Department of Neurology, Erasmus University Medical Center , 3000 CA Rotterdam, The Netherlands.
J Proteome Res. 2014 Jun 6;13(6):2807-17. doi: 10.1021/pr401232e. Epub 2014 May 1.
In biological samples, proteins and peptides are altered by proteolytic activity. The actual ex vivo form of the peptidome or proteome analyzed, therefore, does not always reflect the natural in vivo state. Sample stabilization and sample treatment are thereby decisive for how far these two states diverge. To assess ex vivo formation of peptides, we used enzymatic incorporation of oxygen-18 water during proteolysis (PALeO approach) to label ex-vivo-formed peptides in rodent brain tissue. Rates of ex-vivo-formed peptides were determined in 25 samples that were stabilized and treated by six different protocols, whereby samples were subjected to different conditions such as temperature, urea concentration, and duration of treatment. Samples were measured by nano LC-Orbitrap-MS, and incorporation of oxygen-18 was determined by MS/MS database search and analysis of the precursor isotope pattern. Extent of ex vivo degradations was affected relevantly by the sample treatment protocol applied and stopped almost completely by heat stabilization. Determination of the formation state by oxygen-18 incorporation by MS/MS database search correlated well to more elaborate analysis of the MS isotope pattern. Overall, oxygen-18 labeling in combination with shotgun data-acquisition and MS/MS database search offers an adjuvant and easily applicable tool to monitor sample quality and fidelity in peptide and neuropeptide sample preparations.
在生物样本中，蛋白质和肽会被蛋白水解活性改变。因此，实际分析的肽组或蛋白质组的外显形式并不总是反映自然的体内状态。样品的稳定和处理对于这两种状态的差异程度具有决定性作用。为了评估肽的体外形成，我们使用蛋白酶解过程中的氧-18 水的酶促掺入（PALeO 方法）来标记啮齿动物脑组织中的体外形成的肽。通过 6 种不同方案稳定和处理的 25 个样本中测定了体外形成的肽的速率，其中样本经历了不同的条件，如温度、脲浓度和处理时间。通过纳升 LC-Orbitrap-MS 进行样本测量，并通过 MS/MS 数据库搜索和分析前体同位素模式来确定氧-18 的掺入。体外降解的程度受应用的样品处理方案的影响，通过热稳定几乎完全停止。通过 MS/MS 数据库搜索确定的氧-18 掺入形成状态与对 MS 同位素模式的更详细分析相关性良好。总的来说，氧-18 标记与 shotgun 数据采集和 MS/MS 数据库搜索相结合，为监测肽和神经肽样品制备中的样品质量和保真度提供了一种辅助且易于应用的工具。
