding metal ion movement is induced by binding of the cyclic conformation of either substrates or cyclic inhibitors and not by binding of the extended form of the sugars.

An orientationally-selected electron spin-echo envelope modulation (ESEEM) spectroscopy investigation was performed on VO{sup 2+} introduced into the high-affinity metal-binding site of D-xylose isomerase. The ESEEM spectra clearly reveal the presence of nitrogen ligands with hyperfine coupling A{sup N} {approx} 6 MHz. Detailed analysis includes first- and second-order treatment of the nitrogen basic and combination harmonics in two-pulse ESEEM spectra of the g{sub {parallel}} and g{sub {perpendicular}} components. Complete determination of the hyperfine and quadrupole tensor indicates equatorial coordination of the imine nitrogen of the histidine residue. The presence of Cd{sup 2+} ion in the second, low-affinity metal-binding site does not affect the nitrogen couplings. The protons surrounding the VO{sup 2+} ion have been examined via the proton sum combinations in four-pulse ESEEM. They demonstrate the contribution of two protons probably belonging to the histidine ligand. These investigations strongly support the further application of VO{sup 2+} as a spin probe in conjunction with ESEEM spectroscopy for detailed investigation of nitrogen ligands in the active metal sites of proteins. 41 refs., 8 figs., 2 tabs.

The coordination sphere of the two metal-binding sites/subunit of the homotetrameric D-xylose isomerase from Streptomyces rubiginosus has been probed by the investigation of the Co2(+)-substituted enzyme using electronic absorption, CD and magnetic circular dichroic spectroscopies in the visible region. The spectrum of the high-affinity site (B site) has an absorption coefficient, epsilon 545, of 18 M-1 cm-1, indicating a distorted octahedral complex geometry. The spectrum of the low-affinity site (A site) shows two absorption maxima at 505 nm and 586 nm with epsilon values of 170 M-1 cm-1 and 240 M-1 cm-1, respectively, which indicates a distorted tetrahedral or pentacoordinated complex structure as also observed for the enzyme from Streptomyces violaceoruber [Callens et al. (1988) Biochem. J. 250, 285-290] having the same feature but lower epsilon values. The first 4 mol Co2+ added/mol apoenzyme occupy both sites nearly equally. Subsequently the Co2+ located in the A site slowly moves into the B site. After equilibrium is reached, the next 4 mol Co2+/mol again occupy the A site with its typical spectrum, restoring full activity. Addition of 4 mol Cd2+ or Pb2+/mol Co4-loaded derivative displaces the Co2+ from the B site to form the Pb4/Co4 derivative containing Co2+ in the A site, reducing activity fourfold while the Pb4/Pb4 species is completely inactive. In contrast, Eu3+ displaces Co2+ preferentially from the A site. Thus, the high- and low-affinity sites may be different for different cations. After addition of the substrates D-xylose, D-glucose and D-fructose and the inhibitor xylitol the intense Co2+ A-site spectrum of both the active Co4/Co4 derivative and the less active Pb4/PCo4 derivative decreases, indicating that these compounds are bound to the A site, changing the distorted tetrahedral or pentacoordinated symmetry there to a distorted octahedral complex geometry.

Objective Testing of serum for protein patterns to monitor progression of suspected to definite chronic pancreatitis (CP). Methods Serum samples of CP patients and healthy volunteers were fractionated on anion exchange columns and analyzed by surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry to elucidate CP-related protein alterations and to identify biomarkers for this disease. Potential biomarkers were purified and identified by mass spectrometry. Results In total, 258 protein peaks were found that discriminated between the 2 groups. Analysis revealed 28 most prominent peaks on immobilized metal affinity capture coupled with Cu and CM10 protein chips, covering the m/z range between 3.3 and 33.3 kd. Performing multivariate pattern analysis, the best pattern model was obtained using fraction 6 on immobilized metal affinity capture coupled with Cu arrays with a sensitivity of 96% and a specificity of 84%. Using a combination of matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry and immunodepletion, we identified 14-m/z peaks. The proteins were found to be significantly decreased in CP serum and were identified as retinol-binding protein, serum amyloid-alpha, apolipoprotein A-II (Apo A-II), Apo C-I, Apo C-II, Apo C-III, and transthyretin and truncated forms thereof. Conclusions Distinct protein profile differences exist between normal and CP serum and reflect the metabolic and inflammatory condition in CP patients. The identified protein panel may eventually serve as a diagnostic marker set for CP.

