The Effect of Preanalytical Factors on Stability of the Proteome and Selected Metabolites in Cerebrospinal Fluid (CSF)
The Effect of Preanalytical Factors on Stability of the Proteome and Selected Metabolites in Cerebrospinal Fluid (CSF)
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DOI：
10.1021/pr9005876
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发表时间：
2009-12-01
影响因子：
4.4
通讯作者：
Bischoff, Rainer
中科院分区：
文献类型：
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作者：
Rosenling, Therese;Slim, Christiaan L.;Bischoff, Rainer
为了标准化脑脊液 (CSF) 在生物标志物研究中的使用，对猪样品进行了一系列稳定性研究，以研究常见样品处理程序对蛋白质、肽、代谢物和游离氨基酸的影响。本研究重点关注对蛋白质和肽的影响，通过使用微流体纳米级液相色谱与四极杆飞行时间质谱 (chipLC-MS) 以及基质辅助激光解吸电离傅立叶变换离子回旋共振质谱 (MALDI-FT-ICR-MS) 和 Orbitrap LC-MS/MS 进行无标记定量分析，对胰蛋白酶消化的脑脊液样本进行分析，评估的因素为 30 或收集脑脊液后，在室温下延迟 120 分钟，然后在 -80 摄氏度下储存，以模拟诊所中潜在的延迟（延迟储存），胰蛋白酶消化后在 4 摄氏度下储存，以模拟样品在分析仪冷却自动进样器中保留的时间，并重复冻融循环以模拟实验室中的储存和处理程序。还通过气相色谱质谱法（GC-MS）和液相色谱质谱法（LC-MS）分别分析延迟储存因子中代谢物和游离氨基酸的变化。我们的结果表明，重复冷冻/解冻会导致转甲状腺素蛋白肽水平发生变化。自动进样器中 4 摄氏度下的胰蛋白酶消化样品显示，前列腺素 D-合酶和血清转铁蛋白的肽峰面积随时间而减少。脑脊液的延迟储存导致前列腺素 D-合酶衍生肽的变化以及某些氨基酸和代谢物水平的增加。延迟储存研究中代谢物、氨基酸和蛋白质的变化似乎与剩余的白细胞有关。我们的建议是在收集后立即离心脑脊液样本以去除白细胞，等分，然后将上清液快速冷冻在液氮中，以便在-80°C下储存。最好不要将样本留在自动进样器中超过24小时，并且应尽可能避免冷冻/解冻循环。
To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples, The factors assessed were a 30 or 120 min time delay at room temperature before storage at -80 degrees C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 degrees C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze-thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 degrees C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at -80 degrees C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.