An Approach for Triplex-Isobaric Peptide Termini Labeling (Triplex-IPTL)
An Approach for Triplex-Isobaric Peptide Termini Labeling (Triplex-IPTL)
复制标题
DOI：
10.1021/ac3035508
复制
发表时间：
2013-02-19
影响因子：
7.4
通讯作者：
Thiede, Bernd
中科院分区：
文献类型：
--
作者：
Koehler, Christian J.;Arntzen, Magnus O.;Thiede, Bernd
等压肽末端标记(IPTL)是基于用互补的同位素标记来标记两个多肽末端，从而产生等压肽。IPTL衍生化后的MS/MS分析产生的多肽特异碎片离子分布在整个MS/MS光谱中。因此，可以获得每个肽的几个量化点。在这份报告中，我们提出了三重IPTL，一种IPTL的化学标记策略，允许在一次MS运行中同时定量三个状态。为此，对不同稳定同位素的甲醛和氰化硼进行了N-末端氨基的二甲基化和赖氨酸的二甲基化。通过LC-MS/MS分析，组合样品显示了三个相应的同位素碎片离子系列，定量地反映了多肽的比例。为了支持这种多路标记策略，我们进一步开发了数据分析工具IsobariQ，包括多维VSN归一化、统计推断、三重IPTL数据的图形可视化和蛋白质图谱模式的聚类。通过与激动素EG5抑制剂S-三甲基-L-半胱氨酸孵育的HeLa细胞的时间谱分析，证明了三重IPTL方法与IsobariQ相结合的有效性。结果，根据它们的比率分布找到了量化的蛋白质簇，它们分别与它们在有丝分裂停止和细胞死亡中的基因本体论关联很好地对应。
Isobaric peptide termini labeling (IPTL) is based on labeling of both peptide termini with complementary isotopic labels resulting in isobaric peptides. MS/MS analysis after IPTL derivatization produces peptide-specific fragment ions which are distributed throughout the MS/MS spectrum. Thus, several quantification points can be obtained per peptide. In this report, we present triplex-IPTL, a chemical labeling strategy for IPTL allowing the simultaneous quantification of three states within one MS run. For this purpose, dimethylation of the N-terminal amino group followed by dimethylation of lysines was used with different stable isotopes of formaldehyde and cyanoborohydride. Upon LC-MS/MS analysis, the combined samples revealed three corresponding isotopic fragment ion series reflecting quantitatively the peptide ratios. To support this multiplexing labeling strategy, we have further developed the data analysis tool IsobariQ and included multidimensional VSN normalization, statistical inference, and graphical visualization of triplex-IPTL data and clustering of protein profiling patterns. The power of the triplex-IPTL approach in combination with IsobariQ was demonstrated through temporal profiling of HeLa cells incubated with the kinesin Eg5 inhibitor S-Trityl-L-cysteine (STLC). As a result, clusters of quantified proteins were found by their ratio profiles which corresponded well to their gene ontology association in mitotic arrest and cell death, respectively.