来自中枢神经系统癌症的小儿患者的脑脊液的深度蛋白质组分析
Deep Proteome Analysis of Cerebrospinal Fluid from Pediatric Patients with Central Nervous System Cancer
影响因子:3.60000
分区:生物学2区 / 生化研究方法2区
发表日期:2024 Nov 01
作者:
Christian Mirian, Ole Østergaard, Maria Thastrup, Signe Modvig, Jon Foss-Skiftesvik, Jane Skjøth-Rasmussen, Marianne Berntsen, Josefine Britze, Alex Christian Yde Nielsen, René Mathiasen, Kjeld Schmiegelow, Jesper Velgaard Olsen
摘要
脑脊液（CSF）是发现与预后相关的生物标志物和小儿中枢神经系统恶性肿瘤治疗靶标相关的关键基质。但是，儿童的蛋白质浓度和与年龄相关的差异范围很大，因此具有挑战性。此外，小儿CSF样品通常是稀疏的，首先是出于临床目的的优先级。目前的工作着重于优化蛋白质组分析工作流程的每个步骤，以从可用于研究目的的有限的CSF资源中提取最详细的蛋白质组信息。该策略包括应用顺序超速离心以富含细胞外囊泡（EV）外，除了分析少量的RAW CSF外，从400μlCSF中允许定量1351蛋白（相对于RAW CSF）进行定量。当包括光谱库时，可以量化总共2103种蛋白质（+240％）。该工作流程针对CSF输入量，胰蛋白酶消化方法，梯度长度，质谱数据采集方法和数据库搜索策略进行了优化，以量化尽可能多的蛋白质。全面优化的工作流程包括蛋白质聚集捕获（PAC）消化，与数据无关的采集（DIA，21分钟梯度）配对，并允许仅从400μlCSF中量化2989个独特的蛋白质，与对RAW CSF的A型分析相比，蛋白质增加了340％。
Abstract
The cerebrospinal fluid (CSF) is a key matrix for discovery of biomarkers relevant for prognosis and the development of therapeutic targets in pediatric central nervous system malignancies. However, the wide range of protein concentrations and age-related differences in children makes such discoveries challenging. In addition, pediatric CSF samples are often sparse and first prioritized for clinical purposes. The present work focused on optimizing each step of the proteome analysis workflow to extract the most detailed proteome information possible from the limited CSF resources available for research purposes. The strategy included applying sequential ultracentrifugation to enrich for extracellular vesicles (EV) in addition to analysis of a small volume of raw CSF, which allowed quantification of 1351 proteins (+55% relative to raw CSF) from 400 μL CSF. When including a spectral library, a total of 2103 proteins (+240%) could be quantified. The workflow was optimized for CSF input volume, tryptic digestion method, gradient length, mass spectrometry data acquisition method and database search strategy to quantify as many proteins a possible. The fully optimized workflow included protein aggregation capture (PAC) digestion, paired with data-independent acquisition (DIA, 21 min gradient) and allowed 2989 unique proteins to be quantified from only 400 μL CSF, which is a 340% increase in proteins compared to analysis of a tryptic digest of raw CSF.