Protein biomarkers are essential in assessing pathogenic processes. The impetus for finding new biomarkers has been accelerated by the arrival of the “omics” technologies. However, equally important is the re-discovery of existing biomarkers with these new approaches as novel variants can be discovered that can improve their utility. Presented here is a mass spectrometric immunoassay method for quantitative determination of beta-2-microglobulin - an established biomarker used in the diagnosis of active rheumatoid arthritis and kidney disease, and its structural variant - cleaved at and deficient in lysine-58 (ΔK58-b2m). Beta lactoglobulin was incorporated into the assay as an internal reference standard, serving as normalization point for beta-2-microglobulin quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing beta-2-microglobulin ELISA. The assay was utilized to determine the individual concentration of beta-2-microglobulin and its variant across a larger cohort of samples, demonstrating the ability to simultaneously quantify both proteins.

Transthyretin (TTR, or prealbumin) is a tetrameric protein found in plasma and cerebrospinal fluid. Its major role is to transport thyroid hormones (thyroxin-T4) and retinol (through association with retinol-binding protein). TTR has been studied extensively, due to the great number of point mutations that result in sequence heterogeneity. Many of these variants are associated with pathological conditions that result in extracellular deposition of amyloid fibers in tissues. In this work, we have developed a rapid mass spectrometric immunoassay for determination and quantification of TTR and its variants from human serum and plasma samples. The assay was fully characterized in terms of its precision, linearity and recovery characteristics. The new assay was also compared with a conventional TTR ELISA. Furthermore, we have applied the optimized method to analyze transthyretin and its modifications in 44 human plasma samples, and in the process, optimized a method for TTR proteolytic digestion and identification of point mutations.

Post-translational modifications and genetic variations give rise to protein variants that significantly increase human proteome complexity. Modified proteins also play an important role in biological processes. Mass spectrometry, coupled to immunoaffinity separations, provides an efficient mean for simultaneous detection and quantification of protein variants. In Mass Spectrometric Immunoassays, affinity interactions are utilized to selectively retrieve specific proteins from complex biological fluids, and are followed by mass spectrometric analysis of the targeted proteins. These assays are similar to traditional enzyme immunoassays in that they use antibodies as reagents for affinity retrieval of specific proteins, but instead of enzymatic reaction for indirect protein detection, mass spectrometric analysis is used for direct identification of the proteins and their modifications. Described here are mass spectrometric immunoassays for targeted quantitative proteomics analysis of specific protein modifications. Assays for several protein biomarkers exhibiting posttranslational modifications were developed, including beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin. The following assay performance characteristics were examined (and determined): LOQs (sufficient for quantification of normal/healthy levels of the proteins from human serum or plasma); intra- and inter-assay precision (CVs of <10%), linearity and spiking recovery (90-110% range); specificity (<5% cross reactivity with other proteins), and correlation with ELISA-generated results (acceptable Passing & Bablok fits and Cusum linearity p-values of >0.1). The new assays were then utilized to determine the individual concentration of the specific protein variants across larger cohorts of samples (n=500), demonstrating the ability to fully quantify individual forms of post-translationally modified proteins. The mass spectrometric immunoassays are ideally suited for detection and quantification of novel protein variants, not known a priori. These MS-based assays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5114. doi:10.1158/1538-7445.AM2011-5114