Rapid and Quantitative Enrichment of Peptides from Plasma for Mass Spectrometric Analysis.
Rapid and Quantitative Enrichment of Peptides from Plasma for Mass Spectrometric Analysis.
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快速定量富集血浆中的肽用于质谱分析。
DOI：
10.1007/978-1-0716-2978-9_28
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
George AL
中科院分区：
文献类型：
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作者：
George AL
通过对多肽进行质谱分析，可以确定它们的确切质量，并确认多肽的全部或很大一部分氨基酸序列。LC-MS/MS分析在多肽学研究中被证明是非常有价值的，可以识别新的生物标志物并指定它们的循环浓度，以帮助对疾病过程的研究。然而，由于背景血浆蛋白含量较高，掩盖了天然低丰度循环多肽的存在，因此在进行质谱分析之前从血浆中提取多肽是至关重要的。有机溶剂有效地沉淀了这些高相对分子质量的血浆蛋白，而将小分子多肽留在溶液中，为从污染的血浆蛋白中分离多肽提供了一种快速有效的技术。在LC-MS/MS分析之前，需要进行二次净化步骤，包括固相萃取，以去除脂类和高度疏水的污染物。本章中描述的方法在LC-MS/MS分析之前有效地丰富循环血浆多肽，并已被用于多个多肽研究，以改进多肽的检测和定量。使用这种方法研究的多肽包括胰岛素、C-肽、胰高血糖素、PYY、GIP和其他一些具有挑战性的肠肽激素。用所描述的方法对多肽进行定量分析，与现有的免疫分析方法有很好的相关性。
Mass spectrometric analysis of peptides enables the assignment of their exact mass and confirmation of all or a significant portion of the peptide’s amino acid sequence. LC-MS/MS analysis has proven invaluable in peptidomics research and can identify new biomarkers and assign their circulatory concentrations to aid research into disease processes. However, due to the high background plasma protein content, which masks the presence of the naturally low abundance circulatory peptidome, extraction of peptides from plasma prior to mass spectrometric analysis is therefore crucial. Organic solvents efficiently precipitate these high molecular weight plasma proteins while leaving small molecular weight peptides in solution, providing a rapid and effective technique for separating peptides from the contaminating plasma proteins. A secondary cleanup step involving solid phase extraction is required to remove lipids and highly hydrophobic contaminants before LC-MS/MS analysis. The method described within this chapter is effective at enriching circulatory plasma peptides prior to LC-MS/MS analysis and has been used in multiple peptidomic studies to improve peptide detection and quantification. Peptides studied using this methodology include insulin, C-peptide, glucagon, PYY, GIP, and a number of other challenging gut peptide hormones. Quantitative analyses of peptides using the described method showed good correlation with existing immunoassays.