Sharaireh Aseel, Tierney Anna L, Unwin Richard D
Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK.
Department of Conservative Dentistry, School of Dentistry, University of Jordan, Amman, Jordan.
Methods Mol Biol. 2022;2490:157-177. doi: 10.1007/978-1-0716-2281-0_12.
In this methods chapter, we describe the use of isobaric tags for relative and absolute quantification (iTRAQ) for the differential expression analysis of global proteins between embryonic stem cell samples. This protocol describes how proteins are collected from cell culture, digested and prepared so that peptides are labeled with these isobaric tags. Labeled digests are pooled, fractionated offline, and quantified using liquid chromatography-mass spectrometry (LC-MS). This offline fractionation allows for a greater separation and thus increased identification/quantification of peptides. This combined method enables large-scale, deep penetration into the proteome of embryonic stem cells. During quantification, the relative intensities of label-derived reporter ions represent the relative amount of peptide in each sample. Using search algorithms that integrate the generated data for the identified and quantified peptides allows the relative quantification of proteins in the samples. The isobaric tags can be used in a 4 or 8 multiplexed manner; however, using an 8-plex experimental setup allows for the simultaneous analysis of biological and technical replicates within the same mass spectrometry run, thus minimizing experimental variation and increasing the confidence in any identified expression differences.
在本章方法中，我们描述了使用等压标签相对和绝对定量技术（iTRAQ）对胚胎干细胞样本间的整体蛋白质进行差异表达分析。本方案介绍了如何从细胞培养物中收集蛋白质、进行消化和制备，以便用这些等压标签对肽段进行标记。标记后的消化产物混合后进行离线分级分离，并使用液相色谱 - 质谱联用（LC - MS）进行定量。这种离线分级分离能够实现更大程度的分离，从而增加肽段的鉴定/定量。这种组合方法能够大规模、深入地分析胚胎干细胞的蛋白质组。在定量过程中，标签衍生的报告离子的相对强度代表每个样本中肽段的相对量。使用整合已鉴定和定量肽段的生成数据的搜索算法，可以对样本中的蛋白质进行相对定量。等压标签可以以4重或8重的方式使用；然而，使用8重实验设置允许在同一次质谱运行中同时分析生物学重复和技术重复，从而最大限度地减少实验变异并提高对任何鉴定出的表达差异的置信度。
