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See detailUltraviolet Laser Induced Hydrogen Transfer Reaction: Study of the First Step of MALDI In-Source Decay Mass Spectrometry
Asakawa, Daiki; Calligaris, David ULg; Smargiasso, Nicolas ULg et al

in Journal of Physical Chemistry B (2013), 117(8), 2321-2327

The early mechanisms of matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) are described herein. MALDI-ISD is initiated by the hydrogen transfer from excited matrix molecules to the ... [more ▼]

The early mechanisms of matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) are described herein. MALDI-ISD is initiated by the hydrogen transfer from excited matrix molecules to the carbonyl oxygen of the peptide backbone, which is followed by a radical-induced cleavage, producing the c′/z• fragment pair. As expected, the use of 2,5-DHB or 1,5-DAN was efficient to induce MALDI-ISD, and the strongest intensity of MALDI-ISD fragments was observed when laser shots were performed on matrix crystals. In contrast, the hydrogen radical transfer reaction was suppressed by using ionic liquid and amorphous structure of 2,5-DHB and 1,5-DAN mixture as a matrix. Our results suggest that the hydrogen transfer occurs on the matrix crystal during the dissipation of the laser energy and before desorption, following ISD fragments formed in the MALDI plume. [less ▲]

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See detailQuantitative proteomic analysis to decipher the differential apoptotic response of bortezomib-treated APL cells before and after retinoic acid differentiation reveals involvement of protein toxicity mechanisms.
Uttenweiler-Joseph, Sandrine; Bouyssié, David; Calligaris, David ULg et al

in Proteomics (2013), 13

The ubiquitin-proteasome system allows the targeted degradation of proteins and plays a critical role in the regulation of many cellular processes. Proteasome inhibition is a recent antitumor therapeutic ... [more ▼]

The ubiquitin-proteasome system allows the targeted degradation of proteins and plays a critical role in the regulation of many cellular processes. Proteasome inhibition is a recent antitumor therapeutic strategy and bortezomib was the first proteasome inhibitor approved for clinical use. In this study, we used the NB4 cell line to investigate the effects of bortezomib toward acute promyelocytic leukemia cells before and after retinoic acid-induced differentiation. We showed that apoptosis level after bortezomib treatment is higher in NB4 cells than in differentiated NB4 cells. To compare early protein variations upon bortezomib treatment in both NB4 cell populations, we performed a quantitative proteomic analysis based on iTRAQ peptide labeling followed by data analysis with in-house developed scripts. This strategy revealed the regulation of 14 proteins principally involved in protein stress response and apoptosis in NB4 cells after proteasome inhibition. Altogether, our results suggest that the differential level of apoptosis induced by bortezomib treatment in both NB4 cell populations could result from distinct protein toxicity level. [less ▲]

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See detailTubulin isoforms identified in the brain by MALDI in-source decay
Ait-Belkacem, Rima; Calligaris, David ULg; Sellami, Lina et al

in Journal of Proteomics (2013), 79

Identification of biomarkers is a major issue for enhancement of chemotherapies. The molecular characterization of tissues necessitates the identification of thousands of biomolecules each participating ... [more ▼]

Identification of biomarkers is a major issue for enhancement of chemotherapies. The molecular characterization of tissues necessitates the identification of thousands of biomolecules each participating in physiopathological processes. MALDI in-source decay (ISD) fragmentation has already been proven to be effective for protein characterization. However, the difficulty to identify proteins from complex mixtures such as tissue sections can limit the applications of this technique. In this study, we evidenced that tubulin has an unusual fragmentation pathway in the MALDI source. This striking property allowed the detecting of several mouse brain tubulin isotypes simultaneously by simply using laser fragmentation. Tubulin isoforms are consistent markers of a bad prognosis of solid tumors and could be the target of targeted chemotherapies. Such a direct molecular printout of tubulin in tissues is a milestone that should be useful either at preclinical or clinical stage. [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 detailSéminaire des chercheurs Télévie 2013
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, December 10)

Séminaire des chercheurs Télévie 2013

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See detailDetermination of the molecular players of adaptation to anti-angiogenic therapy in breast cancer by quantitative proteomic and high molecular MALDI Imaging.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, October 13)

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive ... [more ▼]

