肽的质谱分析使得它们的精确质量的分配和肽的氨基酸序列的全部或显著部分的确认成为可能。LC-MS/MS分析已被证明在肽组学研究中具有无价价值,可以识别新的生物标志物并分配其循环浓度以帮助研究疾病过程。然而,由于高背景血浆蛋白含量,它掩盖了天然低丰度循环肽组的存在,因此,在质谱分析之前从血浆中提取肽是至关重要的。有机溶剂有效地沉淀这些高分子量血浆蛋白,同时在溶液中留下小分子量肽,提供了一种快速有效的技术,用于从污染的血浆蛋白中分离肽。在LC-MS/MS分析之前,需要涉及固相萃取的二次净化步骤以除去脂质和高度疏水性污染物。本章中描述的方法对于在LC-MS/MS分析之前富集循环血浆肽是有效的,并且已经用于多个肽研究以改善肽检测和定量。使用这种方法研究的肽包括胰岛素,C-肽,胰高血糖素,PYY,GIP,和许多其他具有挑战性的肠道肽激素。使用所述方法对肽的定量分析显示出与现有免疫测定的良好相关性。
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.