Development and Comparative Evaluation of Endolysosomal Proximity Labeling-Based Proteomic Methods in Human iPSC-Derived Neurons.
Development and Comparative Evaluation of Endolysosomal Proximity Labeling-Based Proteomic Methods in Human iPSC-Derived Neurons.
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DOI：
10.1021/acs.analchem.0c03107
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发表时间：
2020-12-01
影响因子：
7.4
通讯作者：
Hao L
中科院分区：
文献类型：
--
作者：
Frankenfield AM;Fernandopulle MS;Hasan S;Ward ME;Hao L
基于邻近性的原位标记技术提供了一种独特的方法来捕获稳定和瞬时的蛋白质-蛋白质和蛋白质-细胞器相互作用。将该技术与基于质谱 (MS) 的蛋白质组学相结合，使我们能够获得纳米分辨率的分子微环境快照，从而促进复杂动态蛋白质网络的发现。然而，仍然存在许多技术挑战，例如内源生物素化蛋白质和其他高丰度旁观者的干扰，如何选择适当的对照以尽量减少错误发现，以及生物/技术复制之间的实验差异。在这里，我们开发了一种新方法，通过将工程抗坏血酸过氧化物酶 (APEX) 基因敲入 (KI) 溶酶体相关膜蛋白 1 (LAMP1) 的内源基因座来捕获神经元内溶酶体网络的蛋白质组微环境。我们发现，将邻近标记蛋白质组数据标准化为内源生物素化蛋白质（PCCA）可以大大减少变异，并能够在不同批次的 APEX 标记和不同 APEX 探针之间进行公平比较。我们对这种 KI-LAMP1-APEX 方法和我们的两种过表达 LAMP1-APEX 探针进行了比较评估，实现了人类 iPSC 衍生神经元中已知和新的溶酶体膜和溶酶体相互作用蛋白的互补覆盖。总而言之，这项研究展示了表征人类神经元溶酶体功能和微环境的新分析工具，并填补了设计和优化邻近标记蛋白质组实验领域的关键空白。
Proximity-based in situ labeling techniques offer a unique way to capture both stable and transient protein–protein and protein–organelle interactions. Combining this technology with mass spectrometry (MS)-based proteomics allows us to obtain snapshots of molecular microenvironments with nanometer resolution, facilitating the discovery of complex and dynamic protein networks. However, a number of technical challenges still exist, such as interferences from endogenously biotinylated proteins and other highly abundant bystanders, how to select the proper controls to minimize false discoveries, and experimental variations among biological/technical replicates. Here, we developed a new method to capture the proteomic microenvironment of the neuronal endolysosomal network by knocking in (KI) an engineered ascorbate peroxidase (APEX) gene to the endogenous locus of lysosome-associated membrane protein 1 (LAMP1). We found that normalizing proximity labeling proteomics data to the endogenously biotinylated protein (PCCA) can greatly reduce variations and enable fair comparisons among different batches of APEX labeling and different APEX probes. We conducted a comparative evaluation between this KI-LAMP1-APEX method and our two overexpression LAMP1-APEX probes, achieving complementary coverage of both known and new lysosomal membrane and lysosomal-interacting proteins in human iPSC-derived neurons. To summarize, this study demonstrated new analytical tools to characterize lysosomal functions and microenvironment in human neurons and filled critical gaps in the field for designing and optimizing proximity labeling proteomic experiments.
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影响因子：
14.9
作者：
Deutsch EW;Csordas A;Sun Z;Jarnuczak A;Perez-Riverol Y;Ternent T;Campbell DS;Bernal-Llinares M;Okuda S;Kawano S;Moritz RL;Carver JJ;Wang M;Ishihama Y;Bandeira N;Hermjakob H;Vizcaíno JA
通讯作者：
Vizcaíno JA
影响因子：
4.4
作者：
Glatter, Timo;Ludwig, Christina;Schmidt, Alexander
通讯作者：
Schmidt, Alexander
影响因子：
13.3
作者：
Kissing, Sandra;Rudnik, Soenke;Saftig, Paul
通讯作者：
Saftig, Paul
DOI：
10.1007/s13361-011-0237-2
发表时间：
2011-12-01
影响因子：
3.2
作者：
Gokce, Emine;Shuford, Christopher M.;Muddiman, David C.
通讯作者：
Muddiman, David C.
影响因子：
7.7
作者：
Li, Jian;Pfeffer, Suzanne R.
通讯作者：
Pfeffer, Suzanne R.