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and me<x>tastatic disease. To this end, we applied quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. In this project, we first developed a reproducible model of resistance to Sunitinib of human triple negative breast cancer MDA-MB-231 cells expressing luciferase gene. Cells were subcutaneously injected into mice RAG1-/- and divided into four experimental groups including, control mice treated with vehicle or Sunitinib for 30 days and sacrificed 1 days after treatment withdrawal or when tumor reached a volume of 300 mm3. In the second step. Tumors were analyzed using a nanoAcquity UPLC Synapt TM HDMS TM G1 (Waters, Manchester,UK) and Mass Spectrometry Imaging. For quantitative proteomic analyses of tumors, a bioinformatics analysis was used with the Protein lynx global server 2.2.5 software. Imaging mass spectrometry was performed on tissue sections of tumors and organs subsequently colonized by me<x>tastases. Matrix sublimation was used to coat tumor sections (14 µm-tick) with 1.5 Diaminonaphthalene for lipids analysis and Sinapinic acid for entire proteins analysis. Ion cartographies were recorded with a Solarix 9.4T FTMS instrument for lipids and with an Ultraflex II TOF-TOF instrument for entire proteins (Bruker Daltonics, Germany) with a spatial resolution of 100 µm. Global protemic revealed different protein profiles between tumor treated or not with Sunitinib. The Mass Spectrometry Imaging detected differences in intensity and location of some proteins and lipids are also associated with some histological features including inflammatory, necrotic and angiogenic areas. Bioinformatics analysis will be applied to ensure the integration of all data in order to provide the basis for identifying molecular pathways activated during the acquisition of refractoriness to drug treatments. [less ▲]

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See detailAnalysis of the Biocompatibility of Different Intraocular Lens (IOL) Material Using Mass Spectrometry Tisssue Imaging
Bertrand, Virginie ULg; Debois, Delphine ULg; Calligaris, David ULg et al

Conference (2012, September 04)

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract ... [more ▼]

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract. The surgical intervention consists in removing the cloudy lens and to replace it by an artificial intraocular lens (IOL). The in vivo implantation of these synthetic lenses involves the evaluation of several factors as their physico-chemical properties, their capacities to interact with lens epithelial cells and proteins, as well as their biocompatibility. During a previous study, we demonstrated major differences concerning the tackiness (atomic force microscopy), the cellular adhesion and the protein adsorption of various polymer disks intended for the manufacturing of intraocular lenses. The aim of this work was to correlate a histological analysis to a mass spectrometry imaging analysis performed on the same sample. To estimate the biocompatibility of the biomaterials, an animal testing was realized in rabbits. The various polymers were implanted subcutaneously. After one month, the 2 cm x 3 cm pieces of rabbit skin and underlying muscle with a 2 cm thickness were removed, fixed with formaldehyde 10% during six days, treated for the paraffin inclusion and stored at room temperature until use. Slices of 5 µm thickness were performed using a microtome. Paraffin was removed and tissue sections were washed in graded ethanol baths. The slices were then stained with the hematoxylin and eosin dyes. The analysis of stained sections showed different histo-morphological features according to the implanted polymer. For MALDI MSI purposes, on tissue protein digestion was performed using trypsin (1) and the MALDI matrix (α-cyanohydroxycinnamic acid, 5 mg/mL in ACN/0.2% TFA 70:30) was deposited using an ImagePrep automated sprayer (Bruker Daltonics, Bremen, Germany). Experiments were carried out using an UltraFlex II TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). MALDI imaging can show the detection of different proteomic profiles according to the tested biomaterials, which may be considered as biocompatibility markers. The MALDI images of these markers are then correlated with the histo-morphological profiles. Consequently, mass spectrometry imaging can become a powerful tool in the evaluation of the biocompatibility of artificial implants in biomedical application. [less ▲]

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See detailStudy  of  breast  cancer  adaptation  to  anti-­angiogenic  therapies  by   molecular  imaging  on  tissue  slides
Cimino, Jonathan ULg; Calligaris, David ULg; Debois, Delphine ULg et al

Conference (2012, September 04)

Breast   carcinoma   is   the   most   common   and   second   leading   cause   of   cancer   mortality   in   women1.   The   ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣␣␣ ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣ ␣␣␣␣-­‐limiting   ... [more ▼]

