PDF(Pubmed)
Abstract：
Lysine (K) is widely used in the design of lysine-targeted crosslinkers, structural elucidation of protein complexes, and analysis of protein-protein interactions. In \"shotgun\" proteomics, which is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), proteins from complex samples are enzymatically digested, generating thousands of peptides and presenting significant challenges for the direct analysis of K-containing peptides. In view of the lack of effective methods for the enrichment of K-containing peptides, this work developed a method which based on a hydrophobic-tag-labeling reagent C10-S-S-NHS and reversed-phase chromatography (termed as HYTARP) to achieve the efficient enrichment and identification of K-containing peptides from complex samples. The C10-S-S-NHS synthesized in this work successfully labeled standard peptides containing various numbers of K and the labeling efficiency achieved up to 96% for HeLa cell protein tryptic digests. By investigating the retention behavior of these labeled peptides in C18 RP column, we found that most K-labeled peptides were eluted once when acetonitrile percentage reached 57.6% (v/v). Further optimization of the elution gradient enabled the efficient separation and enrichment of the K-labeled peptides in HeLa digests via a stepwise elution gradient. The K-labeled peptides accounted for 90% in the enriched peptides, representing an improvement of 35% compared with the number of peptides without the enrichment. The dynamic range of proteins quantified from the enriched K-containing peptides spans 5-6 orders of magnitude, and realized the detection of low-abundance proteins in the complex sample. In summary, the HYTARP strategy offers a straightforward and effective approach for reducing sample complexity and improving the identification coverage of K-containing peptides and low-abundance proteins.
摘要：
赖氨酸（K）被广泛用于赖氨酸靶向交联剂的设计，蛋白质复合物的结构阐明，以及蛋白质-蛋白质相互作用的分析。在“鸟枪”蛋白质组学中，基于液相色谱-串联质谱（LC-MS/MS），来自复杂样品的蛋白质被酶消化，产生数千种肽，并为直接分析含K肽提出了重大挑战。鉴于目前缺乏有效的含K肽富集方法,这项工作开发了一种基于疏水标记试剂C10-S-S-NHS和反相色谱（称为HYTARP）的方法，以实现对复杂样品中含K肽的有效富集和鉴定。在这项工作中合成的C10-S-S-NHS成功地标记了含有各种数量的K的标准肽,并且对于HeLa细胞蛋白胰蛋白酶消化物,标记效率达到高达96%。通过研究这些标记肽在C18RP柱中的保留行为，我们发现，当乙腈百分比达到57.6％（v/v）时，大多数K标记的肽被洗脱一次。洗脱梯度的进一步优化使得能够通过逐步洗脱梯度有效分离和富集HeLa消化物中的K标记的肽。K标记的肽在富集肽中占90％，表示与没有富集的肽的数量相比提高了35%。从富集的含K肽定量的蛋白质的动态范围跨越5-6个数量级,并实现了复杂样品中低丰度蛋白的检测。总之,HYTARP策略为降低样品复杂性和提高含钾肽和低丰度蛋白质的鉴定覆盖率提供了一种简单有效的方法.
