Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles
Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles
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DOI：
10.1016/j.jpba.2009.10.014
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发表时间：
2010-03-11
影响因子：
3.4
通讯作者：
Bettocchi, C.
中科院分区：
文献类型：
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作者：
Calvano, C. D.;Aresta, A.;Bettocchi, C.
利用质谱法（MS）对生物液体（如尿液）中的蛋白质进行分析，由于样品收集、储存和制备的规程缺乏标准化，仍然存在问题。在这项工作中，研究了这些变量对基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）进行的健康供体人尿蛋白谱分析的影响。对各种尿样预处理程序和不同的MALDI靶样沉积方法进行了筛选。采用主成分分析（PCA）评价了尿液样品保存时间和温度对光谱分布的影响。整个优化过程最终应用于前列腺癌患者尿液样本的MALDI-TOF-MS分析。采用亲水-亲脂平衡（HLB）树脂填充的自制微柱作为样品前处理方法，在质谱中检测到的离子数量和丰度最好；该方法也较便宜，适合于高通量分析。然后，优化了在MALDI靶板上沉积样品的自旋镀膜方法，获得了均匀且可重现的光斑。然后，主成分分析表明，酸化和离心样品的低储存温度以及较短的处理时间可以获得可重复的剖面，而不受实验条件的影响。最后，通过比较健康供者和前列腺癌患者汇集的尿液样本的MALDI-TOF-MS蛋白谱，发现了有趣的差异。结果表明，分析和分析前变量对于尿液分析的成功至关重要，即使患者内部变异性很难避免，也可以获得有意义和可重复的数据。事实证明，汇集尿液样本是一种有趣的方法，可以更容易地比较健康样本和病理样本，并使两组样本之间的蛋白质表达可能存在差异。(C) 2009 Elsevier B.V.版权所有
Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied.A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients.The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients.The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples. (C) 2009 Elsevier B.V. All rights reserved.