Objective: Early detection is the only promising approach to significantly improve the survival of patients with pancreatic cancer (PCa). Noninvasive tools for the diagnosis, prognosis, and monitoring of this disease are of urgent need. The purpose of this study was to identify and validate new biomarkers in PCa patient serum samples. Methods: Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry has been applied to analyze serum protein alterations associated with PCa and to identify sets of potential biomarkers indicative for this disease. A cohort of 96 serum samples from patients undergoing PCa surgery was compared with sera from 96 healthy volunteers as controls. The sera were fractionated by anion exchange chromatography, and 3 of 6 fractions were analyzed onto 2 different chromatographic arrays. Results: Data analysis revealed 24 differentially expressed protein peaks (P < 0.001), of which 21 were downregulated in the PCa samples. The best single marker can predict 92% of the controls and 89% of the cancer samples correctly. In addition, multivariate pattern analysis was performed. The best pattern model using a set of 3 markers was obtained using fraction 6 on immobilized metal affinity capture, loaded with Cu-Cu arrays. With this pattern model, a sensitivity of 100% and a specificity of 98% for the training data set and a sensitivity of 83% and specificity of 77% for the test data set were achieved with the PCa group set as true positive. Several of protein peaks, including the best single marker at 17.27 kd and other proteins from the pattern models, were purified and identified by peptide mapping and postsource decay-matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Apolipoprotein A-II, transthyretin, and apolipoprotein A-I were identified as markers, and these identified proteins were decreased at least 2-fold in PCa serum when compared with the control group. Conclusions: PCa is associated with a specific decrease of distinct serum proteins, which allows a reliable differentiation between pancreatic cancer and healthy controls.

The resistance of the malarial parasite Plasmodium falciparum to chloroquine represents an emerging problem since neither mode of drug action nor mechanisms of resistance are fully elucidated. We describe a protein expression profiling approach by SELDI-TOF-MS as a useful tool for studying the proteome of malarial parasites. Reproducible and complex protein profiles of the P. falciparum strains K1, Dd2, HB3 and 3D7 were measured on four array types. Hierarchical clustering led to a clear separation of the two major subgroups "resistant" and "sensitive" as well as of the four parasite strains. Our study delivers sets of regulated proteins derived from extensive comparative analyses of 64 P. falciparum protein profiles. A group of 12 peaks reflecting proteome changes under chloroquine treatment and a set of 10 potential chloroquine resistance markers were defined. Three of these regulated peaks were preparatively enriched, purified and identified. They were shown to represent the plasmodial EXP-1 protein, also called circumsporozoite-related antigen, as well as the alpha- and beta- (delta-) chains of human hemoglobin.

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.

Novel prognostic biomarkers are imperatively needed to help direct treatment decisions by typing subgroups of node-negative breast cancer patients. The current study has used a proteomic approach of SELDI-TOF-MS screening to identify differentially cytosolic expressed proteins with a prognostic impact in 30 node-negative breast cancer patients with no relapse versus 30 patients with metastatic relapse. The data analysis took into account 73 peaks, among which 2 proved, by means of univariate Cox regression, to have a good cumulative prognostic-informative power. Repeated random sampling (n = 500) was performed to ensure the reliability of the peaks. Optimized thresholds were then computed to use both peaks as risk factors and, adding them to the St. Gallen ones, improve the prognostic classification of node-negative breast cancer patients. Identification of ubiquitin and ferritin light chain (FLC), corresponding to the two peaks of interest, was obtained using ProteinChip LDI-Qq-TOF-MS. Differential expression of the two proteins was further confirmed by Western blotting analyses and immunohistochemistry. SELDI-TOF-MS protein profiling clearly showed that a high level of cytosolic ubiquitin and/or a low level of FLC were associated with a good prognosis in breast cancer.

