Comprehensive Analysis of Protein N-Terminome by Guanidination of Terminal Amines.,Analytical Chemistry

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Comprehensive Analysis of Protein N-Terminome by Guanidination of Terminal Amines.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2019-12-20 , DOI: 10.1021/acs.analchem.9b04141
Mingwei Sun _{1,

2} , Yu Liang ₁ , Yang Li _{1,

3} , Kaiguang Yang ₁ , Baofeng Zhao ₁ , Huiming Yuan ₁ , Xiao Li ₁ , Xiaodan Zhang ₁ , Zhen Liang ₁ , Yichu Shan ₁ , Lihua Zhang ₁ , Yukui Zhang ₁

Affiliation

Protein N-termini and their modifications not only represent different protein isoforms but also relate to the functional annotation and proteolytic activities. Currently, negative selection methods, such as terminal amine isotopic labeling of substrates (TAILS), are the most popular strategy to analyze the protein N-terminome, in which dimethylation or acetylation modification is commonly used to block the free amines of proteome samples. However, after tryptic digestion, the generated long peptides, caused by the missing cleavage of blocked lysine, could hardly be identified by MS, which hindered the deep-coverage analysis of N-terminome. Herein, to solve this problem, we developed an approach, named terminal amine guanidination of substrates (TAGS). 1H-Pyrazole-1-carboxamidine was used to effectively guanidinate lysine ε-amines and N-terminal α-amines, followed by tryptic digestion to generate N-terminal peptides without free amines and internal peptides with free amines. Then, the internal peptides with free amines were removed by hyperbranched polyglycerol-aldehyde polymers (HPG-ALDs) to achieve the negative enrichment of N-terminome. By TAGS, not only the cleavage rate of blocked lysine could be improved, but also the ionization efficiency of tryptic peptides was increased. In comparison, 1814 and 1620 protein N-termini were, respectively, identified by TAGS and TAILS in Saccharomyces cerevisiae (S. cerevisiae). Among them, 1012 N-termini were uniquely identified in TAGS. Furthermore, by the combination of TAGS and the stable isotope labeling with amino acids in cell culture (SILAC)/label-free quantitative method, we not only identified the known N-terminal cleavage fragment of gasdermin D but also identified some new cleavage sites during Val-boroPro-induced pyroptosis. All these results demonstrated that our developed approach, TAGS, might be of great promise for the comprehensive analysis of N-terminome and beneficial for promoting the identification of protein isoforms and studying in-depth the proteolytic activity of proteins.

中文翻译：

通过末端胺的胍化对蛋白质N末端进行综合分析。

N末端蛋白及其修饰不仅代表不同的蛋白同工型，而且与功能注释和蛋白水解活性有关。目前，阴性选择方法，例如底物的末端胺同位素标记（TAILS），是分析蛋白质N端组的最流行策略，其中通常使用二甲基化或乙酰化修饰来阻止蛋白质组样品的游离胺。但是，在胰蛋白酶消化后，MS几乎无法鉴定出由于赖氨酸被封闭的裂解缺失而产生的长肽，这阻碍了N端组的深层分析。在此，为了解决此问题，我们开发了一种方法，称为底物末端胺胍化（TAGS）。1H-吡唑-1-羧car用于有效地胍基赖氨酸ε-胺和N-末端α-胺，然后进行胰蛋白酶消化以产生不含游离胺的N末端肽和含游离胺的内部肽。然后，通过超支化聚甘油-醛聚合物（HPG-ALDs）去除带有游离胺的内部肽，以实现N末端组的负富集。通过TAGS，不仅可以提高封闭赖氨酸的裂解率，而且可以提高胰蛋白酶肽的电离效率。相比之下，在酿酒酵母（S.cerevisiae）中，分别通过TAGS和TAILS鉴定了1814和1620蛋白N末端。其中，在TAGS中唯一鉴定出1012个N末端。此外，通过TAGS结合稳定的同位素标记和细胞培养中的氨基酸（SILAC）/无标记定量方法，我们不仅鉴定了Gasdermin D的已知N末端裂解片段，而且还鉴定了Val-过程中的一些新裂解位点boroPro诱导的细胞凋亡。所有这些结果表明，我们开发的方法TAGS可能对N端基因组的综合分析具有广阔的前景，并且有利于促进蛋白质同工型的鉴定和深入研究蛋白质的蛋白水解活性。

更新日期：2019-12-20

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