Breast   carcinoma   is   the   most   common   and   second   leading   cause   of   cancer   mortality   in   women1.   The   ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣␣␣ ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣ ␣␣␣␣-­‐limiting   secondary   step   in   tumorigenesis   led   to   extensive   pre-­‐clinical   researches   on   angiogenesis   and   finally   the   approval   of   VEGF-­‐neutralizing   antibodies   (bevacizumab)  and  VEGF  receptor  tyrosine  kinase  inhibitors  (RTKs:sunitinib).  The  Sunitinib  has  been  used   clinically   in   patients   with   breast   cancer   refractory   to   other   therapeutic   agents2.   Unfortunately,   like   the   cytotoxic   therapies,   these   drugs   do   not   produce   lasting   effects   and   resistance   to   treatment   appeared   clinically3.   Recently,   independent   laboratories   have   reported   experimental   data   demonstrating   that   anti-­‐ angiogenic   treatments   inhibit   tumor   growth,   but   also   stimulate   the   formation   of   lung   metastases   after   treatment   discontinuation4.   The   field   of   imaging   mass   spectrometry   provides   new   tools   to   visualize   and   study  the  profiles  of  proteins  and  small  molecules  associated  with  biomedical  problems5.   To  this  aim,  we  conducted  a  series  of  experiments  to  setup  a  reproductible  model  of  resistance  to  sunitinib.   The   cells   MDA-­‐MB-­‐231   triple   negative,   from   human   breast   cancer   and   expressing   luciferase   are   injected   subcutaneously  into  mice  RAG1-­‐/-­‐.  The  mice  were  divided  into  four  experimental  groups  including,  on  the   one  hand,  control  mice  treated  with  placebo  (Carboxymethyl  cellulose,  CMC)  sacrificed  on  day  30  (group  1)   or  when  the  tumor  reached  a  volume  of  300  mm3  (group  2).    On  the  other  hand,  Sunitinib-­‐treated  mice  (LC   Laboratories,   40mg/kg/day),   sacrificed   at   day   30   (group   3),   or   when   the   tumor   reached   a   volume   of   300   mm3  (group  4).  MALDI  mass  spectrometry  imaging  was  performed  on  tissue  sections  of  tumors  and  organs   subsequently   colonized   by   metastases.   Matrix   sublimation   was   used   to   coat   tumor   sections   (14   μm-­‐tick)   with   1.5   Diaminonaphthalene   (1.5   DAN)   for   lipids   analysis   and   Sinapinic   acid   (SA)   for   entire   proteins   analysis.   Ion   cartographies   were   recorded   with   a   Solarix9.4T   FTMS   instrument   for   lipids   and   with   an   Ultraflex   II   TOF-­‐TOF   instrument   for   entire   proteins   (BrukerDaltonics,   Bremen,   Germany)   with   a   spatial   resolution  of  100  μm.     The  analysis  of  differential  protein/lipid  profiles  with  high  mass  accuracy  and  broadband  resolution  allows   detection   of   intense   signals   from   lipid   families   such   as   Phosphatidylcholine   (PC),   Triglyceride   (TAG),   Sphingomyelin   (SM)   and   precise   lipid   droplets   or   tumor   cells   differentiated   location   in   the   Sunitinib   resistant   tumor   cells   compared   to   control   cells.The   protein   profiles   of   the   4   groups   of   mice   show   differences   in   intensity   and   location,   enabling   a   correlation   to   inflammatory   (highlighted   by   histological   staining)  and  angiogenic  phenomenon.   [less ▲]

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See detailMass spectrometry imaging is moving toward drug protein co-localization
Ait-Belkacem, Rima; Sellami, Lyna; Villard, Claude et al

in Trends in Biotechnology (2012), 30(9), 466-474

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution ... [more ▼]

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution can be achieved down to the cellular level (10–20 mm) for conventional matrix-assisted laser desorption/ionization (MALDI) imaging, or even to the subcellular level for more complex technologies such as secondary ionization mass spectrometry (SIMS) imaging. One question remains: are we going to be able to investigate functional relationships between drugs and proteins and compare with localized phenomena? This review describes the various spatial levels of investigation offered by mass spectrometry imaging (MSI), and the advantages and disadvantages compared with other labeling technologies. [less ▲]

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See detailMolecular imaging through in combinaison with quantitative proteomic approaches unraveling the molecular players of breast cancer adaptation to anti-angiogenic therapy.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, June 22)

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive ... [more ▼]

