Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation.
Fiche publication
Date publication
janvier 2015
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MAGDALOU Jacques, Pr MAINARD Didier, Dr VINCOURT Jean-Baptiste
Tous les auteurs :
Riffault M, Moulin D, Grossin L, Mainard D, Magdalou J, Vincourt JB
Lien Pubmed
Résumé
Proteomics users enjoy the rapid development of LC-MS-based label-free relative quantification methods but in practice these remain restricted to mass spectrometers using electrospray ionization. Here, tools dedicated to ion chromatogram extraction, time alignment, signal normalization and statistical analysis were used to interpret label-free relative difference between primary human chondrocyte secretomes and dilutions thereof, analyzed successively by LC-MALDI. The analysis of secretomes diluted into culture medium demonstrated that abundant proteins could be relatively quantified within 1.5-20-fold changes with satisfactory statistics. In addition, comparison of multiple samples requires analyzing most samples in TOF mode only, saving considerable machine-time usage. The method allowed identification and quantification of most secreted proteins relevant to the chondrocyte phenotype and evidenced their up- or down regulations by TGFbeta1 and patient-to-patient differential expression. Novel targets of TGFbeta1 were evidenced, such as pro-collagen C-proteinase enhancer protein 1, Metalloproteinase inhibitor 1, Fibulin-3, Tetranectin and Cartilage Intermediate Layer Protein 1, while others match previous findings. Several were verified by Western blot. This whole workflow is non-invasive, compatible with many cell culture protocols, technically straightforward and rapid, particularly regarding mass spectrometer time usage and could make label-free LC-MALDI analysis of low-complexity proteomes a major tool for routine cell culture characterization. BIOLOGICAL SIGNIFICANCE: The present work presents the adaptation of label free relative protein quantification principles to LC-MALDI data to rapidly measure protein fold-changes between samples of relative complexity and its utility to characterize the secreted proteome of human primary chondrocytes. The method was employed to characterize the chondrocyte secretome regulation by TGFbeta1 and is proposed as a routine tool to assess the quality of biomaterials designed for cartilage repair and to quantitatively investigate the influence of environmental factors upon it.
Référence
J Proteomics. 2015 Jan 30;114:263-73