Quantitation of peptides from non-invasive skin tapings using isotope dilution and tandem mass spectrometry.

Quantitation of peptides from non-invasive skin tapings using isotope dilution and tandem mass spectrometry.
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DOI:
10.1016/j.jchromb.2018.03.031
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发表时间:
2018-05-01
期刊:
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
影响因子:
--
通讯作者:
Reisdorph R
Reisdorph R
中科院分区:
其他
文献类型:
--
作者:
Reisdorph N;Armstrong M;Powell R;Quinn K;Legg K;Leung D;Reisdorph R

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我们实验室以前的工作利用了一种新的皮肤贴敷方法和基于质谱的蛋白质组学来发现皮肤疾病的临床生物标志物;这些包括特应性皮炎,金黄色葡萄球菌定植和疱疹性湿疹。虽然适合于发现目的,但半定量蛋白质组学通常耗时且昂贵。此外,根据所使用的方法,基于发现的蛋白质组学可能导致高变异和检测低丰度肽的灵敏度不足。因此,我们致力于开发一种快速,灵敏,可重复的方法来定量疾病相关的蛋白质从皮肤胶带。我们利用同位素标记的肽和串联质谱法获得绝对定量值的14肽从7种蛋白质,这些蛋白质已显示出以前的重要性,在皮肤疾病。当在0.05-2.5 pmol范围内分析n=3个标准品时,该方法表现出良好的重现性、动态范围和线性(R2 > 0.993)。该方法被用来确定是否存在差异皮肤蛋白质之间的一小群特应性与非特应性个体(n=12)。虽然仅发现最小的差异,但在所有样品中均检测到肽,并且7种蛋白质中的5种的肽之间表现出良好的相关性(R2 = 0.71 - 0.98)。这种方法可以应用于更大的群体,以进一步建立这些蛋白质与皮肤病的关系。
Previous work from our laboratories utilized a novel skin taping method and mass spectrometry-based proteomics to discover clinical biomarkers of skin conditions; these included atopic dermatitis, Staphylococcus aureus colonization, and eczema herpeticum. While suitable for discovery purposes, semi-quantitative proteomics is generally time-consuming and expensive. Furthermore, depending on the method used, discovery-based proteomics can result in high variation and inadequate sensitivity to detect low abundant peptides. Therefore, we strove to develop a rapid, sensitive, and reproducible method to quantitate disease-related proteins from skin tapings. We utilized isotopically-labeled peptides and tandem mass spectrometry to obtain absolute quantitation values on 14 peptides from 7 proteins; these proteins had shown previous importance in skin disease. The method demonstrated good reproducibility, dynamic range, and linearity (R2 > 0.993) when n=3 standards were analyzed across 0.05-2.5 pmol. The method was used to determine if differences exist between skin proteins in a small group of atopic versus non-atopic individuals (n=12). While only minimal differences were found, peptides were detected in all samples and exhibited good correlation between peptides for 5 of the 7 proteins (R2 = 0.71 – 0.98). This method can be applied to larger cohorts to further establish the relationships of these proteins to skin disease.
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