References of "Turtoi, Andrei"
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See detailOrganized Proteomic Heterogeneity in Colorectal Cancer Liver Metastases and Implications for Therapies
Turtoi, Andrei ULg; Blomme, Arnaud; Debois, Delphine et al

in Hepatology (Baltimore, Md.) (2013)

Tumor heterogeneity is a major obstacle for developing effective anticancer treatments. Recent studies have pointed to large stochastic genetic heterogeneity within cancer lesions, where no pattern seems ... [more ▼]

Tumor heterogeneity is a major obstacle for developing effective anticancer treatments. Recent studies have pointed to large stochastic genetic heterogeneity within cancer lesions, where no pattern seems to exist that would enable a more structured targeted therapy approach. Because to date no similar information is available at the protein (phenotype) level, we employed matrix assisted laser desorption ionization (MALDI) image-guided proteomics and explored the heterogeneity of extracellular and membrane subproteome in a unique collection of eight fresh human colorectal carcinoma (CRC) liver metastases. Monitoring the spatial distribution of over 1,000 proteins, we found unexpectedly that all liver metastasis lesions displayed a reproducible, zonally delineated pattern of functional and therapeutic biomarker heterogeneity. The peritumoral region featured elevated lipid metabolism and protein synthesis, the rim of the metastasis dis- played increased cellular growth, movement, and drug metabolism, whereas the center of the lesion was characterized by elevated carbohydrate metabolism and DNA-repair activity. From the aspect of therapeutic targeting, zonal expression of known and novel biomarkers was evident, reinforcing the need to select several targets in order to achieve optimal coverage of the lesion. Finally, we highlight two novel antigens, LTBP2 and TGFBI, whose expression is a consistent feature of CRC liver metastasis. We demon- strate their in vivo antibody-based targeting and highlight their potential usefulness for clinical applications. Conclusion: The proteome heterogeneity of human CRC liver metastases has a distinct, organized pattern. This particular hallmark can now be used as part of the strategy for developing rational therapies based on multiple sets of target- able antigens. [less ▲]

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See detailSelected Protein Monitoring in Histological Sections by Targeted MALDI-FTICR in-source decay Imaging.
Calligaris, David ULg; Longuespée, Rémi ULg; Debois, Delphine ULg et al

in Analytical Chemistry (2013), 85(4), 2117-26

MALDI mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of ... [more ▼]

MALDI mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of spectral pattern to define regions of interest. However, the identification and the differential quantitative analysis of proteins require off line or in situ proteomic methods using enzymatic digestion. The rapid identification of biomarkers holds great promise for diagnostic research but the major obstacle is the absence of rapid and direct method to detect and identify with a sufficient dynamic range a set of specific biomarkers. In the current work, we present a proof of concept for a method allowing identifying simultaneously a set of selected biomarkers on histological slices with minimal sample treatment using in-source decay (ISD) MSI and MALDI-Fourier transform ion cyclotron resonance (FTICR). In the proposed method, known biomarkers are spotted next to the tissue of interest, the whole MALDI plate being coated with 1,5-DAN matrix. The latter enhances MALDI radical-induced ISD, providing large tags of the amino acid sequences. Comparative analysis of ISD fragments between the reference spots and the specimen in imaging mode allows for unambiguous identification of the selected biomarker while preserving full spatial resolution. Moreover, the high resolution/high mass accuracy provided by FTICR mass spectrometry allows the identification of proteins. Well-resolved peaks and precise measurements of masses and mass differences allow the construction of reliable sequence tags for proteins identification. The method will allow the use MALDI-FTICR MSI as method for rapid targeted biomarker detection in complement to classical histology. [less ▲]

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See detailSecretion and maturation of conotoxins in the venom ducts of Conus textile
Dobson, Rowan ULg; Collodoro, Mike; Gilles, Nicolas et al

in Toxicon (2012), 60(8), 1370-1379

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See detailIn Ovo PET Imaging Of A Human Colorectal Carcinoma Model In Chicken Chorioallantoic Membrane
Warnock, Geoffrey ULg; Turtoi, Andrei ULg; Blomme, Arnaud ULg et al

Poster (2012, October)

Aim. The objective of this study was to use in vivo PET/CT imaging as a validation tool for a novel human colorectal carcinoma model being developed in chicken chorioallantoic membrane (CAM). For this ... [more ▼]

