School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore.
J Proteome Res. 2011 Dec 2;10(12):5568-74. doi: 10.1021/pr2007686. Epub 2011 Nov 15.
The iTRAQ technique is popular for the comparative analysis of proteins in different complex samples. To increase the dynamic range and sensitivity of peptide identification in shotgun proteomics, SCX chromatography is generally used for the fractionation of iTRAQ-labeled peptides before LC-MS/MS analysis. However, SCX suffers from clustering of similarly charged peptides and the need to desalt fractions. In this report, SCX is compared with the alternative ERLIC method for fractionating iTRAQ-labeled peptides. The simultaneous effect of electrostatic repulsion and hydrophilic interaction in ERLIC results in peptide elution in order of decreasing pI and GRAVY values (increasing polarity). Volatile solvents can be used. We applied ERLIC to iTRAQ-labeled peptides from rat liver tissue, and 2745 proteins and 30,016 unique peptides were identified with high confidence from three technical replicates. This was 12.9 and 49.4% higher, respectively, than was obtained using SCX. In addition, ERLIC is appreciably better at the identification of highly hydrophobic peptides. The results indicate that ERLIC is a more convenient and more effective alternative to SCX for the fractionation of iTRAQ-labeled peptides. Quantification data show that both SCX and ERLIC fractionation have no significant effect on protein quantification by iTRAQ.
iTRAQ 技术常用于不同复杂样本中蛋白质的比较分析。为了提高 shotgun 蛋白质组学中肽鉴定的动态范围和灵敏度，通常在 LC-MS/MS 分析前使用 SCX 色谱法对 iTRAQ 标记的肽进行分级。然而，SCX 存在相似电荷肽的聚类问题，并且需要脱盐级分。在本报告中，将 SCX 与替代的 ERLIC 方法进行了比较，用于分级 iTRAQ 标记的肽。ERLIC 中静电排斥和亲水相互作用的同时作用导致肽按 pI 和 GRAVY 值（增加极性）递减的顺序洗脱。可以使用挥发性溶剂。我们将 ERLIC 应用于来自大鼠肝组织的 iTRAQ 标记的肽，从三个技术重复中以高置信度鉴定了 2745 种蛋白质和 30016 种独特肽。与 SCX 相比，分别提高了 12.9%和 49.4%。此外，ERLIC 在鉴定高度疏水性肽方面表现出色。结果表明，ERLIC 是一种更方便、更有效的替代 SCX 用于 iTRAQ 标记肽分级的方法。定量数据表明，SCX 和 ERLIC 分级对 iTRAQ 的蛋白质定量均无显著影响。
