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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 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 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 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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See detailMicrotubule targeting agents: from biophysics to proteomics
Calligaris, David ULg; Verdier-Pinard, Pascal; Devred, François et al

in Cellular and Molecular Life Sciences : CMLS (2010), 67(7), 1089-1104

This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin ... [more ▼]

This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin polymerization and microtubule targeting agent binding, we then highlight how the three-dimensional structures of drug-tubulin complexes obtained on stabilized tubulin are seeded by precise biological and biophysical data. New avenues opened by thermodynamics analysis, high throughput screening, and proteomics for the molecular pharmacology of these drugs are presented. The amount of data generated by biophysical, proteomic and cellular techniques shed more light onto the microtubule-tubulin equilibrium and tubulin-drug interaction. Combining these approaches provides new insight into the mechanism of action of known microtubule interacting agents and rapid in-depth characterization of next generation molecules targeting the interaction between microtubules and associated modulators of their dynamics. This will facilitate the design of improved and/or alternative chemotherapies targeting the microtubule cytoskeleton. [less ▲]

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