Aim. The objective of this study was to use in vivo PET/CT imaging as a validation tool for a novel human colorectal carcinoma model being developed in chicken chorioallantoic membrane (CAM). For this initial pilot study a cell line modeling colon cancer was selected and imaged using [18F]fluorodeoxyglucose (FDG). <br />Materials and methods. A window was made in the shell of fertilized chicken eggs and 3x106 SW1222 human colorectal carcinoma cells were implanted at day 10 post-fertilization. On day 17 the shell window was enlarged to allow direct injection of FDG (12.2 ± 4.5 MBq/egg) into a CAM blood vessel. During injection the egg was warmed on a heating pad. A mixture of ketamine/medetomidine (50 :1 mg/ml, 0.2 ml/egg) was injected into the albumin in some eggs to assess the effect of anesthesia. After FDG injection the egg was returned to the incubator for a 45 min uptake period before imaging. Imaging was performed on a Siemens Focus 120 microPET with structural CT on a General Electric eXplore CT120. A Minerve cell system allowed reproducible positioning between modalities. PET data was acquired in list mode before histogramming into a single 10 min frame for reconstruction using a 3D maximum a posteriori (MAP) method with all corrections except scatter. A standard 100 µm (theoretical) image resolution protocol (70 kV, 50 mA, 32 ms, 220 views) was used to obtain structural CT data. Image coregistration was performed in PMOD version 3.3. In a separate egg, the influence of added contrast on the CT data was investigated by adding iodinated contrast agent (Iobitridol 35 mgI/ml) to the albumin. <br />Results. FDG uptake was clear in chick and tumor, with notably high uptake at the major joints. Tumors were identified by localization of FDG uptake on the surface of the CAM. A lack of soft tissue contrast between tumor, CAM and albumin made precise structural identification of the tumor difficult. Anesthesia was crucial to image quality in both PET and CT. CT contrast between the soft tissues of the chick and surrounding albumin/structures was improved by addition of contrast agent. <br />Conclusion. For the first time we demonstrate successful imaging of FDG uptake in a human colorectal carcinoma chicken CAM model in ovo. Methods to improve structural data are under investigation and will be used in further studies. With such improvement, this model could be of great value to PET oncology imaging. [less ▲]

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See detailImaging Guided Proteomics Unveils Heterogeneity in Colorectal Carcinoma Liver Metastases – Implications for Targeted Therapies
Blomme, Arnaud ULg; Turtoi, Andrei ULg; Castronovo, Vincenzo ULg

Conference (2012, September)

Patients suffering from liver metastases are diagnosed late and have a poor outcome. Targeted therapies are promising treatment options, however the malignant lesions are heterogeneous in nature offering ... [more ▼]

Patients suffering from liver metastases are diagnosed late and have a poor outcome. Targeted therapies are promising treatment options, however the malignant lesions are heterogeneous in nature offering niches for cancer cells to survive and regrow. A rational strategy is needed to select targetable antigens that would overcome this intra-tumoral heterogeneity. MALDI-MS imaging is an emerging tool to study the distribution of biomolecules in tissue samples and is a good base for defining the regions of interest (ROI) that deserve further in-depth analysis. We employed MALDI-MS imaging of colorectal liver metastasis to identify ROI and guide the proteomic analysis for a more in-depth picture of modulated proteins. The focus was laid on cell membrane and extracellular proteins as these have enhanced potential to be used for targeted therapy and clinical imaging applications. Four defined ROI were further analyzed employing 2D-Nano-UPLC-MSe methodology. Over 1500 unique proteins were statistically divided into different patterns of expression, generating a quantitative picture of the proteome heterogeneity in liver metastases. The results offered insight into novel targets but also antigens against which the antibodies are already involved in cancer clinical trials. Following immunohistochemistry based validation experiments, certain proteins demonstrated the potential to homogeneously cover the metastatic lesion and become better targets. Two such antigens, LTBP2 and TGFBI were selected for in vivo functional/ tumor targeting studies in colorectal carcinoma animal model. Importantly, we were able to demonstrate the “targetable” nature of these antigens for homing antibodies injected i.v. Functionally, TGFBI showed an additional potential to target the tumor via it’s ability to affect migration and growth of cancer cells, hence taking the influence on the process of tumorigenesis. In conclusion, liver metastases display a significant heterogeneity in terms of targetable biomarkers and these findings should flow in the future development of targeted therapies aiming to cure the patient. [less ▲]

