Abstract：
The recent increase in peptidomimetic-based medications and the growing interest in peptide hormones has brought new attention to the quantification of peptides for diagnostic purposes. Indeed, the circulating concentrations of peptide hormones in the blood provide a snapshot of the state of the body and could eventually lead to detecting a particular health condition. Although extremely useful, the quantification of such molecules, preferably by liquid chromatography coupled to mass spectrometry, might be quite tricky. First, peptides are subjected to hydrolysis, oxidation, and other post-translational modifications, and, most importantly, they are substrates of specific and nonspecific proteases in biological matrixes. All these events might continue after sampling, changing the peptide hormone concentrations. Second, because they include positively and negatively charged groups and hydrophilic and hydrophobic residues, they interact with their environment; these interactions might lead to a local change in the measured concentrations. A phenomenon such as nonspecific adsorption to lab glassware or materials has often a tremendous effect on the concentration and needs to be controlled with particular care. Finally, the circulating levels of peptides might be low (pico- or femtomolar range), increasing the impact of the aforementioned effects and inducing the need for highly sensitive instruments and well-optimized methods. Thus, despite the extreme diversity of these peptides and their matrixes, there is a common challenge for all the assays: the need to keep concentrations unchanged from sampling to analysis. While significant efforts are often placed on optimizing the analysis, few studies consider in depth the impact of pre-analytical steps on the results. By working through practical examples, this solution-oriented tutorial review addresses typical pre-analytical challenges encountered during the development of a peptide assay from the standpoint of a clinical laboratory. We provide tips and tricks to avoid pitfalls as well as strategies to guide all new developments. Our ultimate goal is to increase pre-analytical awareness to ensure that newly developed peptide assays produce robust and accurate results.
摘要：
最近基于肽模拟物的药物的增加和对肽激素的日益增长的兴趣带来了对用于诊断目的的肽的定量的新关注。的确,血液中肽激素的循环浓度提供了身体状态的快照，并可能最终导致检测特定的健康状况。虽然非常有用，这些分子的量化，优选通过液相色谱与质谱联用，可能很棘手。首先,肽进行水解，氧化，和其他翻译后修饰，and,最重要的是,它们是生物基质中特异性和非特异性蛋白酶的底物。所有这些事件可能会在采样后继续，改变肽激素浓度。第二,因为它们包括带正电荷和负电荷的基团以及亲水和疏水残基，它们与环境相互作用；这些相互作用可能导致测量浓度的局部变化。诸如对实验室玻璃器皿或材料的非特异性吸附之类的现象通常对浓度有巨大影响，需要特别小心地控制。最后,肽的循环水平可能很低（皮卡或毫摩尔范围），增加上述影响的影响，并诱导需要高度敏感的仪器和优化的方法。因此，尽管这些肽和它们的基质极其多样，所有的分析都有一个共同的挑战:从取样到分析都需要保持浓度不变。虽然通常在优化分析上付出了巨大的努力，很少有研究深入考虑分析前步骤对结果的影响。通过实际例子，这篇面向解决方案的教程综述从临床实验室的角度解决了肽检测方法开发过程中遇到的典型分析前挑战.我们提供提示和技巧，以避免陷阱以及策略，以指导所有新的发展。我们的最终目标是提高分析前意识，以确保新开发的肽测定产生可靠和准确的结果。
