Koehler Christian J, Arntzen Magnus O, Treumann Achim, Thiede Bernd
The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway.
Methods Mol Biol. 2012;893:129-41. doi: 10.1007/978-1-61779-885-6_10.
Isobaric peptide termini labeling (IPTL) is a recently introduced approach to the chemical labeling of peptides with isotopic reagents. Peptides derived from two different samples are labeled at the N terminus and at the C terminus with isotopically labeled reagents that have identical mass differences. To obtain isobaric peptides, labeling is carried out such that the introduced mass increase at one terminus will exactly match the mass decrease at the other terminus (and the other way around). This results in product ion spectra that display the quantitative difference of the peptide signal derived from the two samples for every b-ion and y-ion in the spectrum. The original IPTL approach required the selective modification of lysines followed by C-18 micropurification of modified peptides and reaction of the N termini. Here, we describe a new approach for IPTL that is based on the selective modification of the peptide N termini with succinic anhydride and subsequent reductive amination of C-terminal lysines with formaldehyde and cyanoborohydride. Both reactions can be carried out in one pot within 10 min and without C-18 micropurification. In addition, we present the software package IsobariQ for straightforward data analysis.
等压肽端标记（IPTL）是一种最近引入的用同位素试剂对肽进行化学标记的方法。来自两个不同样品的肽在N端和C端用具有相同质量差的同位素标记试剂进行标记。为了获得等压肽，标记过程要使得在一个末端引入的质量增加与在另一个末端的质量减少完全匹配（反之亦然）。这会产生产物离子光谱，该光谱显示了光谱中每个b离子和y离子源自两个样品的肽信号的定量差异。最初的IPTL方法需要对赖氨酸进行选择性修饰，然后对修饰后的肽进行C-18微纯化以及N端的反应。在此，我们描述了一种新的IPTL方法，该方法基于用琥珀酸酐对肽N端进行选择性修饰，随后用甲醛和氰基硼氢化钠对C端赖氨酸进行还原胺化。这两个反应都可以在一个反应容器中在10分钟内完成，且无需C-18微纯化。此外，我们还展示了用于直接数据分析的软件包IsobariQ。
