title: 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.
  
  
  CONCLUSIONS
  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.affiliation: The Biodesign Institute at Arizona State University, Tempe, AZ 85287, United States. Electronic address: olgica.trenchevska@asu.edu.
authors.forename: Olgica
authors.initials: O
authors.lastname: Trenchevska
authors.affiliation: The Biodesign Institute at Arizona State University, Tempe, AZ 85287, United States.
authors.forename: Nisha D
authors.initials: ND
authors.lastname: Sherma
authors.affiliation: The Biodesign Institute at Arizona State University, Tempe, AZ 85287, United States.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Oran
authors.affiliation: Phoenix VA Health Care System, United States.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Reaven
authors.affiliation: The Biodesign Institute at Arizona State University, Tempe, AZ 85287, United States.
authors.forename: Randall W
authors.initials: RW
authors.lastname: Nelson
authors.affiliation: The Biodesign Institute at Arizona State University, Tempe, AZ 85287, United States.
authors.forename: Dobrin
authors.initials: D
authors.lastname: Nedelkov
mesh_terms: D018946:Chemokine CCL5 / Q000032:analysis* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism
chemical_list: C483024:CCL5 protein, human; D018946:Chemokine CCL5; D020033:Protein Isoforms
keywords: Chemokine; Mass spectrometric immunoassay; Quantitative analysis; RANTES