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See detailA Promising Perspective for Pathologies Diagnosis by MALDI In-Source Decay Imaging with a FTMS System.
Calligaris, David ULg; Debois, Delphine ULg; Turtoi, Andrei ULg et al

Poster (2012, May 23)

Introduction MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular diagnosis could be done directly ... [more ▼]

Introduction MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular diagnosis could be done directly on tissue sections in the environment of the diseased area. The use of in-source decay (ISD), that does allow fast and reliable sequences assignments of proteins termini, is a crucial tool for the identification of known biomarkers during MALDI imaging experiments. Combined with ultra-high mass resolution and high mass measurement accuracy of Fourier transform ion-cyclotron (FTICR) mass spectrometry, it is possible to unambiguously assign sequences of proteins present in tissue slices. In this study, we have shown that FTICR mass spectrometry could be a powerful tool to diagnose pathologies by MALDI-ISD imaging. Methods All measurements were carried out on a SolariX FTMS (9.4 tesla) equipped with a Dual Source including smartbeamTMII laser which includes a robust solid state 1 kHz laser with advanced optics for molecular imaging (Bruker Daltonics). Lysozyme (14.3-kDa) or Human Serum Albumin (66.3-kDa) solution (1 mg/ml in 0.1 % TFA) was mixed with 1,5-diaminonaphthalene (DAN) and analyzed by MALDI-ISD and MALDI-ISD imaging. Mouse brain and rabbit eye tissue slices were washed (fixed) to obtain optimal sensitivity and high-quality ion. Before DAN application with an ImagePrep (Bruker Daltonics) and MALDI-ISD imaging analyzes, spots of myelin and crystalline were deposited near mouse brain or rabbit eye tissues, respectively. Results were interpreted using BioToolsTM 3.2 in combination with MascotTM (Matrix Science) for ISD spectra and FlexImagingTM 2.1 for MALDI-ISD imaging experiments. α Preliminary data The studies were carried out by MALDI-ISD and MALDI-ISD imaging analyses to evidence the interest on FTICR mass spectrometer for proteins identification in the field of biomarkers characterization. It is demonstrated that protein ISD leads to the same pattern of fragmentation observed during MALDI-TOF analyzes. Fragmentation generates cn- and zn-series ions of lysozyme and HSA in presence of DAN. Supplementary an-, bn-, xn- and yn-series ions can also be observed. The internal calibration of all the data provides a mass accuracy neighboring 2.5 ppm over the m/z range of interest (300-2500 Da) and a mass resolution of 70000 at m/z 400 Da. It allows the assignment of ISD fragments of proteins, in the low mass range (m/z between 300 and 900), whether from pure solutions or included in tissue slices. Moreover, spots of pure proteins solution (myelin or crystalline) near tissue slices allows to unambiguously validate the proteins identification during MALDI ISD imaging experiments. Novel aspect This study evidences the main input of FTICR mass spectrometer for pathologies diagnosis based on biomarkers localization and identification by MALDI-ISD imaging. [less ▲]

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See detailImaging Guided Proteomics Unveils Heterogeniety in Colorectal Carcinoma Liver Metastases – Implications for Targeted Therapies.
blomme, Arnaud; Turtoi, Andrei ULg; Delvaux, David ULg et al

in Proceedings Giga Day 2012 (2012, May 04)

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See detailINTRA-TUMORAL HETEROGENEITY AND RATIONAL SELECTION OF ANTIGENS FOR TARGETED THERAPY OF LIVER METASTASES
Turtoi, Andrei ULg; Blomme, Arnaud ULg; Delvaux, David ULg et al

in Acta Chirurgica Belgica (2012, May), 112(3), 8953

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer ... [more ▼]

