Gegner Hagen M, Naake Thomas, Dugourd Aurélien, Müller Torsten, Czernilofsky Felix, Kliewer Georg, Jäger Evelyn, Helm Barbara, Kunze-Rohrbach Nina, Klingmüller Ursula, Hopf Carsten, Müller-Tidow Carsten, Dietrich Sascha, Saez-Rodriguez Julio, Huber Wolfgang, Hell Rüdiger, Poschet Gernot, Krijgsveld Jeroen
Centre for Organismal Studies (COS), Metabolomics Core Technology Platform, University of Heidelberg, Heidelberg, Germany.
Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
Front Mol Biosci. 2022 Dec 20;9:961448. doi: 10.3389/fmolb.2022.961448. eCollection 2022.
Metabolomic and proteomic analyses of human plasma and serum samples harbor the power to advance our understanding of disease biology. Pre-analytical factors may contribute to variability and bias in the detection of analytes, especially when multiple labs are involved, caused by sample handling, processing time, and differing operating procedures. To better understand the impact of pre-analytical factors that are relevant to implementing a unified proteomic and metabolomic approach in a clinical setting, we assessed the influence of temperature, sitting times, and centrifugation speed on the plasma and serum metabolomes and proteomes from six healthy volunteers. We used targeted metabolic profiling (497 metabolites) and data-independent acquisition (DIA) proteomics (572 proteins) on the same samples generated with well-defined pre-analytical conditions to evaluate criteria for pre-analytical SOPs for plasma and serum samples. Time and temperature showed the strongest influence on the integrity of plasma and serum proteome and metabolome. While rapid handling and low temperatures (4°C) are imperative for metabolic profiling, the analyzed proteomics data set showed variability when exposed to temperatures of 4°C for more than 2 h, highlighting the need for compromises in a combined analysis. We formalized a quality control scoring system to objectively rate sample stability and tested this score using external data sets from other pre-analytical studies. Stringent and harmonized standard operating procedures (SOPs) are required for pre-analytical sample handling when combining proteomics and metabolomics of clinical samples to yield robust and interpretable data on a longitudinal scale and across different clinics. To ensure an adequate level of practicability in a clinical routine for metabolomics and proteomics studies, we suggest keeping blood samples up to 2 h on ice (4°C) prior to snap-freezing as a compromise between stability and operability. Finally, we provide the methodology as an open-source R package allowing the systematic scoring of proteomics and metabolomics data sets to assess the stability of plasma and serum samples.
对人体血浆和血清样本进行代谢组学和蛋白质组学分析，有助于深化我们对疾病生物学的理解。分析前因素可能导致分析物检测的变异性和偏差，尤其是在涉及多个实验室时，这是由样本处理、处理时间和不同的操作程序引起的。为了更好地理解与在临床环境中实施统一的蛋白质组学和代谢组学方法相关的分析前因素的影响，我们评估了温度、静置时间和离心速度对六名健康志愿者的血浆和血清代谢组及蛋白质组的影响。我们在具有明确分析前条件的相同样本上使用靶向代谢谱分析（497种代谢物）和数据非依赖采集（DIA）蛋白质组学（572种蛋白质），以评估血浆和血清样本分析前标准操作规程（SOP）的标准。时间和温度对血浆和血清蛋白质组及代谢组的完整性影响最大。虽然快速处理和低温（4°C）对于代谢谱分析至关重要，但分析的蛋白质组学数据集在暴露于4°C温度超过2小时时显示出变异性，这突出了在联合分析中需要做出妥协。我们制定了一个质量控制评分系统，以客观地评估样本稳定性，并使用来自其他分析前研究的外部数据集对该评分进行了测试。在对临床样本进行蛋白质组学和代谢组学联合分析时，为了在纵向尺度上和不同诊所获得可靠且可解释的数据，分析前样本处理需要严格且统一的标准操作规程（SOP）。为了确保代谢组学和蛋白质组学研究在临床常规操作中有足够的实用性，我们建议在速冻之前将血样在冰上（4°C）保存长达2小时，这是稳定性和可操作性之间的一种折衷。最后，我们将该方法作为一个开源R包提供，允许对蛋白质组学和代谢组学数据集进行系统评分，以评估血浆和血清样本的稳定性。
