High dynamic range characterization of the trauma patient plasma proteome
High dynamic range characterization of the trauma patient plasma proteome
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DOI：
10.1074/mcp.m600068-mcp200
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发表时间：
2006-10-01
影响因子：
7
通讯作者：
Smith, Richard D.
中科院分区：
文献类型：
--
作者：
Liu, Tao;Qian, Wei-Jun;Smith, Richard D.
尽管人血浆代表了用于疾病生物标志物发现的有吸引力的样品，但蛋白质浓度的极端复杂性和大的动态范围对表征、候选生物标志物发现和验证提出了重大挑战。在这里，我们描述了一种策略，结合免疫亲和减法和随后的化学分馏的基础上半胱氨酰肽和N-糖肽捕获与二维LC-MS/MS，以增加血浆分析的动态范围。将这种“分而治之”策略应用于创伤患者血浆显著提高了检测的总体动态范围，并导致从四种不同的肽群（半胱氨酰肽、非半胱氨酰肽、N-糖肽和非糖肽）中可靠地鉴定出22，267种独特的肽，所述肽群覆盖3654种不同的蛋白质，其中1494种蛋白质由多种肽鉴定。鉴定了许多低丰度蛋白质，例如78种“经典”细胞因子和细胞因子受体以及136种人类细胞分化分子。此外，共鉴定了2910种不同的N-糖肽，对应于662个N-糖蛋白和1553个N-糖基化位点。在这项研究中鉴定的一组蛋白质已知与炎症和免疫反应有关。这项研究建立了一个广泛的参考蛋白质数据库创伤患者，提供了一个基础，为未来的高通量定量血浆蛋白质组学研究，旨在阐明全身炎症反应的机制。
Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3654 different proteins with 1494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.