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and metastatic disease. To this end, we applied quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. In this project, we first developed a reproducible model of resistance to Sunitinib of human triple negative breast cancer MDA-MB-231 cells expressing luciferase gene. Cells were subcutaneously injected into mice RAG1-/- and divided into four experimental groups including, control mice treated with vehicle or Sunitinib for 30 days and sacrificed 1 days after treatment withdrawal or when tumor reached a volume of 300 mm3. In the second step. Tumors were analyzed using a nanoAcquity UPLC Synapt TM HDMS TM G1 (Waters, Manchester,UK) and Mass Spectrometry Imaging. For quantitative proteomic analyses of tumors, a bioinformatics analysis was used with the Protein lynx global server 2.2.5 software. Imaging mass spectrometry was performed on tissue sections of tumors and organs subsequently colonized by metastases. Matrix sublimation was used to coat tumor sections (14 µm-tick) with 1.5 Diaminonaphthalene for lipids analysis and Sinapinic acid for entire proteins analysis. Ion cartographies were recorded with a Solarix 9.4T FTMS instrument for lipids and with an Ultraflex II TOF-TOF instrument for entire proteins (Bruker Daltonics, Germany) with a spatial resolution of 100 µm. Global protemic revealed different protein profiles between tumor treated or not with Sunitinib. The Mass Spectrometry Imaging detected differences in intensity and location of some proteins and lipids are also associated with some histological features including inflammatory, necrotic and angiogenic areas. Bioinformatics analysis will be applied to ensure the integration of all data in order to provide the basis for identifying molecular pathways activated during the acquisition of refractoriness to drug treatments. [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 detailIn situ protein identification in imaging mass spectrometry
Calligaris, David ULg; Debois, Delphine ULg; De Pauw, Edwin ULg

Scientific conference (2012, May 04)

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an emerging tool for clinical research. MALDI MSI can be used to elucidate the relative abundance and spatial ... [more ▼]

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an emerging tool for clinical research. MALDI MSI can be used to elucidate the relative abundance and spatial localization of peptides and proteins throughout a tissue section. For this, a matrix is applied on the tissue in either a spotted array or a homogenous coating. Acquisition of mass spectra is then carried out by performing a raster with a laser across the tissue section in a defined pattern. The spectra acquired from each position on the tissue section contain molecular weight and intensity information representative of the biomolecules at that position. One can plot the intensity of any measured ion as a function of individual pixel locations to generate m/z specific images. But, if protein desorption/ionization and subsequent MS analyses provides a measurement of molecular weight, no protein identification is performed. To achieve this, several methods have been developed. In this talk, I will first present the methods inspired by classical proteomics techniques that are regularly used to identify proteins. Bottom-up and top-down approaches have been used directly from a tissue slice, leading to the identification of some of the most abundant proteins present within the tissue slice. Then, I will present the new developments led in our lab for imaging and especially for in situ protein identification. The first example will deal with the exceptional features of FT-ICR mass spectrometry for in-source decay (ISD)-based protein identification. The benefit of mass accuracy and high mass resolution allow unequivocal assignment of ISD fragments of proteins, in the low mass range (m/z between 400 and 900), whether from pure solutions or from tissue slices. The next example is the use of a matrix “cleaning” software that reduce/remove matrix peaks thus facilitating ISD spectra analyses. Finally, proteins identification by localization and MALDI-ISD profile matching will also be a really simplistic and interesting method that will complement the immunohistological techniques commonly used to validate expression of known biomarkers within diseased tissues. [less ▲]

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See detailApplication of molecular imaging in combination with quantitative proteomic approaches to determine the molecular players of adaptation to anti-angiogenic therapy in breast cancer.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, May 04)

The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing ... [more ▼]

The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and metastatic disease. To this end, we applied a quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. [less ▲]

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See detailNew Advances for In Situ Protein Identification by MALDI In-Source Decay FTMS Imaging
Calligaris, David ULg; Zimmerman, Tyler ULg; Debois, Delphine ULg et al

Poster (2012, April 18)

