Department of Human Morphology and Applied Biology, University of Pisa, Via Roma 55, 56126 Pisa, Italy.
Proteome Sci. 2010 Mar 24;8:15. doi: 10.1186/1477-5956-8-15.
The use of chromatography coupled with mass spectrometry (MS) analysis is a powerful approach to identify proteins, owing to its capacity to fractionate molecules according to different chemical features. The first protein expression map of vascular smooth muscle cells (VSMC) was published in 2001 and since then other papers have been produced. The most detailed two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map was presented by Mayr et al who identified 235 proteins, corresponding to the 154 most abundant unique proteins in mouse aortic VSMC. A chromatographic approach aimed at fractionating the VSMC proteome has never been used before.
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.
色谱法与质谱（MS）分析相结合是鉴定蛋白质的有力方法，因为它能够根据不同的化学特征对分子进行分离。2001 年发表了第一篇血管平滑肌细胞（VSMC）的蛋白质表达图谱，此后又发表了其他论文。Mayr 等人发表了最详细的二维聚丙烯酰胺凝胶电泳（2D-PAGE）图谱，他们鉴定了 235 种蛋白质，对应于小鼠主动脉 VSMC 中 154 种最丰富的独特蛋白质。以前从未使用过针对 VSMC 蛋白质组进行分离的色谱方法。
本文描述了一种研究 VSMC 蛋白质组的策略。我们的方法基于离子交换色谱的预分离，结合基质辅助激光解吸飞行时间质谱分析，并辅助液相色谱（LC-MALDI-TOF/TOF）。离子交换色谱是一种很好的策略，旨在简化细胞提取物的复杂性，并鉴定大量蛋白质。如果根据蛋白质 pI 评估，基于离子交换化学特征的选择性是足够的。LC-MALDI 方法被证明具有高度的重现性和灵敏度，因为我们能够鉴定多达 815 种蛋白质，浓度动态范围为 7 个数量级。
在我们看来，大量鉴定出的蛋白质和有前途的定量重现性使这种方法成为一种强大的方法，可以高通量分析复杂的蛋白质混合物，并获得用于发现参与 VSMC 激活的关键因素和分析无标记差异蛋白质表达的统计数据。
