2-D LC-MS/MS Analysis of secreted proteins from HepG2 cells: Combination with various sample preparation methods before in-solution trypsin digestion

Ryo Yamashita, Y. Fujiwara, Xunmei Yuan, K. Yasuda, Y. Kaburagi
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引用次数: 3

Abstract

Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2-D LC-MS/MS) technique has high capability of resolving peptides, and is used for analysis of tryptic peptides derived from highly complex protein mixtures such as plasma. However, the detection of low-abundant proteins and low-molecular-weight proteins is often very difficult in this system, because major peptides from high-abundant proteins such as albumin mostly disturb the separation of minor peptides from low-abundant proteins. To resolve these problems, different methods of sample preparation for in-solution trypsin digestion were tested, and the optimized method was applied to the 2-D LC-MS/MS analysis of proteins secreted from human hepatoma cell line, HepG2. We could identify 247 and 142 proteins from the chemically and thermally denatured samples, respectively. Among them, 71 proteins were identified in common to both methods, while most of the proteins were identified using either of the two procedures. In addition to these denaturation methods, the molecular-mass cutoff via ultrafiltration improved the efficiency in identifying low-molecular-weight proteins. Finally, we could identify 478 secreted proteins in total using the combination of these processes. These data indicate that in sample preparation the combination of various denaturation methods, as well as molecular-mass cutoff, are very critical for the identification of a wider range of low-abundant proteins via 2-D LC-MS/MS analysis.
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HepG2细胞分泌蛋白的二维LC-MS/MS分析:溶液胰酶消化前不同样品制备方法的结合
二维液相色谱-串联质谱(2-D LC-MS/MS)技术具有较高的肽分辨能力,可用于分析血浆等高度复杂蛋白质混合物中衍生的色氨酸。然而,在这个系统中,低丰度蛋白质和低分子量蛋白质的检测通常是非常困难的,因为来自高丰度蛋白质(如白蛋白)的主要肽通常会干扰从低丰度蛋白质中分离的次要肽。为了解决这些问题,我们对不同的溶液胰酶消化样品制备方法进行了测试,并将优化后的方法应用于人肝癌细胞株HepG2分泌蛋白的二维LC-MS/MS分析。我们分别从化学变性和热变性样品中鉴定出247种和142种蛋白质。其中,两种方法共鉴定出71种蛋白,而大多数蛋白均使用两种方法中的一种进行鉴定。除了这些变性方法,通过超滤的分子质量切断提高了识别低分子量蛋白质的效率。最后,我们利用这些过程的组合,共鉴定出478种分泌蛋白。这些数据表明,在样品制备中,各种变性方法的结合以及分子质量切断对于通过2-D LC-MS/MS分析鉴定更广泛的低丰度蛋白质是非常关键的。
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