Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS San Raffaele Pisana, Rome, Italy.
J Proteomics. 2010 Jan 3;73(3):667-77. doi: 10.1016/j.jprot.2009.09.006. Epub 2009 Sep 18.
Biological specimen collection and storage are an integral component of serum proteomics research. Although many efforts have been posed to address the effects of pre-analytical procedures, standardized protocols for collection and storage of samples for Low Molecular Weight (LMW) proteome profiling are still needed. Here we report a systematic analysis on the influence of pre-analytical factors [clotting times, temperature and time storage, addition of protease inhibitor (PI)] on serum LMW proteome profiling. Moreover, a comparison between manual versus automated peptide purification by functionalized magnetic bead-based MALDI-MS approach was performed. The results demonstrated best serum LMW proteins recovery and stability using a clotting time between 1 and 2h, with serum stored up to 2h either at room temperature or at 4 degrees C, independently of PI addition. PI addition to whole blood resulted in a lower number of LMW peaks detected. Finally, minimal effects on serum proteome profiles were observed after 1-month storage at -80 degrees C, independently of PI addition on whole blood and/or serum. In conclusion, the use of standardized pre-analytical and storage procedures together with an automated peptide purification might minimize potential bias on serum LMW profiling results, thus allowing a better homogeneity and reproducibility in future proteomics studies.
生物标本的采集和储存是血清蛋白质组学研究的一个组成部分。尽管已经做出了许多努力来解决分析前程序的影响，但仍需要标准化的协议来收集和储存用于低分子量（LMW）蛋白质组分析的样本。在这里，我们报告了一项关于分析前因素（凝血时间、温度和储存时间、蛋白酶抑制剂（PI）的添加）对血清 LMW 蛋白质组分析的影响的系统分析。此外，还比较了基于功能化磁性珠的 MALDI-MS 方法的手动与自动肽纯化。结果表明，在 1 到 2 小时的凝血时间内，使用凝血时间为 1 到 2 小时，可以获得最佳的血清 LMW 蛋白质回收率和稳定性，血清可以在室温下或 4 摄氏度下储存长达 2 小时，而无需添加 PI。PI 添加到全血中会导致检测到的 LMW 峰数量减少。最后，在 -80 摄氏度下储存 1 个月后，血清蛋白质组图谱几乎没有变化，无论全血和/或血清中是否添加 PI。总之，使用标准化的分析前和储存程序以及自动肽纯化可以最大限度地减少血清 LMW 分析结果中的潜在偏差，从而在未来的蛋白质组学研究中实现更好的均一性和重现性。
