Cai Jiaxi, Quan Yun, Zhang Cindy Yuxuan, Wang Ziyi, Hinshaw Stephen M, Zhou Huilin, Suhandynata Raymond T
https://ror.org/0168r3w48 Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, CA, USA.
https://ror.org/0168r3w48 Department of Bioengineering, University of California, San Diego, San Diego, CA, USA.
Life Sci Alliance. 2024 Dec 31;8(3). doi: 10.26508/lsa.202403007. Print 2025 Mar.
Large multiprotein machines are central to many biological processes. However, stoichiometric determination of protein complex subunits in their native states presents a significant challenge. This study addresses the limitations of current tools in accuracy and precision by introducing concatemer-assisted stoichiometry analysis (CASA). CASA leverages stable isotope-labeled concatemers and liquid chromatography-parallel reaction monitoring-mass spectrometry (LC-PRM-MS) to achieve robust quantification of proteins with sub-femtomole sensitivity. As a proof of concept, CASA was applied to study budding yeast kinetochores. Stoichiometries were determined for ex vivo reconstituted kinetochore components, including the canonical H3 nucleosomes, centromeric (Cse4) nucleosomes, centromere proximal factors (Cbf1 and CBF3 complex), inner kinetochore proteins (Mif2, Ctf19 complex), and outer kinetochore proteins (KMN network). Absolute quantification by CASA revealed Cse4 as a cell cycle-controlled limiting factor for kinetochore assembly. These findings demonstrate that CASA is applicable for stoichiometry analysis of multiprotein assemblies.
大型多蛋白机器在许多生物过程中起着核心作用。然而，在天然状态下对蛋白质复合物亚基进行化学计量测定面临重大挑战。本研究通过引入串联体辅助化学计量分析（CASA）解决了当前工具在准确性和精密度方面的局限性。CASA利用稳定同位素标记的串联体和液相色谱 - 平行反应监测 - 质谱（LC - PRM - MS）实现对亚飞摩尔灵敏度蛋白质的可靠定量。作为概念验证，CASA被应用于研究出芽酵母动粒。确定了体外重组动粒组分的化学计量，包括典型的H3核小体、着丝粒（Cse4）核小体、着丝粒近端因子（Cbf1和CBF3复合物）、内动粒蛋白（Mif2、Ctf19复合物）和外动粒蛋白（KMN网络）。通过CASA进行的绝对定量揭示Cse4是动粒组装的细胞周期控制限制因子。这些发现表明CASA适用于多蛋白组装体的化学计量分析。
