Profiling Proteins and Phosphorylation Sites During T Cell Activation Using an Integrated Thermal Shift Assay.

IF 6.1 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Molecular & Cellular Proteomics Pub Date : 2024-07-01 Epub Date: 2024-06-15 DOI:10.1016/j.mcpro.2024.100801

Brandon M Gassaway, Edward L Huttlin, Emily M Huntsman, Tomer M Yaron-Barir, Jared L Johnson, Kiran Kurmi, Lewis C Cantley, Joao A Paulo, Alison E Ringel, Steven P Gygi, Marcia C Haigis

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Abstract

T cell activation is a complex biological process of naive cells maturing into effector cells. Proteomic and phospho-proteomic approaches have provided critical insights into this process, yet it is not always clear how changes in individual proteins or phosphorylation sites have functional significance. Here, we developed the Phosphorylation Integrated Thermal Shift Assay (PITSA) that combines the measurement of protein or phosphorylation site abundance and thermal stability into a single tandem mass tags experiment and apply this method to study T cell activation. We quantified the abundance and thermal stability of over 7500 proteins and 5000 phosphorylation sites and identified significant differences in chromatin-related, TCR signaling, DNA repair, and proliferative phosphoproteins. PITSA may be applied to a wide range of biological contexts to generate hypotheses as to which proteins or phosphorylation sites are functionally regulated in a given system as well as the mechanisms by which this regulation may occur.

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利用集成热转移分析法剖析 T 细胞活化过程中的蛋白质和磷酸化位点

T 细胞活化是幼稚细胞成熟为效应细胞的复杂生物过程。蛋白质组学和磷酸化蛋白质组学方法为这一过程提供了重要的洞察力，但并不总是清楚单个蛋白质或磷酸化位点的变化具有怎样的功能意义。在这里，我们开发了磷酸化整合热转移测定（PITSA），它将蛋白质或磷酸化位点丰度和热稳定性的测量结合到一个单一的 TMT 实验中，并将这种方法应用于研究 T 细胞活化。我们量化了 7,500 多种蛋白质和 5,000 个磷酸化位点的丰度和热稳定性，并确定了染色质相关、TCR 信号转导、DNA 修复和增殖性磷酸化蛋白的显著差异。PITSA 可广泛应用于各种生物环境，就特定系统中哪些蛋白质或磷酸化位点受到功能调控以及这种调控可能发生的机制提出假设。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Molecular & Cellular Proteomics 生物-生化研究方法

CiteScore

11.50

自引率

4.30%

发文量

131

审稿时长

84 days

期刊介绍： The mission of MCP is to foster the development and applications of proteomics in both basic and translational research. MCP will publish manuscripts that report significant new biological or clinical discoveries underpinned by proteomic observations across all kingdoms of life. Manuscripts must define the biological roles played by the proteins investigated or their mechanisms of action. The journal also emphasizes articles that describe innovative new computational methods and technological advancements that will enable future discoveries. Manuscripts describing such approaches do not have to include a solution to a biological problem, but must demonstrate that the technology works as described, is reproducible and is appropriate to uncover yet unknown protein/proteome function or properties using relevant model systems or publicly available data. Scope: -Fundamental studies in biology, including integrative "omics" studies, that provide mechanistic insights -Novel experimental and computational technologies -Proteogenomic data integration and analysis that enable greater understanding of physiology and disease processes -Pathway and network analyses of signaling that focus on the roles of post-translational modifications -Studies of proteome dynamics and quality controls, and their roles in disease -Studies of evolutionary processes effecting proteome dynamics, quality and regulation -Chemical proteomics, including mechanisms of drug action -Proteomics of the immune system and antigen presentation/recognition -Microbiome proteomics, host-microbe and host-pathogen interactions, and their roles in health and disease -Clinical and translational studies of human diseases -Metabolomics to understand functional connections between genes, proteins and phenotypes