Novel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis

[en] 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.

Research Center/Unit :

Giga-Systems Biology and Chemical Biology - ULiège
Giga-Cancer - ULiège

Disciplines :

Oncology
Biochemistry, biophysics & molecular biology

Author, co-author :

Fleron, Maximilien ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Histologie - Cytologie

Greffe, Yannick ; Université de Liège - ULiège > Doct. sc. bioméd. & pharma. (Bologne)

Musmeci, Davide ; Université de Liège - ULiège > Doct. sc. bioméd. & pharma. (Bologne)

Massart, Anne-Cécile ; Université de Liège - ULiège > Center for Analytical Research and Technology (CART)

Hennequière, Vincent ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biologie générale et cellulaire

Mazzucchelli, Gabriel ; Université de Liège - ULiège > Center for Analytical Research and Technology (CART)

Waltregny, David ; Université de Liège - ULiège > Département des sciences cliniques > Urologie

De Pauw-Gillet, Marie-Claire ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Histologie - Cytologie

De Pauw, Edwin ; Université de Liège - ULiège > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.)

Castronovo, Vincenzo ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biologie générale et cellulaire

Turtoi, Andrei ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > GIGA-R : Labo de recherche sur les métastases

Language :

English

Title :

Novel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis

Publication date :

10 September 2010

Journal title :

Journal of Proteomics

ISSN :

1874-3919

eISSN :

1876-7737

Publisher :

Elsevier, Amsterdam, Netherlands

Volume :

Issue :

Pages :

1986-2005

Peer reviewed :

Peer Reviewed verified by ORBi

Additional URL :

http://www.sciencedirect.com/science/article/pii/S1874391910001818

Funders :

FRIA - Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture

Available on ORBi :

since 28 June 2011

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