A gel-free approach in vascular smooth muscle cell proteome: perspectives for a better insight into activation

This paper describes a strategy for the study of the VSMC proteome. Our approach was based on pre-fractionation with ion exchange chromatography coupled with matrix assisted laser desorption-time of flight mass spectrometry analysis assisted by a liquid chromatography (LC-MALDI-TOF/TOF). Ion exchange chromatography resulted in a good strategy designed to simplify the complexity of the cellular extract and to identify a large number of proteins. Selectivity based on the ion-exchange chemical features was adequate if evaluated on the basis of protein pI. The LC-MALDI approach proved to be highly reproducible and sensitive since we were able to identify up to 815 proteins with a concentration dynamic range of 7 orders of magnitude.In our opinion, the large number of identified proteins and the promising quantitative reproducibility made this approach a powerful method to analyze complex protein mixtures in a high throughput way and to obtain statistical data for the discovery of key factors involved in VSMC activation and to analyze a label-free differential protein expression.The use of chromatography coupled with MS analysis is a powerful approach for the identification of proteins, owing to its capacity to fractionate molecules with different chemical features [1-4]. Furthermore, LC-MALDI-TOF/TOF analysis combined with preliminary fractionation of a total protein extract is a potential tool for biomarker discovery because of its high sensitivity and high throughput capacity [5].However, the use of LC-MALDI analysis still needs to be optimized and evaluated [6,7]. To obtain useful information for comparative analysis of samples and differential protein expression using a label-free approach in LC-MALDI techniques, the reproducibility in measuring m/z abundances (peak intensity) and a linear relation between intensity and marker concentration are essential [8-10]. Moreover, although LC-MALDI MS/MS analysis is a high mass precision technique, it is time consuming, espe