Department of Biosciences, University of Oslo, Oslo, Norway.
Methods Mol Biol. 2021;2228:133-144. doi: 10.1007/978-1-0716-1024-4_10.
Isobaric peptide termini labeling (IPTL) is an approach for quantitative proteomics based on crosswise isotopic labeling of peptides at the N- and C-terminus. The labeling reagents are chosen in isotopic variations that the resulting mass of all labels per peptide is isobaric, but the individual label on each peptide terminus is different. Therefore, the quantitative difference of the peptide signal can be determined by the fragment ions of the corresponding MS2 spectra. Here, we describe an approach for triplex-IPTL to allow the comparison of three proteomes. This approach is based on digestion of the proteins by endoproteinase Lys-C, followed by three combinations of selective dimethylation of the peptide N-termini and subsequent dimethylation of the lysine residues at the C-termini. Data analysis is performed using Mascot for database searches and the freely available software package IsobariQ for quantification.
等压肽端标记 (IPTL) 是一种基于肽的 N 端和 C 端的交叉同位素标记的定量蛋白质组学方法。标记试剂的选择具有同位素变化，使得每个肽的所有标记的质量是等压的，但每个肽末端的单个标记是不同的。因此，通过相应 MS2 谱的碎片离子可以确定肽信号的定量差异。在这里，我们描述了一种三联 IPTL 方法，以允许比较三种蛋白质组。该方法基于内切蛋白酶 Lys-C 对蛋白质的消化，然后进行三种组合的肽 N 端选择性二甲化和随后的 C 端赖氨酸残基的二甲化。数据分析使用 Mascot 进行数据库搜索，使用免费的 IsobariQ 软件包进行定量分析。
