Pumford Andrew D, Arul Albert B, Ford Katarena I, Robinson Renã A S
Vanderbilt Undergrad Res J. 2021 Apr 30;11:43-51. doi: 10.15695/vurj.v11i1.5096.
S-Nitrosylation (SNO) is a cysteine post-translational modification that increases with normal aging and is present in Alzheimer's disease and other aging-related illnesses. Detection of SNO-modified proteins can be challenging; however, we previously developed a robust quantitative proteomics approach termed "Oxidized Cysteine-Selective combined precursor isobaric labeling and isobaric tagging (OxcyscPILOT)" that allows for detection of endogenous SNO-modified proteins. OxcyscPILOT involves enrichment of SNO-modified proteins using a thiol-based resin. This enrichment is performed manually, and wash steps with the resin require numerous stages and buffer reagents. The goal of this study is to transfer the manual protocol to an automated liquid handler system in order to reduce wash steps, increase sample throughput, and minimize experimental error. In order to accomplish this, we evaluated the Biomek i7 liquid handler automated workstation and a Positive Pressure ALP (PPA) apparatus to conduct automated on-resin enrichment. Our findings provide starting pressure conditions for the use of PPA in an automated OxcyscPILOT proteomics workflow that could be transferred to other robotic liquid handling systems.
S-亚硝基化（SNO）是一种半胱氨酸的翻译后修饰，会随着正常衰老而增加，并且存在于阿尔茨海默病和其他与衰老相关的疾病中。检测SNO修饰的蛋白质可能具有挑战性；然而，我们之前开发了一种强大的定量蛋白质组学方法，称为“氧化半胱氨酸选择性联合前体等压标记和等压标签（OxcyscPILOT）”，该方法能够检测内源性SNO修饰的蛋白质。OxcyscPILOT包括使用基于硫醇的树脂富集SNO修饰的蛋白质。这种富集是手动进行的，并且用树脂进行的洗涤步骤需要多个阶段和缓冲试剂。本研究的目的是将手动方案转移到自动液体处理系统中，以减少洗涤步骤、提高样品通量并将实验误差降至最低。为了实现这一点，我们评估了Biomek i7液体处理自动工作站和正压碱性磷酸酶（PPA）装置，以进行树脂上的自动富集。我们的研究结果为在自动化OxcyscPILOT蛋白质组学工作流程中使用PPA提供了起始压力条件，该工作流程可转移到其他机器人液体处理系统中。
