色谱2024，Vol.42Issue(7)：721-729,9.DOI:10.3724/SP.J.1123.2024.02017
一种基于疏水基团标记和反相色谱分离的富集策略及其在含赖氨酸多肽分析中的应用
An enrichment strategy based on hydrophobic tagging and reversed-phase chromatographic separation for the analysis of lysine-containing peptides
摘要
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 pep-tides 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 de-veloped a method which based on a hydrophobic-tag-labeling reagent C10-S-S-NHS and re-versed-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 labe-ling 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 optimi-zation 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-abun-dance proteins in the complex sample.In summary,the HYTARP strategy offers a straightfor-ward and effective approach for reducing sample complexity and improving the identification coverage of K-containing peptides and low-abundance proteins.关键词
疏水基团标记/富集/液相色谱-串联质谱/含赖氨酸多肽Key words
hydrophobic tagging/enrichment/liquid chromatography-tandem mass spectrome-try(LC-MS/MS)/lysine-containing peptides分类
化学化工引用本文复制引用
何宇,单亦初,张丽华,张振宾,李洋..一种基于疏水基团标记和反相色谱分离的富集策略及其在含赖氨酸多肽分析中的应用[J].色谱,2024,42(7):721-729,9.基金项目
宁波市顶尖人才项目(215-432094250) （215-432094250）
宁波大学王宽诚幸福基金.Ningbo Top Talent Project(No.215-432094250) （No.215-432094250）
the K.C.Wong Magna Fund in Ningbo University. （）
