Università degli Studi di Bari, Centro Interdipartimentale di Ricerca S.M.A.R.T., Dipartimento di Chimica, Via Orabona, 4, 70100 Bari, Italy.
J Pharm Biomed Anal. 2010 Mar 11;51(4):907-14. doi: 10.1016/j.jpba.2009.10.014. Epub 2009 Oct 30.
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.
生物体液（如尿液）中的蛋白质分析采用质谱法（MS），但在样本采集、储存和制备方面的方案缺乏标准化。在这项工作中，研究了这些变量对健康供体人尿液蛋白质谱分析的影响，采用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）。对各种尿液样本预处理程序和不同样本沉积方法对 MALDI 靶的影响进行了筛选。通过主成分分析（PCA）评估了尿液样本储存时间和温度对光谱谱图的影响。最终，将整个优化程序应用于前列腺癌患者的人尿液样本的 MALDI-TOF-MS 分析。采用自制的亲水-亲脂平衡（HLB）树脂填充微柱作为样品预处理方法，获得了数量最多的检测离子和 MS 谱图中的丰度，效果最佳；该方法还更便宜且适合高通量分析。然后，对 MALDI 靶板上样品沉积的旋转涂覆方法进行了优化，获得了均匀且可重现的斑点。然后，PCA 表明，酸化和离心后的样品低温储存，以及短的处理时间，可在不引入实验条件artifact 的情况下获得重现性好的图谱。最后，通过比较健康供体和前列腺癌患者的混合尿液样本的 MALDI-TOF-MS 蛋白质谱，发现了有趣的差异。结果表明，分析和前分析变量对于尿液分析的成功至关重要，以获得有意义且可重现的数据，即使患者内的变异性很难避免。已经证明，混合尿液样本可以是一种很有意义的方法，可以简化健康和病理样本之间的比较，并确定两组样本之间蛋白质表达的可能差异。
