Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates
Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates
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DOI：
10.1038/nprot.2011.382
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发表时间：
2011-10-01
期刊：
影响因子：
14.8
通讯作者：
Overall, Christopher M.
中科院分区：
文献类型：
--
作者：
Kleifeld, Oded;Doucet, Alain;Overall, Christopher M.
分析蛋白质N末端的序列和性质有许多应用。在人类蛋白质组计划中，确定蛋白质组注释的蛋白质末端变得越来越重要。对降解物中的蛋白酶裂解位点进行终端组学分析以发现底物是一个关键的新应用。在这里，我们描述了进行底物(尾部)末端胺同位素标记的分步过程，这是一种2-到3-D(取决于标记方法)的高通量方法，可以高置信度地识别和区分蛋白酶产生的neo-N末端和成熟蛋白N末端。TAILS使用负选择来丰富所有N末端的多肽，并使用伯胺标记的定量作为区分因子。标记具有多种用途，适合于许多应用，包括体外生化和细胞培养分析；使用动物和人类来源的组织样本进行体内分析也很容易进行。在蛋白质水平上，N末端和赖氨酸胺被二甲基化(甲醛/氰化硼氢化钠)阻断，并通过在细胞培养标签中加入重和轻二甲基化试剂或用氨基酸进行稳定同位素标记来进行同位素标记。或者，可以使用胺封闭和等压标记进行相对和绝对定量，也称为iTRAQ，从而实现简单的多重样品分析。在胰酶消化后，使用高相对分子质量的树状聚甘油醛聚合物实现N-末端多肽的分离，该聚合物结合内部的胰酶多肽和现在具有N-末端阿尔法胺的C-末端多肽。通过超滤回收未结合的天然封闭的(乙酰化、环化、甲基化等)或标记的成熟N末端和neo-N末端多肽，并用串联质谱仪(MS/MS)进行分析。分级底物筛选通过多肽同位素定量和生物信息学搜索标准将底物与背景蛋白降解产物和未切割的蛋白质区分开来。
Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.