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer cells causing treatment resistance and relapse. Therefore, a rational strategy is needed to select targetable antigens that would overcome this intra-tumoral heterogeneity. Methods: After ethical committee approval, 48 fresh liver metastases of colorectal origin were prospectively collected from patients undergoing liver resection. Here we macroscopically divided the lesion in different zones and generated a unique quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. Particular focus was laid on accessible proteins, a protein subclass comprising cell membrane associated and extracellular proteins. Accordingly, the tissues were ex-vivo biotinylated, affinity purified and analyzed for each zone separately using nano-UPLC-MSe proteomics technique. In total over 1500 unique proteins were statistically divided into different patterns of expression. Results: We have generated a quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. The study offers insight into novel targets but also antigens against which the antibodies are already involved in clinical trials or treatment of liver metastases. Extensive clustering and validation experiments highlight novel markers that offer the potential to homogeneously cover the metastatic lesion and become better targets. Conclusions: Two such antigens, LTBP2 and TGFBI were selected for functional analysis in colorectal carcinoma cells. In vitro and in vivo experiments showed that in particular TGFBI is relevant for migration and proliferation capacity of colorectal cancer cells. The suppression of this protein led to significant inhibition of tumor growth, crystalizing it as bona fide target for the development of anti-metastases therapies. [less ▲]

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See detailDifferential proteomic analysis of a human breast tumor and its matched bone metastasis identifies cell membrane and extracellular proteins associated with bone metastasis
Dumont, Bruno ULg; Castronovo, Vincenzo ULg; Peulen, Olivier ULg et al

in Journal of Proteome Research (2012)

The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface ... [more ▼]

The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface and in the extracellular space, establishing the first contacts with the target tissue. In this study, we had the rare opportunity to analyze a bone metastatic lesion and its corresponding breast primary tumor obtained simultaneously from the same patient. Using mass spectrometry, we undertook a proteomic study on cell surface and extracellular protein-enriched material. We provide a repertoire of significantly modulated proteins, some with yet unknown roles in the bone metastatic process as well as proteins notably involved in cancer cell invasiveness and in bone metabolism. The comparison of these clinical data with those previously obtained using a human osteotropic breast cancer cell line highlighted an overlapping group of proteins. Certain differentially expressed proteins are validated in the present study using immunohistochemistry on a retrospective collection of breast tumors and matched bone metastases. Our exclusive set of selected proteins supports the set-up of further investigations on both clinical samples and experimental bone metastasis models that will help to reveal the finely coordinated expression of proteins that favor the development of metastases in the bone microenvironment. [less ▲]

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See detailSparc-like protein 1 is a new marker of human glioma progression.
Turtoi, Andrei ULg; Musmeci, Davide; Naccarato, Antonio Giuseppe et al

in Journal of Proteome Research (2012), 11(10), 5011-21

High-grade gliomas (glioblastomas) are the most common and deadly brain tumors in adults, currently with no satisfactory treatment available. Apart from de novo glioblastoma, it is currently accepted that ... [more ▼]

High-grade gliomas (glioblastomas) are the most common and deadly brain tumors in adults, currently with no satisfactory treatment available. Apart from de novo glioblastoma, it is currently accepted that these malignancies mainly progress from lower grade glial tumors. However, the molecular entities governing the progression of gliomas are poorly understood. Extracellular and membrane proteins are key biomolecules found at the cell-to-cell communication interface and hence are a promising proteome subpopulation that could help understand the development of glioma. Accordingly, the current study aims at identifying new protein markers of human glioma progression. For this purpose, we used glial tumors generated orthotopically with T98G and U373 human glioma cells in nude mice. This setup allowed also to discriminate the protein origin, namely, human (tumor) or mouse (host). Extracellular and membrane proteins were selectively purified using biotinylation followed by streptavidin affinity chromatography. Isolated proteins were digested and then identified and quantified employing 2D-nano-HPLC-MS/MS analysis. A total of 23 and 27 up-regulated extracellular and membrane proteins were identified in the T98G and U373 models, respectively. Approximately two-thirds of these were predominantly produced by the tumor, whereas the remaining proteins appeared to be mainly overexpressed by the host tissue. Following extensive validation, we have focused our attention on sparc-like protein 1. This protein was further investigated using immunohistochemistry in a large collection of human glioma samples of different grades. The results showed that sparc-like protein 1 expression correlates with glioma grade, suggesting the possible role for this protein in the progression of this malignancy. [less ▲]

