References of "Greffe, Yannick"
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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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See detailNovel Relative ICPL Based Quantitative Phospho- and Glycoproteome Analysis Method
Fleron, Maximilien ULg; Greffe, Yannick ULg; Massart, Anne-Cécile ULg et al

Poster (2010, April 16)

Large scale proteomic analysis remains challenging partially because proteins are inhomogeneous and often influenced by a variety of structural modifications. In particular, these specific chemical ... [more ▼]

Large scale proteomic analysis remains challenging partially because proteins are inhomogeneous and often influenced by a variety of structural modifications. In particular, these specific chemical modifications called posttranslational modifications (PTM) are crucial determinants for the protein function and biological role. Up to now there have been a growing number of studies describing the enrichment and identification of PTM. However, a significant dearth of data offering a reliable methodology for PTM quantification does exist. The present work aims at developing a label based protein PTM quantification strategy and demonstrating its value on comparative analysis of cells originating from two distinct prostate metastasis sites. PC3 and LNCaP cells isolated from bone and lymph node prostate cancer metastasis sites respectively, were lysed and spiked with three non-human proteins serving as internal standards. Following this, the samples were reduced and alkylated, digested with trypsin and subjected to peptide ICPL (isotope coded protein label) labeling. The two peptide containing samples were joined together followed by the affinity isolation of phospho- (using TiO2 metal affinity chromatography) and glycopeptides (oxidized glycans were bound on hydrazide resin). The enriched fraction as well as the flow-through were analyzed on a 2D-(SCX and C18-RP)-nano-HPLC system. The peptide identification and quantification was conducted using electrospray ion-trap mass spectrometer (Bruker, HCT-ultra). Validation of the differentially modulated proteins was conducted in several biological and technical replicates using the label free MSe based quantification strategy. This PTM based, novel relative protein quantification using post-digest ICPL has detected over 598 individual proteins. Of these more than 95 % have been successfully quantified. PTM enrichment methodologies allowed an isolation rate of 91 % and 50 % for phosphorylated and glycosylated proteins respectively. The detailed comparison of PC3 and LNCaP cells has shown specific overexpression of selected proteins indicating differences between these two prostate metastatic cell lines. Several of these modulated proteins have been previously described to be related to prostate cancer (e.g. annexin A2 and vimentin) while others could be considered as potentially novel. These proteins might be implicated in the fundamental process related to metastasis dissemination. However, because of the known discrepancy between cell systems and clinical material, the present study can be regarded only as a step towards elucidation of these complex interactions. [less ▲]

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See detailVersican overexpression in human breast cancer lesions: Known and new isoforms for stromal tumor targeting.
Kischel, Philippe ULg; Waltregny, David ULg; Dumont, Bruno ULg et al

in International Journal of Cancer = Journal International du Cancer (2010), 126(3), 640-50

Proteoglycans play a key role in cancer development and progression by participating in the constitution of a specific fertile tumor microenvironment. As they are largely overexpressed in the malignant ... [more ▼]

Proteoglycans play a key role in cancer development and progression by participating in the constitution of a specific fertile tumor microenvironment. As they are largely overexpressed in the malignant stroma, proteoglycans provide a reservoir of potential new targets for anticancer therapies, because they can serve to convey toxic payloads in the close proximity of cancer cells and subsequently destroy them. In this context, versican, a proteoglycan largely overexpressed in several solid cancers, bears the potential to be such an ideal target. As 4 main versican isoforms have been characterized, we sought to determine which isoform could represent the best target in human breast cancer. We used a series of 10 primary breast cancer lesions that were characterized as overexpressing the versican protein, when compared with matched normal breast tissues, using shotgun mass spectrometry and immunohistochemistry experiments. Quantitative polymerase chain reaction and western-blotting experiments were used to evaluate versican isoform expression in breast cancer/normal tissue pairs for which ARN quality was excellent. All known isoforms were significantly overexpressed in the malignant lesions, both at the mRNA and at the protein levels. In the course of this study, we also identified and cloned a new alternatively spliced versican isoform, referred to as V4, which was also found to be upregulated in human breast cancer. This study provides for the first time a comprehensive mRNA and protein analysis of versican isoforms expression in human breast tissues, and offers insights into which therapeutic strategy would be best suited to target versican in human breast cancer lesions. [less ▲]

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