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 -200 ng/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.

Development of quantitative mass spectrometric immunoassay for serum amyloid A

“…The proteoforms were, thus identified due to the following: 1) knowledge of the amino acid sequence of the SAA and 2) knowledge/estimation of the PTMs for SAA. We have done such assignments in numerous previously published studies (Trenchevska et al, 2011, Trenchevska et al, 2015a, Trenchevska et al, 2015b). Using this approach we determined the SAA protein profile and concentrations of several SAA proteoforms in 170 human plasma samples.…”

Mass Spectrometric Immunoassays in Characterization of Clinically Significant Proteoforms

“…The IRS should also produce signals in the mass spectra that are in close proximity of the targeted protein signal, so that the same MS acquisition parameters can be used for both proteins. Another approach is to use homologous proteins from other animal species (such as equine protein) [49], or protein derivatives (His-tag modified proteins, or methionine (Met) modified proteins) [50,51] as IRS. These homologs are recognized by the anti-human protein antibody and are spiked into the analytical sample.…”

Mass Spectrometric Immunoassays for Discovery, Screening and Quantification of Clinically Relevant Proteoforms

“…We have recently developed a mass spectrometric immunoassay for analysis of the RANTES proteoforms [39]. Representative mass spectrum resulting from the application of the assay to a human plasma sample is shown in Figure 3.…”

“…The quantitative RANTES mass spectrometric immunoassay was applied to analysis of plasma samples obtained from 297 individuals with Type 2 diabetes (not treated with DPP IV inhibitor medication) [39]. The assays were performed on an automated high-throughput platform, 96 samples at a time.…”