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

MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular analysis could be performed directly from tissue sections in the region of the diseased area. The use of in-source decay (ISD), allowing fast and reliable sequences assignments of proteins termini, has proven to be a crucial tool for proteins identification in solution and tissue slices. However, it is necessary to develop additional tools that allow unambiguous assignment of proteins sequences in complex tissue slices. The development of bioinformatic tools and the use of ultra-high mass resolution and high mass accuracy of Fourier transform ion-cyclotron (FTICR) mass spectrometry are ideal for this purpose. In this study, we show that FTICR mass spectrometry combined with data filtering with a software that subtracts matrix peaks aid protein identification. All measurements were carried out on a SolariX FTMS (9.4 Tesla) equipped with a Dual Source with a smartbeamTMII laser (Bruker Daltonics). Mouse brain tissue slices of 14 µm thickness were rinsed to obtain optimal sensitivity and high-quality ions. Before matrix application, a spot of myelin was deposited near mouse brain. 1,5-Diaminionaphtalene was sprayed using an ImagePrep (Bruker Daltonics). Results were interpreted using BioToolsTM 3.2 in combination with MascotTM (Matrix Science) for ISD spectra and FlexImagingTM 3.0 for MALDI-ISD imaging experiments. Matrix peaks were subtracted using an in-house written Java code that sequentially scans all peak lists from acquired spectra against the DAN mass list. Then, another Java code allows to create 2D ion images at selected m/z ratios. The studies were carried out by MALDI-ISD imaging to create 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 myelin in presence of DAN. 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) that is unambiguously validated by the “ISD signal” recorded from the spots of pure protein solution (myelin) near tissue slice. Moreover, the use of our software “cleans” MS imaging data by reducing/eliminating MALDI matrix peaks that are isobaric to an analyte peak. 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 detailContribution of high mass resolution and accuracy of FTMS to molecular imaging
Debois, Delphine ULg; Calligaris, David ULg; Cimino, Jonathan ULg et al

Conference (2012, April 04)

Since its first implementation in 1997, MALDI Mass Spectrometry Imaging (MALDI MSI) has become an important tool in the proteomic arsenal, especially for biomarker hunting. First dedicated to high ... [more ▼]

Since its first implementation in 1997, MALDI Mass Spectrometry Imaging (MALDI MSI) has become an important tool in the proteomic arsenal, especially for biomarker hunting. First dedicated to high molecular weight, MALDI MSI is more and more used to map the distribution of small molecules too (lipids, drugs and metabolites,…). Last developments tend to improve the sample treatments to obtain the best spatial resolution as possible. From this perspective, great efforts have been made on the MALDI matrix deposition methods. Now, one of the remaining challenges for MALDI-MSI users consists of identification of detected molecules. For high molecular weight, methods inspired by classical proteomics techniques, are regularly used. Bottom-Up (PMF obtained after in situ trypsin digestion) and Top-Down (in situ In-Source Decay) approaches have been used directly from a tissue slice, leading to the identification of some of the most abundant proteins present at the surface of the tissue. When small molecules are analyzed, the identification is more straightforward. Indeed, tandem mass spectrometry can easily be used, leading to the fragmentation of the detected compounds which allows their unambiguous identification. This identification is even more reliable when high resolution exact mass measurements can be performed. In this talk, I will present how in our lab, we profit of the exceptional features of FT-ICR mass spectrometry for imaging and especially for identification purposes. The first example will deal with the benefit of high mass accuracy and high mass resolution for ISD-based protein identification. The mass accuracy and high mass resolution coupled with the use of a “cleaning” software allow unequivocal assignment of ISD fragments of proteins, in the low mass range (m/z between 300 and 900), whether from pure solutions or from tissue slices. The next examples will deal with the imaging of small molecules. The identification of drugs and their metabolites is facilitated with high mass accuracy. In our lab, we work on the localization of methadone and its first metabolite, EDDP in necrophagous fly larvae. In the mass range of these compounds (278-310 m/z), many matrix ion peaks are detected and the unique features of FT-ICR allows for unambiguous identification thanks to exact mass measurements. We also use MALDI Imaging to map the messenger molecules between plant roots and beneficial bacteria. The comparison of spectra recorded with a TOF/TOF instrument and with a FT-ICR demonstrates that high resolution allows for detecting molecules which could have been missed otherwise. It also allows to distinguish unknown compounds from alkali adducts of known molecules. [less ▲]

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See detailTyrosine-dependent capture of CAP-Gly domain‐containing proteins in complex mixture by EB1 C-terminal peptidic probes
Calligaris, David ULg; Manatschal, Cristina; Marcellin, Marlène et al

in Journal of Proteomics (2012), 75

Microtubule dynamics is regulated by an array of microtubule associated proteins of which the microtubule plus-end tracking proteins (+TIPs) are prominent examples. +TIPs form dynamic interaction networks ... [more ▼]