The diagnosis of brucellosis is mainly based on the detection of anti-LPS antibodies. Due to substantial similarity of the O-polysaccharide of Brucella LPS to that of various other Gram-negative bacteria, serological tests of samples containing high amounts of LPS lack specificity. Hence, the development of assays based on more specific protein antigens is an essential subject in brucellosis research. The aim of this study was proteomic characterization of various antigen preparations of the diagnostic reference strain Brucella abortus 1119-3 and the identification of immunogenic proteins suitable for serological assays. Seventeen out of 383 protein spots of B. abortus 1119-3 were identified to be immunogenic by 2-D immunoblotting. These immunogenic spots were assigned to 6 proteins by MALDI-MS and nLC-ESI-MS/MS: Cu-Zn SOD, BCSP31, L7/L12, GroEL, GroES, and DnaK. All immunogenic proteins were present in three different antigen preparations investigated, i.e. native antigen, standard agglutination and commercially available agglutination antigen. 2-D immunoblotting of bacteria cross-reacting with Brucellae in agglutination tests proved that cross-reactivity of proteins is negligible. Surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) spectra also differentiated B. abortus clearly from cross-reacting bacteria. The combination of SELDI-MS analysis with the specificity of antibody binding will improve the identification of Brucella specific immunogenic proteins.

Background Laser microdissection allows precise isolation of specific cell types and compartments from complex tissues. To analyse proteins from small cell numbers, we combine laser-microdissection and manipulation (LMM) with mass spectrometry techniques. Results Hemalaun stained mouse lung sections were used to isolate 500–2,000 cells, enough material for complex protein profiles by SELDI-TOF MS (surface enhanced laser desorption and ionization/time of flight mass spectrometry), employing different chromatographic ProteinChip® Arrays. Initially, to establish the principle, we identified specific protein peaks from 20,000 laser-microdissected cells, combining column chromatography, SDS-PAGE, tryptic digestion, SELDI technology and Tandem MS/MS using a ProteinChip® Tandem MS Interface. Secondly, our aim was to reduce the labour requirements of microdissecting several thousand cells. Therefore, we first defined target proteins in a few microdissected cells, then recovered in whole tissue section homogenates from the same lung and applied to these analytical techniques. Both approaches resulted in a successful identification of the selected peaks. Conclusion Laser-microdissection may thus be combined with SELDI-TOF MS for generation of protein marker profiles in a cell-type- or compartment-specific manner in complex tissues, linked with mass fingerprinting and peptide sequencing by Tandem MS/MS for definite characterization.

Head and neck cancer is a frequent malignancy with a complex, and up to now not clear etiology. Therefore, despite of improvements in diagnosis and therapy, the survival rate with head and neck squamous-cell carcinomas is poor. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, we have analyzed changes of protein expression between microdissected normal pharyngeal epithelium and tumor tissue by ProteinChip® technology. For this, cryostat sections from head and neck tumors (n = 57) and adjacent mucosa (n = 44) were laser-microdissected and analyzed on ProteinChip arrays. The derived mass spectrometry profiles exhibited numerous statistical differences. One peak significantly higher expressed in the tumor (p = 0.000029) was isolated by two-dimensional gel electrophoresis and identified as annexin V by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immuno-deplete assay. The relevance of this single marker protein was further evaluated by immunohistochemistry. Annexin-positive tissue areas were re-analyzed on ProteinChip arrays to confirm the identity of this protein. In this study, we could show that biomarker in head and neck cancer can be found, identified, and assessed by combination of ProteinChip technology, two-dimensional gel electrophoresis, and immunohistochemistry. In our experience, however, such studies only make sense if a relatively pure microdissected tumor tissue is used. Only then minute changes in protein expression between normal pharyngeal epithelium and tumor tissue can be detected, and it will become possible to educe a tumor-associated protein pattern that might be used as a marker for tumorigenesis and progression.

