Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum

Abstract

Objectives The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. Design and methods The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. Results In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67–0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. Conclusions We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.

Clinical translation of MS-based, quantitative plasma proteomics: status, challenges, requirements, and potential

“…In an example of the former, anti-protein antibodies were covalently bound to a monolithic column (as opposed to magnetic beads) [50] for protein enrichment and detection of the target proteins or peptides in a top-down [54] or bottom-up [55] manner, respectively. This immunoenrichment approach, referred to as a mass spectrometric immunoassay (MSIA ™ ), enabled the multiplexed targeted quantification of 16 plasma proteins (related to 7 different disease states, e.g., cardiovascular, Alzheimer's, and cancer) at concentrations ranging from mg/mL to pg/mL [50]. In a recent study, the MSIA-SRM approach was used to robustly and sensitively quantify several domains of PCSK9 (proprotein convertase subtilisin kexin 9) gainof-function mutations in which are responsible for some cases of familial hypercholesterolemia [56].…”

“…In general, anti-protein antibodies are incorporated at the beginning of an analytical workflow for protein enrichment [39,49,50], while anti-peptide antibodies are employed post-digestion for tryptic peptide enrichment [51][52][53] (see Figure 2). In an example of the former, anti-protein antibodies were covalently bound to a monolithic column (as opposed to magnetic beads) [50] for protein enrichment and detection of the target proteins or peptides in a top-down [54] or bottom-up [55] manner, respectively. This immunoenrichment approach, referred to as a mass spectrometric immunoassay (MSIA ™ ), enabled the multiplexed targeted quantification of 16 plasma proteins (related to 7 different disease states, e.g., cardiovascular, Alzheimer's, and cancer) at concentrations ranging from mg/mL to pg/mL [50].…”

The clinical utility of mass spectrometry based protein assays

“…The selectivity of the mass spectrometer has been employed to quantitate heterogeneous large molecules which might exist in multiple isoforms or contain a variable number of post-translational modifications [19]. Other factors which might drive the use of MS include the ability to use stable isotope labeled molecules as internal standards and the relatively straight-forward ability to develop multiplexed assays [20,21]. MS-based assays do not require the use of antibody reagents (although recently, the combination of antibody reagents and MS has proven to be a powerful technique) and therefore can be more easily employed and adopted when antibody reagents are not available or not trusted.…”

Applications of targeted proteomics in systems biology and translational medicine

“…Using an approach known as mass spectrometric immunoassay (MSIA), Krastins and coworkers rapidly developed assays for 16 different target proteins and their isoforms across seven different clinically important areas and ranging in concentration from pg/ml to ng/ml in bona fide clinical samples 112. Recently, Peterman and coworkers applied the MSIA approach to detect insulin and its analogues using a pan‐insulin antibody over a range from 1.5 to 960 pM 113.…”