Microtubule dynamics is regulated by an array of microtubule associated proteins of which the microtubule plus-end tracking proteins (+TIPs) are prominent examples. +TIPs form dynamic interaction networks at growing microtubule ends in an EB1-dependent manner. The interaction between the C-terminal domain of EB1 and the CAP-Gly domains of the +TIP CLIP-170 depends on the last tyrosine residue of EB1. In the present study, we generated peptidic probes corresponding to the C-terminal tail of EB1 to affinity-capture binding partners from cell lysates. Using an MS-based approach, we showed that the last 15 amino-acid residues of EB1, either free or immobilized on beads, bound recombinant CAP-Gly domains of CLIP-170. We further demonstrate that this binding was prevented when the C-terminal tyrosine of EB1 was absent in the peptidic probes. Western blotting in combination with a label-free quantitative proteomic analysis revealed that the peptidic probe harboring the C-terminal tyrosine of EB1 effectively pulled-down proteins with CAP-Gly domains from endothelial cell extracts. Additional proteins known to interact directly or indirectly with EB1 and the microtubule cytoskeletonwere also identified. Our peptidic probes represent valuable tools to detect changes induced in EB1-dependent +TIP networks by external cues such as growth factors and small molecules. [less ▲]

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See detailAdvances in top-down proteomics for disease biomarker discovery.
Calligaris, David ULg; Villard, Claude; Lafitte, Daniel

in Journal of Proteomics (2011), 74(7), 920-934

Top-down mass spectrometry strategies allow identification and characterization of proteins and protein networks by direct fragmentation. These analytical processes involve a panel of fragmentation ... [more ▼]

Top-down mass spectrometry strategies allow identification and characterization of proteins and protein networks by direct fragmentation. These analytical processes involve a panel of fragmentation mechanisms, some of which preserve protein post-translational modifications. Thus top-down is of special interest in clinical biochemistry to probe modified proteins as potential disease biomarkers. This review describes separating methods, mass spectrometry instrumentation, bioinformatics, and theoretical aspects of fragmentation mechanisms used for top-down analysis. The biological interest of this strategy is extensively reported regarding the characterization of post-translational modifications in biochemical pathways and the discovery of biomarkers. One has to bear in mind that quantitative aspects that are beyond the focus of this review are also of critical important for biomarker discovery. The constant evolution of technologies makes top-down strategies crucial players in clinical and basic proteomics. [less ▲]

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See detailChemical inhibitors: the challenge of finding the right target.
Calligaris, David ULg; Lafitte, Daniel

in Chemistry & Biology (2011), 18(5), 555-557

A paper in this issue of Chemistry & Biology shows that the diaryl oxazole compound UA62784 that targets pancreatic cancer cells interacts with tubulin near the colchicine binding site (Tcherniuk et al ... [more ▼]

A paper in this issue of Chemistry & Biology shows that the diaryl oxazole compound UA62784 that targets pancreatic cancer cells interacts with tubulin near the colchicine binding site (Tcherniuk et al., 2011). These findings differ from previous observations, highlighting the challenges of identifying the biological target for chemical inhibitors. [less ▲]

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See detailMALDI in-source decay of high mass protein isoforms: application to alpha- and beta-tubulin variants.
Calligaris, David ULg; Villard, Claude; Terras, Lionel et al

in Analytical Chemistry (2010), 82(14), 6176-6184

Tubulin is one of the major targets in cancer chemotherapy and the target of more than twenty percent of the cancer chemotherapic agents. The modulation of isoform content has been hypothesized as being a ... [more ▼]

Tubulin is one of the major targets in cancer chemotherapy and the target of more than twenty percent of the cancer chemotherapic agents. The modulation of isoform content has been hypothesized as being a cause of resistance to treatment. Isoform differences lie mostly in the C-terminus part of the protein. Extensive characterization of this polypeptide region is therefore of critical importance. MALDI-TOF fragmentation of tubulin C-terminal domains was tested using synthetic peptides. Then, isotypes from HeLa cells were successfully characterized for the first time by in-source decay (ISD) fragmentation of their C-terminus coupled to a pseudo MS3 technique named T3-sequencing. The fragmentation occurred in-source, preferentially generating yn-series ions. This approach required guanidination for the characterization of the βIII-tubulin C-terminus peptide. This study is, to our knowledge, the first example of reflectron in-source decay (reISD) of the C-terminus of a 50 kDa protein. This potentially occurs via a CID-like mechanism occurring in the MALDI plume. There are now new avenues for top-down characterization of important clinical biomarkers such as βIII-tubulin isotypes, a potential marker of drug resistance and tumor progression. This paper raises the challenge of protein isotypes characterization for early cancer detection and treatment monitoring. [less ▲]

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