Publisher Summary This chapter discusses affinity chromatography using an immobilized phenylarsine oxide derivative coupled to Sepharose for the purification of vicinal dithiolcontaining proteins. This methodology uses a phenylarsine oxide (PAO) resin to selectively bind, elute, and identify proteins that might be involved in redox regulation and to test a possible disulfide formation as a regulatory mechanism in oxidative stress. Because such conditions are found in activated polymorphonuclear leukocytes (PMN), a cytosolic fraction from PMN is used to isolate potential target proteins. This way the isolation and identification of six proteins with affinity for PAO can be achieved: thioredoxin (Trx), calcineurin (CAN), glutathione S-transferase (GST), calgranulin, L-plastin, and cofilin. From these candidates, a more detailed characterization of the interaction of PAO and oxidants with the serine-threonine phosphatase CaN (also known as protein phosphatase 2B) can be explored, which strongly suggests a dithiol-disulfide equilibrium in the enzyme as a mechanism for redox regulation.

Following the discovery of the neuronal growth inhibitory factor, classified as metallothionein3 (MT-3), by Uchida et. al. 1991 [1] we have focussed our research on the structure-function analysis of this protein. MT-3 is a central nervous system (CNS) specific metallothionein-like protein, which has been linked to Alzheimer’s disease (AD) [1]. The amino acid sequence of human MT-3 (68 amino acids) exhibits 70% sequence identity to mammalian metallothioneins (MT-1 and MT-2) (61 amino acids), including the preserved array of 20 Cys residues. In MT-1 and MT-2 these 20 Cys residues are involved in the formation of two metal-thiolate clusters, i.e. MeII 3(Cys)9 and MeII 4(Cys)11 [2]. Relative to mammalian MT-1 and MT-2 sequences, MT-3 contains two conserved inserts, a single Thr in the N-terminal and a Glu-rich hexapeptide in the C-terminal region (see Fig. 1). Despite their high sequence identity only MT-3 exhibits a growth inhibitory activity in neuronal cell culture studies, indicating that the — so far unknown — structure of MT-3 must differ from that of the other MTs. Furthermore, it has been demonstrated that the N-terminal metal binding domain MT-3(1–32) is sufficient for the growth inhibitory activity [3, 6]. In contrast to MTs, which usually contain 7 Zn(II) ions, native MT-3 isolated from bovine brain possesses an unusual metal ion composition, i.e. 4-5 Cu(I) and 2-2.5 Zn(II) per polypeptide chain, suggesting the presence of different cluster structures in this protein [4].

After introducing the 3D structure of mammalian metallothioneins (MTs), a class of low molecular weight metal-binding proteins, spectroscopic evidence for the metal selectivity of clusters and their structural changes upon binding of different divalent metal ions is presented. An interplay between both the chemistry of metal ions and the steric requirements of the polypeptide chain as the cause of the diversity of the metal-thiolate cluster structures in MT is discussed. Moreover, a metal-assisted ligand (apoMT) preorganization as a general concept for the formation of MT structure has been proposed.

Urine has long been a "favored" biofluid among metabolomics researchers. It is sterile, easy-to-obtain in large volumes, largely free from interfering proteins or lipids and chemically complex. However, this chemical complexity has also made urine a particularly difficult substrate to fully understand. As a biological waste material, urine typically contains metabolic breakdown products from a wide range of foods, drinks, drugs, environmental contaminants, endogenous waste metabolites and bacterial by-products. Many of these compounds are poorly characterized and poorly understood. In an effort to improve our understanding of this biofluid we have undertaken a comprehensive, quantitative, metabolome-wide characterization of human urine. This involved both computer-aided literature mining and comprehensive, quantitative experimental assessment/validation. The experimental portion employed NMR spectroscopy, gas chromatography mass spectrometry (GC-MS), direct flow injection mass spectrometry (DFI/LC-MS/MS), inductively coupled plasma mass spectrometry (ICP-MS) and high performance liquid chromatography (HPLC) experiments performed on multiple human urine samples. This multi-platform metabolomic analysis allowed us to identify 445 and quantify 378 unique urine metabolites or metabolite species. The different analytical platforms were able to identify (quantify) a total of: 209 (209) by NMR, 179 (85) by GC-MS, 127 (127) by DFI/LC-MS/MS, 40 (40) by ICP-MS and 10 (10) by HPLC. Our use of multiple metabolomics platforms and technologies allowed us to identify several previously unknown urine metabolites and to substantially enhance the level of metabolome coverage. It also allowed us to critically assess the relative strengths and weaknesses of different platforms or technologies. The literature review led to the identification and annotation of another 2206 urinary compounds and was used to help guide the subsequent experimental studies. An online database containing the complete set of 2651 confirmed human urine metabolite species, their structures (3079 in total), concentrations, related literature references and links to their known disease associations are freely available at http://www.urinemetabolome.ca. © 2013 Bouatra et al.