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See detailIsotopically labeled proteome as an internal standard for multiple reaction monitoring-based biomarker quantification.
Turtoi, Andrei ULg; Castronovo, Vincenzo ULg

in Expert Review of Proteomics (2012), 9(3), 245-8

Multiple reaction monitoring is a mass spectrometry technology used to selectively identify and quantify a known molecule in a complex mixture. The technology has gained favor in proteomic applications ... [more ▼]

Multiple reaction monitoring is a mass spectrometry technology used to selectively identify and quantify a known molecule in a complex mixture. The technology has gained favor in proteomic applications, especially for biomarker quantification in human samples. For this purpose, employment of internal standard consisting of isotopically (heavy) labeled proteins is currently considered the best way of normalizing sample preparation and correcting for different ionization efficiencies. However, synthesis of heavy-labeled proteins is considered laborious and expensive. The work outlined here presents an efficient strategy of utilizing isotope-labeled amino acids in cell culture to produce heavy-labeled proteins. These are then spiked into serum and serve as internal standards to relatively quantify a large number of target proteins. The method has been applied to quantify 72 proteins in the sera of pancreatic cancer patients with remarkable efficiency and accuracy. [less ▲]

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See detailThe angiogenesis suppressor gene AKAP12 is under the epigenetic control of HDAC7 in endothelial cells.
Turtoi, Andrei ULg; Mottet, Denis ULg; Matheus, Nicolas ULg et al

in Angiogenesis (2012)

Histone deacetylases (HDACs) are a family of 18 enzymes that deacetylate lysine residues of both histone and nonhistone proteins and to a large extent govern the process of angiogenesis. Previous studies ... [more ▼]

Histone deacetylases (HDACs) are a family of 18 enzymes that deacetylate lysine residues of both histone and nonhistone proteins and to a large extent govern the process of angiogenesis. Previous studies have shown that specific inhibition of HDAC7 blocks angiogenesis both in vitro and in vivo. However, the underlying molecular mechanisms are not fully understood and hence preclude any meaningful development of suitable therapeutic modalities. The goal of the present study was to further the understanding of HDAC7 epigenetic control of angiogenesis in human endothelial cells using the proteomic approach. The underlying problem was approached through siRNA-mediated gene-expression silencing of HDAC7 in human umbilical vein endothelial cells (HUVECs). To this end, HUVEC proteins were extracted and proteomically analyzed. The emphasis was placed on up-regulated proteins, as these may represent potential direct epigenetic targets of HDAC7. Among several proteins, A-kinase anchor protein 12 (AKAP12) was the most reproducibly up-regulated protein following HDAC7 depletion. This overexpression of AKAP12 was responsible for the inhibition of migration and tube formation in HDAC7-depleted HUVEC. Mechanistically, H3 histones associated with AKAP12 promoter were acetylated following the removal of HDAC7, leading to an increase in its mRNA and protein levels. AKAP12 is responsible for protein kinase C mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 increasingly binds to the chromatin and AKAP12 promoter and is necessary for maintaining the elevated levels of AKAP12 following HDAC7 knockdown. We demonstrated for the first time that AKAP12 tumor/angiogenesis suppressor gene is an epigenetic target of HDAC7, whose elevated levels lead to a negative regulation of HUVEC migration and inhibit formation of tube-like structures. [less ▲]

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See detailIdentification of novel accessible proteins bearing diagnostic and therapeutic potential in human pancreatic ductal adenocarcinoma
Turtoi, Andrei ULg; Musmeci, Davide; Wang, Yinghong et al

in Journal of Proteome Research (2011), 10(9), 4302-13

Pancreas ductal adenocarcinoma (PDAC) remains a deadly malignancy with poor early diagnostic and no effective therapy. Although several proteomic studies have performed comparative analysis between normal ... [more ▼]

