Zelle Sarah R, McDonald W Hayes, Rose Kristie L, Mchaourab Hassane S, Schey Kevin L
Chemical and Physical Biology Program, Vanderbilt University, Nashville, Tennessee.
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee.
bioRxiv. 2025 Mar 15:2025.03.13.642445. doi: 10.1101/2025.03.13.642445.
Ocular lens fiber cells degrade their organelles during differentiation to prevent light scattering. Organelle degradation occurs continuously throughout an individual's lifespan, creating a spatial gradient of young cortical fiber cells in the lens periphery to older nuclear fiber cells in the center of the lens. Therefore, separation of cortical and nuclear regions enables examination of protein aging. Previously, the human lens cortex and nucleus have been studied using data-independent acquisition (DIA) proteomics, allowing for the identification of low-abundance protein groups. In this study, we employed data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) proteomics on a timsTOF HT instrument to study the zebrafish lens proteome and compared results to a standard DIA method employed on an Orbitrap Exploris 480. Using the additional ion mobility gas phase separation of diaPASEF, peptide and protein group identifications increased by over 200% relative to an orbitrap DIA method in the zebrafish lens. With diaPASEF, we identified 13,721 and 11,996 unique peptides in the zebrafish lens cortex and nucleus, respectively, which correspond to 1,537 and 1,389 protein groups. Thus, separation of the zebrafish lens into cortical and nuclear regions followed by diaPASEF analysis produced the most comprehensive zebrafish lens proteomic dataset to date.
眼晶状体纤维细胞在分化过程中会降解其细胞器以防止光散射。细胞器降解在个体的整个生命周期中持续发生，在晶状体周边形成从年轻的皮质纤维细胞到晶状体中心较老的核纤维细胞的空间梯度。因此，皮质和核区域的分离使得能够研究蛋白质老化。此前，已使用数据非依赖采集（DIA）蛋白质组学对人晶状体皮质和核进行了研究，从而能够鉴定低丰度蛋白质组。在本研究中，我们在timsTOF HT仪器上采用数据非依赖采集平行累积-串联碎裂（diaPASEF）蛋白质组学来研究斑马鱼晶状体蛋白质组，并将结果与在Orbitrap Exploris 480上采用的标准DIA方法进行比较。利用diaPASEF额外的离子淌度气相分离，相对于斑马鱼晶状体中的轨道阱DIA方法，肽段和蛋白质组的鉴定增加了200%以上。通过diaPASEF，我们在斑马鱼晶状体皮质和核中分别鉴定出13,721个和11,996个独特肽段，分别对应1,537个和1,389个蛋白质组。因此，将斑马鱼晶状体分离为皮质和核区域，然后进行diaPASEF分析，产生了迄今为止最全面的斑马鱼晶状体蛋白质组数据集。
