Quantitative mass spectrometric immunoassay for the chemokine RANTES and its variants.

Abstract

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.

BIOLOGICAL SIGNIFICANCE:

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.

• Authors: Olgica Trenchevska, Nisha D Sherma, Paul E Oran, Peter D Reaven, Randall W Nelson, Dobrin Nedelkov
• Journal: Journal of proteomics (J Proteomics) Vol. 116 Pg. 15-23 (Feb 26 2015) ISSN: 1876-7737 [Electronic] Netherlands
• PMID: 25549571 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: Published by Elsevier B.V.

Chemical References

• CCL5 protein, human
• Chemokine CCL5
• Protein Isoforms

Topics

• Chemokine CCL5 (analysis, metabolism)
• Enzyme-Linked Immunosorbent Assay (methods)
• Humans
• Mass Spectrometry (methods)
• Protein Isoforms (analysis, metabolism)