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

doi:10.3724/SP.J.1123.2024.02017

摘要/Abstract

摘要：

赖氨酸(K)已被广泛用于靶向赖氨酸的交联剂设计、蛋白质复合物的结构解析以及蛋白质-蛋白质相互作用等研究领域。在基于液相色谱-串联质谱(LC-MS/MS)联用技术的“鸟枪法”蛋白质组学研究中,复杂生物样品中的蛋白质被酶切成上万条肽段,为直接分析含有K的肽段带来了巨大挑战。鉴于目前缺乏针对含K多肽的有效富集方法,本工作发展了一种基于疏水标记试剂C10-S-S-NHS和反相色谱分离的方法(简称HYTARP),实现对复杂样品中含K多肽的高效富集和鉴定。合成的C10-S-S-NHS试剂可以高效标记含不同数目K的标准肽段,且对HeLa细胞蛋白酶解肽段的标记效率高达96%。通过考察标记肽段在反相色谱中的保留行为,发现大部分被标记的含K肽段在流动相中乙腈比例升高至约57.6%(v/v)时开始洗脱。进一步优化色谱洗脱梯度,发现阶梯式洗脱能够实现对复杂样品酶解肽段中标记的K肽段的高效分离和富集。富集后样品中含K肽段的占比>90%,较富集前提高了35%。富集的含K肽段对应蛋白质的丰度动态范围跨越了5~6个数量级,实现了对复杂样品中低丰度蛋白质的鉴定。综上,本工作发展的HYTARP策略为降低样品复杂度、提高含K肽段及低丰度蛋白质的鉴定覆盖率提供了一种简单、高效的方法。

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

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.

Key words: hydrophobic tagging, enrichment, liquid chromatography-tandem mass spectrometry (LC-MS/MS), lysine-containing peptides

中图分类号:

O658

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.

HE Yu, SHAN Yichu, ZHANG Lihua, ZHANG Zhenbin, LI Yang. An enrichment strategy based on hydrophobic tagging and reversed-phase chromatographic separation for the analysis of lysine-containing peptides[J]. Chinese Journal of Chromatography, 2024, 42(7): 721-729.

图/表 6

图 1 基于HYTARP策略的含K多肽的富集流程示意图

Fig. 1 Workflow of lysine-containing peptide enrichment using the hydrophobic tagging and reversed-phase chromatographic separation (HYTARP) strategy

图 2 标记试剂C10-S-S-NHS的合成

Fig. 2 Synthesis of 2,5-dioxopyrrolidin-1-yl-3-(decyldisulfanyl)propanoate (C10-S-S-NHS) EDC: N-(3-dimethylaminopropyl)-N'-ethylcarbo.

图 3 标准肽段被C10-S-S-NHS标记前后的MALDI/TOF MS图

Fig. 3 MALDI/TOF MS profiles of standard peptides before and after C10-S-S-NHS labeling a, b. peptide of LA-10 (sequence: LVVSTQTALA) before (a, m/z 1024.8) and after (b, m/z 1284.5) labeling; c, d. peptide of MK-7 (sequence: MIFVGIK) before (c, m/z 807.7) and after (d, m/z 1350.2, Na+) labeling; e, f. peptide of KK-9 (sequence: KSLSLSPGK) before (e, m/z 916.7) and after (f, m/z 1720.1, Na+) labeling.

图 4 HeLa酶解肽段标记0、1、2和3个C10烷基链的百分比分布

Fig. 4 Percentage distribution of HeLa digests labeled by zero, one, two, and three C10 alkyl chains w/o: without.

图 5 (a) HeLa酶解肽段被C10-S-S-NHS标记后的反相分离色谱图, (b)各级分中被标记0个、1个和≥2个 C10链的肽段数(堆积柱形图)以及含K肽段所占百分比(折线图)

Fig. 5 (a) RP separation chromatogram of HeLa digests after C10-S-S-NHS labeling, (b) number of peptides labeled by zero, one, two or more C10 chains (stacked bar chart) and percentage of peptides containing K (line chart) in each fraction Separation gradient: 0-15 min, 60%B; 15-25 min, 60%B-70%B; 25-55 min, 70%B-80%B; 55-70 min, 80%B-95%B; 70-80 min, 95%B (mobile phase A: 2% (v/v) acetonitrile aqueous solution containing 0.1% trifluoroacetic acid (TFA); mobile phase B: 80% (v/v) acetonitrile aqueous solution containing 0.1% TFA); C18 column: 250 mm×4.6 mm, 5 μm; flow rate: 1 mL/min; detection wavelength: 214 nm.

图 6 (a)各级分中被标记0个、1个、2个和2个以上C10链的肽段数(堆积柱形图)以及含K肽段所占百分比(折线图), (b)各级分间含K肽段的鉴定结果Venn图,(c)富集的含K肽段的序列特征,(d)定量到的全部(蓝色圆圈)以及含有被C10标记的K的(红色圆圈)Hela细胞蛋白质的丰度范围

Fig. 6 (a) Number of peptides labeled by zero, one, two or more C10 chains (stacked bar chart) and percentage of peptides containing K (line chart) in each fraction, (b) Venn diagram of K-containing peptides identified in each fraction, (c) sequence logo of enriched K-containing peptides, (d) dynamic range of all quantified HeLa cell proteins (blue circles) and K-containing proteins labeled by C10 (red circles) iBAQ: intensity based absolute quantification.

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