Pancreas ductal adenocarcinoma (PDAC) remains a deadly malignancy with poor early diagnostic and no effective therapy. Although several proteomic studies have performed comparative analysis between normal and malignant tissues, there is a lack of clear characterization of proteins that could be of clinical value. Systemically reachable ("potentially accessible") proteins, suitable for imaging technologies and targeted therapies represent a major group of interest. The current study explores potentially accessible proteins overexpressed in PDAC, employing innovative proteomics technologies. In the discovery phase, potentially accessible proteins from fresh human normal and PDAC tissues were ex vivo biotinylated, isolated and identified using 2D-nano-HPLC-MS/MS method. The analysis revealed 422 up-regulated proteins in the tumor, of which 83 (including protein isoforms) were evaluated as potentially accessible. Eleven selected candidates were further confirmed as up-regulated using Western blot and multiple reaction monitoring protein quantification. Of these, transforming growth factor beta-induced (TGFBI), latent transforming growth factor beta binding 2 (LTBP2), and asporin (ASPN) were further investigated by employing large scale immunohistochemistry-based validations. They were found to be significantly expressed in a large group of clinical PDAC samples compared to corresponding normal and inflammatory tissues. In conclusion, TGFBI, LTBP2, and ASPN are novel, overexpressed, and potentially accessible proteins in human PDAC. They bear the potential to be of clinical value for diagnostic and therapeutic applications and merit further studies using in vivo models. [less ▲]

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See detailNovel comprehensive approach for accessible biomarker identification and absolute quantification from precious human tissues
Turtoi, Andrei ULg; Dumont, Bruno ULg; Greffe, Yannick et al

in Journal of Proteome Research (2011), 10(7), 3160-82

The identification of specific biomarkers obtained directly from human pathological lesions remains a major challenge, because the amount of tissue available is often very limited. We have developed a ... [more ▼]

The identification of specific biomarkers obtained directly from human pathological lesions remains a major challenge, because the amount of tissue available is often very limited. We have developed a novel, comprehensive, and efficient method permitting the identification and absolute quantification of potentially accessible proteins in such precious samples. This protein subclass comprises cell membrane associated and extracellular proteins, which are reachable by systemically deliverable substances and hence especially suitable for diagnosis and targeted therapy applications. To isolate such proteins, we exploited the ability of chemically modified biotin to label ex vivo accessible proteins and the fact that most of these proteins are glycosylated. This approach consists of three successive steps involving first the linkage of potentially accessible proteins to biotin molecules followed by their purification. The remaining proteins are then subjected to glycopeptide isolation. Finally, the analysis of the nonglycosylated peptides and their involvement in an in silico method increased the confident identification of glycoproteins. The value of the technique was demonstrated on human breast cancer tissue samples originating from 5 individuals. Altogether, the method delivered quantitative data on more than 400 potentially accessible proteins (per sample and replicate). In comparison to biotinylation or glycoprotein analysis alone, the sequential method significantly increased the number (≥30% and ≥50% respectively) of potentially therapeutically and diagnostically valuable proteins. The sequential method led to the identification of 93 differentially modulated proteins, among which several were not reported to be associated with the breast cancer. One of these novel potential biomarkers was CD276, a cell membrane-associated glycoprotein. The immunohistochemistry analysis showed that CD276 is significantly differentially expressed in a series of breast cancer lesions. Due to the fact that our technology is applicable to any type of tissue biopsy, it bears the ability to accelerate the discovery of new relevant biomarkers in a broad spectrum of pathologies. [less ▲]

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See detailInnovative Proteomics for the Discovery of Systemically Accessible Cancer Biomarkers Suitable for Imaging and Targeted Therapies
Turtoi, Andrei ULg; De Pauw, Edwin ULg; Castronovo, Vincenzo ULg

in American Journal of Pathology (2011), 178(1), 12-18

The discovery of biomarkers that are readily accessible through the circulating blood and are selectively overexpressed in pathological tissues has become a major research objective, particularly in the ... [more ▼]

The discovery of biomarkers that are readily accessible through the circulating blood and are selectively overexpressed in pathological tissues has become a major research objective, particularly in the field of oncology. Indisputably, this group of molecules has a high potential to serve as an innovative tool for effective imaging and targeted cancer therapy approaches. In this attractive therapeutic concept, specific cancer proteins are reached by intravenously administered ligands that are coupled to cytotoxic drugs. Such compounds are able to induce cancer destruction while sparing normal tissues. Owing to the performance of mass spectrometry technology, current high-throughput proteomic analysis allows for the identification of a high number of proteins that are differentially expressed in the cancerous tissues. However, such approaches provide no information regarding the effective accessibility of the biomarkers and, therefore, the possibility for these discovered proteins to be targeted. To bypass this major limitation, which clearly slows the discovery of such biomarkers, innovative methodological strategies have been developed to enrich the clinical specimens before the mass spectrometry analysis. The focus is laid on the group of proteins that are necessarily located either at the exterior face of the plasma membrane or in the extracellular matrix. The present review addresses the current technologies meant for the discovery and analysis of accessible antigens from clinically relevant samples. [less ▲]

