Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A.
Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A.
复制标题
DOI：
10.1074/mcp.ra118.001043
复制
发表时间：
2019-01
期刊：
影响因子：
--
通讯作者：
Tate EW
中科院分区：
文献类型：
--
作者：
Goya Grocin A;Serwa RA;Morales Sanfrutos J;Ritzefeld M;Tate EW
一种新的方法来量化细胞豆蔻酰化在生理水平和响应N-豆蔻酰转移酶抑制，并验证。分选酶A是标记整个蛋白质组中蛋白质甘氨酸N-末端的有效工具，并且其特异性在此背景下确定。描述了对生物素/抗生物素蛋白亲和富集方案的普遍适用的改进，其有效地消除了抗生物素蛋白衍生的胰蛋白酶肽污染物。应用分选酶A标记整个蛋白质组的蛋白质N-末端。新的凝胶，蛋白质组学和ELISA为基础的方法，以确定N-豆蔻酰化的蛋白质在细胞中，没有代谢标记。通过两种互补方法对蛋白质N-豆蔻酰化的变化进行并排质谱定量。改进的生物素-中性抗生物素蛋白亲和富集方案。N-肉豆蔻酰化是通过N-肉豆蔻酰转移酶（NMT）将14-碳饱和脂肪酸（肉豆蔻酸酯）共价添加到特定蛋白质底物的N-末端甘氨酸，并且通过控制定位、稳定性和相互作用在蛋白质调节中起重要作用。我们开发了一种新的方法，通过标记S。Aureus分选酶A（SrtA），并将其用于评估NMT抑制剂的靶标接合。用工程化生物素化缩酚肽SrtA底物（生物素-ALPET-Haa，Haa = 2-羟基乙酰胺）分析SrtA标记模式，能够通过nanoLC-MS/MS蛋白质组学对响应NMT抑制的从头生成的N-末端Gly蛋白进行全蛋白质组鉴定和定量，并通过基于凝胶的分析和ELISA确认了多个细胞系中的特异性底物。为了实现最佳的信号背景噪声，我们引入了一种新的和普遍适用的改进，生物素/亲和素亲和富集步骤，通过化学二甲基化商业中性亲和素树脂，并结合这两步LysC珠上/胰蛋白酶脱离珠消化，有效地消除亲和素衍生的胰蛋白酶肽，并提高富集肽的鉴定。我们还报告SrtA底物特异性的全细胞裂解物的第一次，确认SrtA混杂超出其公认的偏好N-末端甘氨酸，其有用性作为一种工具，无偏见的标记N-末端甘氨酸含蛋白质。我们的新方法是补充代谢标记策略，提供了第一种方法的整个蛋白质组增益的信号读出NMT抑制复杂的样品是不适合代谢标记。
A new method to quantify cellular myristoylation at physiological levels and in response to N-myristoyltransferase inhibition is presented and validated. Sortase A is an effective tool to label protein glycine N-termini across the whole proteome, and its specificity is determined in this context. Generally applicable improvements to the biotin/avidin affinity enrichment protocol are described that effectively eliminate avidin-derived tryptic peptide contaminants. Highlights Application of Sortase A to label protein N-termini across the whole proteome. Novel gel, proteomic and ELISA-based methods to determine N-myristoylation of proteins in cells, without metabolic labelling. Side by side mass spectrometric quantification of changes in protein N-myristoylation by two complementary methods. Improved Biotin-Neutravidin affinity enrichment protocol. N-myristoylation is the covalent addition of a 14-carbon saturated fatty acid (myristate) to the N-terminal glycine of specific protein substrates by N-myristoyltransferase (NMT) and plays an important role in protein regulation by controlling localization, stability, and interactions. We developed a novel method for whole-proteome profiling of free N-terminal glycines through labeling with S. Aureus sortase A (SrtA) and used it for assessment of target engagement by an NMT inhibitor. Analysis of the SrtA-labeling pattern with an engineered biotinylated depsipeptide SrtA substrate (Biotin-ALPET-Haa, Haa = 2-hydroxyacetamide) enabled whole proteome identification and quantification of de novo generated N-terminal Gly proteins in response to NMT inhibition by nanoLC-MS/MS proteomics, and was confirmed for specific substrates across multiple cell lines by gel-based analyses and ELISA. To achieve optimal signal over background noise we introduce a novel and generally applicable improvement to the biotin/avidin affinity enrichment step by chemically dimethylating commercial NeutrAvidin resin and combining this with two-step LysC on-bead/trypsin off-bead digestion, effectively eliminating avidin-derived tryptic peptides and enhancing identification of enriched peptides. We also report SrtA substrate specificity in whole-cell lysates for the first time, confirming SrtA promiscuity beyond its recognized preference for N-terminal glycine, and its usefulness as a tool for unbiased labeling of N-terminal glycine-containing proteins. Our new methodology is complementary to metabolic tagging strategies, providing the first approach for whole proteome gain-of signal readout for NMT inhibition in complex samples which are not amenable to metabolic tagging.