快速 shotgun 磷酸化蛋白质组学分析。

Rapid Shotgun Phosphoproteomics Analysis.

机构信息

Department of Food Technology, Institute of Marine Research (IIM), Spanish National Research Council (CSIC), Vigo, Spain.

Complutense University of Madrid (UCM), Madrid, Spain.

出版信息

Methods Mol Biol. 2021;2259:259-268. doi: 10.1007/978-1-0716-1178-4_17.

DOI:10.1007/978-1-0716-1178-4_17

PMID:33687721

Abstract

In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15 h.

摘要

在本章中，我们描述了一种用于鸟枪法全局磷酸化蛋白质组学分析的快速工作流程。该策略基于在高强度聚焦超声（HIFU）下通过超声场加速溶液中的胰蛋白酶消化，结合二氧化钛（TiO）选择性磷酸肽富集、强阳离子交换色谱（SCX）的分级以及在高分辨率质谱仪（LTQ-Orbitrap XL）中进行液相色谱-串联质谱（LC-MS/MS）分析。该策略针对 Jurkat T 细胞的全局磷酸化蛋白质组进行了优化。使用这种加速工作流程，HIFU-TiO-SCX-LC-MS/MS，在不到 15 小时内可从属于 3163 种不同磷酸化蛋白质的 13029 种不同磷酸肽中有效鉴定出 15367 个磷酸化位点。