An acidic zinc- and heme-containing protein was isolated from the soluble fraction of bovine brain and has been purified to homogeneity. The zinc-heme protein is a monomeric globular protein with a molecular mass of 31200 Da as determined by electrospray mass spectrometry. The protein was isolated with 0.90±0.05 zinc per protein and with substoichiometric amounts of heme. Amino acid sequences of four peptides (ca. 20% of the protein) were determined and the comparison of these sequences with those of protein and gene sequence databases revealed no significant correlation with any known protein. Thus, it is concluded that it is a novel protein of currently unknown biological function.

Neuronal growth-inhibitory factor (GIF), a central-nervous-system-specific metallothionein-like protein, has been isolated by means of an improved isolation procedure from bovine brain. The native protein contains 4-5 Cu+ and 2-2.5 Zn2+, which results in an overall stoichiometry of 6-7 mol metal ions/mol protein. Native Cu, ZN-GIF and the Zn2+ -substituted and Cd2+-substituted metalloforms have been characterized by means of electronic-absorption, CD, magnetic-circular-dichroism (MCD) and low-temperature (77 K) Cu(I)-luminescence spectroscopy. Analysis of the metal-induced-charge-transfer transitions below 300 nm in the electronic-absorption and CD spectra of Cu, ZN-GIF revealed spectral features characteristic of metal-thiolate coordination. The presence of formally spin-forbidden 3d --> 4s Cu(I)-cluster-centered transitions, above 300 nm in the corresponding CD and MCD spectra indicate the existence of a Cu(I) cluster. The 77-K luminescence spectrum of Cu, ZN-GIF revealed two emissive bands at approximately 420 nm and 570 nm, which were reported also for CU4 clusters in mammalian Cu8-metallothionein. By analogy with Cu8-metallothionein, we propose the presence of a Cu4 cluster with similar electronic structure in native GIF. However, the determined Cys/Cu+ ratio of approximately 2:1 in Cu, Zn-GIF is higher than the ratio found in mammalian Cu(I)-metallothionein forms (approximately 1.6:1 ), which implies that the coordination geometry of CU+-binding sites is different in the CU4 Cluster. The spectroscopic characterization of Zn2+-substituted and Cd2+-substituted GIF (6-7 metal ions/protein) showed CD and MCD features at positions identical to those reported for the well-characterized mammalian Zn7-metallothionein and Cd7-metallothionein. Therefore, it is inferred that the cluster organization in GIF with divalent metal ions is comparable to that found in mammalian metallothioneins. The effect of metal ions on the protein structure with regard to the biological function of GIF is discussed.

