Trenchevska Olgica, Sherma Nisha D, Oran Paul E, Reaven Peter D, Nelson Randall W, Nedelkov Dobrin
The Biodesign Institute at Arizona State University, Tempe, AZ 85287, United States.
The Biodesign Institute at Arizona State University, Tempe, AZ 85287, United States.
J Proteomics. 2015 Feb 26;116:15-23. doi: 10.1016/j.jprot.2014.12.011. Epub 2014 Dec 27.
The chemokine RANTES plays a key role in inflammation, cell recruitment and T cell activation. RANTES is heterogenic and exists as multiple variants in vivo. Herein we describe the development and characterization of a fully quantitative mass spectrometric immunoassay (MSIA) for analysis of intact RANTES and its proteoforms in human serum and plasma samples. The assay exhibits linearity over a wide concentration range (1.56-200ng/mL), intra- and inter-assay precision with CVs <10%, and good linearity and recovery correlations. The assay was tested in different biological matrices, and it was benchmarked against an existing RANTES ELISA. The new RANTES MSIA was used to analyze RANTES and its proteoforms in a small clinical cohort, revealing the quantitative distribution and frequency of the native and truncated RANTES proteoforms.
In the last two decades, RANTES has been studied extensively due to its association with numerous clinical conditions, including kidney-related, autoimmune, cardiovascular, viral and metabolic pathologies. Although a single gene product, RANTES is expressed in a range of cells and tissues presenting with different endogenously produced variants and PTMs. The structural variety and population diversity that has been identified for RANTES necessitate developing advanced methodologies that can provide insight into the protein heterogeneity and its function and regulation in disease. In this work we present a simple, efficient and high-throughput mass spectrometric immunoassay (MSIA) method for analysis of RANTES proteoforms. RANTES MSIA can detect and analyze RANTES proteoforms and provide an insight into the endogenous protein modifications.
趋化因子RANTES在炎症、细胞募集和T细胞活化中起关键作用。RANTES具有异质性，在体内以多种变体形式存在。在此，我们描述了一种用于分析人血清和血浆样本中完整RANTES及其蛋白变体的全定量质谱免疫分析（MSIA）方法的开发与特性。该分析方法在较宽浓度范围（1.56 - 200ng/mL）内呈线性，批内和批间精密度的变异系数（CV）<10%，且具有良好的线性和回收率相关性。该分析方法在不同生物基质中进行了测试，并与现有的RANTES酶联免疫吸附测定（ELISA）进行了比对。新的RANTES MSIA用于分析一个小型临床队列中的RANTES及其蛋白变体，揭示了天然和截短的RANTES蛋白变体的定量分布和频率。
在过去二十年中，由于RANTES与多种临床病症相关，包括肾脏相关、自身免疫、心血管、病毒和代谢性疾病，因此对其进行了广泛研究。尽管RANTES是单一基因产物，但它在一系列细胞和组织中表达，呈现出不同的内源性产生的变体和翻译后修饰（PTM）。已确定的RANTES的结构多样性和群体多样性使得有必要开发先进的方法，以深入了解蛋白质异质性及其在疾病中的功能和调控。在这项工作中，我们提出了一种简单、高效且高通量的质谱免疫分析（MSIA）方法来分析RANTES蛋白变体。RANTES MSIA可以检测和分析RANTES蛋白变体，并深入了解内源性蛋白质修饰。
