West-Nørager Mikkel, Kelstrup Christian Dahl, Schou Christian, Høgdall Estrid V, Høgdall Claus K, Heegaard Niels H H
Department of Autoimmunology, Statens Serum Institut, DK-2300 Copenhagen S, Denmark.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Feb 15;847(1):30-7. doi: 10.1016/j.jchromb.2006.09.048. Epub 2006 Nov 16.
The use of mass spectrometry (MS) for analysing low-molecular weight proteins and peptides from biological fluids has a great, yet not fully realized, potential for biomarker discovery. To prune MS-data as much as possible for non-relevant non-biological variation the development of standardized protocols for handling and processing the samples before MS and adjusting data after MS to compensate for method-induced variability are warranted. This calls for knowledge about how different variables contribute to MS-based proteome analyses. In addition, identification of the peptides involved in pre-analytical variation will be helpful in evaluating the clinical significance of predictive models derived from MS data. Using human sera, extraction by weak cation-exchange magnetic beads, and analysis by MALDI-TOF MS we here evaluated pre-analytical variation and identify peptides involved in this. The influences of humidity, temperature, and time for preparation of sera on spectral changes were evaluated. Also, the reproducibility of the methods and the effect of a baseline correction procedure were examined. Low temperatures, short handling times, and a baseline correction procedure minimize the contribution of artifacts to sample variability as observed by MS. The complement split product C3f and fragments thereof appear to be sensitive indicators of sample handling induced modifications. Other peptides that are indicative of such variability are fibrin and kininogen fragments. Using strict experimental guidelines as well as standardized sample collection procedures it is possible to obtain reproducible peak intensities and positions in serum mass profiling using magnetic bead-based fractionation and MALDI-TOF MS.
使用质谱法（MS）分析生物体液中的低分子量蛋白质和肽段，在生物标志物发现方面具有巨大潜力，但尚未得到充分发挥。为尽可能去除与生物无关的非相关变异的质谱数据，制定标准化方案来处理和加工质谱分析前的样本，并在质谱分析后调整数据以补偿方法诱导的变异性是很有必要的。这需要了解不同变量如何影响基于质谱的蛋白质组分析。此外，识别参与分析前变异的肽段将有助于评估从质谱数据得出的预测模型的临床意义。我们使用人血清、通过弱阳离子交换磁珠进行提取，并采用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行分析，在此评估了分析前的变异并识别了其中涉及的肽段。评估了湿度、温度和血清制备时间对光谱变化的影响。此外，还检查了方法的重现性以及基线校正程序的效果。低温、较短的处理时间和基线校正程序可将质谱观察到的假象对样本变异性的影响降至最低。补体裂解产物C3f及其片段似乎是样本处理诱导修饰的敏感指标。其他指示这种变异性的肽段是纤维蛋白和激肽原片段。使用严格的实验指南以及标准化的样本采集程序，通过基于磁珠的分级分离和MALDI-TOF MS进行血清质谱分析时，有可能获得可重现的峰强度和峰位置。
