School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Sep 15;905:96-104. doi: 10.1016/j.jchromb.2012.08.008. Epub 2012 Aug 14.
The fractionation of complex samples at the protein level prior to shotgun proteomics analysis is an efficient means to more comprehensive analysis of samples. A mixed-bed ion-exchange (IEX) column, packed with both weak anion exchange (WAX) and weak cation exchange (WCX) materials, was used for the first dimensional separation of complex samples at the protein level using volatile solvents. The peptides from digestion of each fraction were then identified by 2D SCX-RP-LC-MS/MS. We applied this 3D strategy to mouse mammary tumor 4T1 cell lysate and identified a total of 3084 proteins in a typical experiment. The moderate separation performance of the mixed-bed IEX column facilitated the in-depth identification of the proteins in the complex sample. There were some acceptable inter-fraction overlaps. Nearly half (45.8%) of the proteins were only identified in single fractions, while 82.3% were identified in no more than 3 fractions. The identified proteins covered a broad range of pI, size and grand average hydrophobicity (GRAVY) values. Detailed analysis of proteins identified in each fraction elucidated the separation characteristics of mixed-bed IEX. Retention on mixed-bed IEX was associated, but not restricted to the extreme pI values (pI<5, pI>10) and to the percentage of charged residues of both signs. In conclusion, we have exploited the mixed-bed IEX column to establish an efficient and comprehensive identification method for complex samples.
在进行鸟枪法蛋白质组学分析之前，对复杂样品进行蛋白质水平的分级分离是更全面地分析样品的有效手段。本文采用混合床离子交换（IEX）柱，填充弱阴离子交换（WAX）和弱阳离子交换（WCX）材料，用挥发性溶剂对复杂样品进行蛋白质水平的一维分离。然后，用二维 SCX-RP-LC-MS/MS 对每个馏分的肽进行鉴定。我们将这种 3D 策略应用于小鼠乳腺癌 4T1 细胞裂解物，在一个典型的实验中总共鉴定了 3084 种蛋白质。混合床 IEX 柱具有中等的分离性能，有助于深入鉴定复杂样品中的蛋白质。各馏分之间存在一些可接受的重叠。将近一半（45.8%）的蛋白质仅在单个馏分中被鉴定，而 82.3%的蛋白质在不超过 3 个馏分中被鉴定。鉴定的蛋白质涵盖了广泛的等电点（pI）、大小和平均疏水性（GRAVY）值范围。对每个馏分中鉴定的蛋白质进行详细分析，阐明了混合床 IEX 的分离特性。在混合床 IEX 上的保留时间与极端 pI 值（pI<5，pI>10）和两种符号带电荷残基的百分比有关，但并不受其限制。总之，我们利用混合床 IEX 柱建立了一种用于复杂样品的高效、全面的鉴定方法。