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See detailProteomic and genomic modulations induced by gamma-irradiation of human blood lymphocytes.
Turtoi, Andrei ULg; Sharan, Rajesh; Srivastava, Alok et al

in International Journal of Radiation Biology (2010), 86(10), 888-904

PURPOSE: Quantitative evaluation of early response proteins (ERPRO) and early response genes (ERG) following γ-irradiation of human lymphocytes; identification of specific proteins and genes as candidate ... [more ▼]

PURPOSE: Quantitative evaluation of early response proteins (ERPRO) and early response genes (ERG) following γ-irradiation of human lymphocytes; identification of specific proteins and genes as candidate biomarkers for the development of a novel biodosimeter. MATERIALS AND METHODS: Human peripheral blood lymphocytes were exposed to clinically relevant doses (1, 2 and 4 Gy) of γ-radiation ex-vivo. Analyses of protein and gene expression modulation were conducted 2 h post-irradiation. Global modulations were monitored using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and DNA microarray analyses of the samples originating from one human donor. On the proteome level, both phosphorylated and non-phosphorylated proteins were considered. Proteins and genes of specific interest were further targeted using Western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR) techniques, employing samples from several human donors (n=3). RESULTS: A set of ERPRO and ERG showing significant alterations 2 h post-γ-irradiation have been identified in human lymphocytes. The most radiation responsive genes and proteins indicated alterations of cellular structure (ß-actin, talin-1 [TLN1], talin-2, zyxin-2), immune and defence reactions (major histocompatibility complex binding protein-2 [MBP2], interleukin-17E and interferon-γ), cell cycle control (cyclin-dependent kinase inhibitor-1A [CDKN1A], mouse double minute-2, annexin-A6 [ANXA6], growth arrest and DNA-damage-inducible protein-α [GADD45A], proliferating cell nuclear antigen [PCNA], dual specificity phosphatase-2 and 8 [DUSP8]) as well as detoxification processes (peroxin-1) and apoptosis (B-cell lymphoma-2 binding component-3 [BBC3]). SUMMARY: The estimations of protein concentration modulation of TLN1 and CDKN1A, phosphorylation status of ANXA6 (dose range 0-2 Gy) and MBP2 as well as the alterations in the level of gene expressions of BBC3, DUSP8, GADD45A and PCNA appears to be of potential value for future biodosimetric applications. [less ▲]

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See detailNovel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis
Fleron, Maximilien ULg; Greffe, Yannick ULg; Musmeci, Davide ULg et al

in Journal of Proteomics (2010), 73(10), 1986-2005

In the field of proteomics there is an apparent lack of reliable methodology for quantification of posttranslational modifications. Present study offers a novel post-digest ICPL quantification strategy ... [more ▼]

In the field of proteomics there is an apparent lack of reliable methodology for quantification of posttranslational modifications. Present study offers a novel post-digest ICPL quantification strategy directed towards characterization of phosphorylated and glycosylated proteins. The value of the method is demonstrated based on the comparison of two prostate related metastatic cell lines originating from two distinct metastasis sites (PC3 and LNCaP). The method consists of protein digestion, ICPL labeling, mixing of the samples, PTM enrichment and MS-analysis. Phosphorylated peptides were isolated using TiO(2), whereas the enrichment of glycosylated peptides was performed using hydrazide based chemistry. Isolated PTM peptides were analyzed along with non enriched sample using 2D-(SCX-RP)-Nano-HPLC-MS/MS instrumentation. Taken together the novel ICPL labeling method offered a significant improvement of the number of identified (∼600 individual proteins) and quantified proteins (>95%) in comparison to the classical ICPL method. The results were validated using alternative protein quantification strategies as well as label-free MS quantification method. On the biological level, the comparison of PC3 and LNCaP cells has shown specific modulation of proteins implicated in the fundamental process related to metastasis dissemination. Finally, a preliminary study involving clinically relevant autopsy cases reiterated the potential biological value of the discovered proteins. [less ▲]

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