The two metal-binding sites (A and B)/subunit of the homotetrameric D-xylose isomerase (Xyl isomerase) from Streptomyces rubiginosus have been studied with Mn(2+)-EPR spectroscopy at X-band and Q-band frequencies and with electronic spectroscopy. Displacement studies in the visible absorbance range showed that Mn2+ have a higher affinity for the B site. With the low-affinity A site unoccupied, the coordination sphere of Mn2+ in the B site is quite distorted giving rise to a highly anisotropic X-band EPR spectrum. Simulation of the Q-band spectrum reveals a zero field splitting (zfs) D of about 45-48 mT and a rhombicity parameter E/D between 0.2 and 0.3. Occupation of both binding sites with Mn2+ induces a significant shift towards a higher symmetry in the coordination sphere of the B site resulting in similar zfs parameters for both binding sites. The change in A-site environment caused by B-site occupation was analysed in mixed Xyl isomerase derivatives, in which the B site is loaded with Co2+, Cd2+ or Pb2+ and the A site with Mn2+. In the Co2+/Mn2+ Xyl isomerase the Mn2+ has a relatively symmetric ligand environment with small zfs parameters (D = 12 mT, E/D < 0.15). Substituting Co2+ with Cd2+ or Pb2+ in the B site leads to a drastic increase in the zfs parameters of Mn2+ in the A site. The distortions are directly linked to the ionic radii of the ions bound to the B site and may be mediated by the carboxylate group of Glu216 that bridges the metal-binding sites. The EPR spectra also reflect the catalytic activity of the mixed metal samples. With the larger Cd2+ or Pb2+ in the B site, which are strongly influencing the stereochemistry of the A site, the catalytic activity is lost, whereas Co2+ and Mn2+ render the enzyme in an active state, so that the mutual influence on catalysis depends on the complex geometry of both metal-binding sites.

The two metal-binding sites of the D-xylose isomerase from Streptomyces rubiginosus were studied using VO2+ as a sensor for the ligand environment. Titration of the tetrameric enzyme with VO2+, followed by EPR spectroscopy and inhibition studies, show that the first four VO2+ equivalents occupy, in analogy to Co2, Cd2+ and Pb2+, the binding site B. The visible absorption data and the EPR parameters indicate that a nitrogen ligand is involved in the ligand sphere of the high-affinity B site. The low-affinity A site could be studied selectively by blocking the B site with visible and EPR-silent Cd2+. The visible data and EPR parameters for this site are consistent with a ligand environment composed of oxygen donors without nitrogen ligation. The nitrogen coordination in the high-affinity site could be demonstrated by electron nuclear double-resonance (ENDOR) studies of the 4VO2+ enzyme, and was assigned to a histidine ligand. The 14N resonances are interpreted in terms of a quartet with a coupling value of 13.2 MHz. 1H-ENDOR coupling of 1.7 MHz, exchangeable in D2O, has been assigned to the N–H proton of the histidine. Additional proton ENDOR couplings, which are not exchangeable, are due to protons bound to the carbon atoms of the histidine. For the low-affinity binding site, a nitrogen coordination could be definitely excluded by the ENDOR measurements. Exchangeable 1H-ENDOR couplings observed in this sample were assigned to H2O ligands in the vicinity of VO2+. The results closely relate to what is known from X-ray structure. However, the relative affinities for the two binding sites seem not to be the same for different bivalent cations. In mixed metal samples with four VO2+ and four Co2+ equivalents, the VO2+ is distributed between both binding sites. Small changes in the complex geometry of the A site, indicated by different EPR features, seem to occur if the B site is occupied by Co2+ or by Cd2+.

Laser microdissection allows precise isolation of specific cell types and compartments from complex tissues. To analyse proteins from small cell numbers, we combine laser-microdissection and manipulation (LMM) with mass spectrometry techniques. Hemalaun stained mouse lung sections were used to isolate 500–2,000 cells, enough material for complex protein profiles by SELDI-TOF MS (surface enhanced laser desorption and ionization/time of flight mass spectrometry), employing different chromatographic ProteinChip® Arrays. Initially, to establish the principle, we identified specific protein peaks from 20,000 laser-microdissected cells, combining column chromatography, SDS-PAGE, tryptic digestion, SELDI technology and Tandem MS/MS using a ProteinChip® Tandem MS Interface. Secondly, our aim was to reduce the labour requirements of microdissecting several thousand cells. Therefore, we first defined target proteins in a few microdissected cells, then recovered in whole tissue section homogenates from the same lung and applied to these analytical techniques. Both approaches resulted in a successful identification of the selected peaks. Laser-microdissection may thus be combined with SELDI-TOF MS for generation of protein marker profiles in a cell-type- or compartment-specific manner in complex tissues, linked with mass fingerprinting and peptide sequencing by Tandem MS/MS for definite characterization.