An Automated and Multiplexed Method for High Throughput Peptide Immunoaffinity Enrichment and Multiple Reaction Monitoring Mass Spectrometry-based Quantification of Protein Biomarkers

There is an urgent need for quantitative assays in verifying and validating the large numbers of protein biomarker candidates produced in modern “-omics” experiments. Stable isotope standards with capture by anti-peptide antibodies (SISCAPA) has shown tremendous potential to meet this need by combining peptide immunoaffinity enrichment with quantitative mass spectrometry. In this study, we describe three significant advances to the SISCAPA technique. First, we develop a method for an automated magnetic bead-based platform capable of high throughput processing. Second, we implement the automated method in a multiplexed SISCAPA assay (nine targets in one assay) and assess the performance characteristics of the multiplexed assay. Using the automated, multiplexed platform, we demonstrate detection limits in the physiologically relevant ng/ml range (from 10 μl of plasma) with sufficient precision (median coefficient of variation, 12.6%) for quantifying biomarkers. Third, we demonstrate that enrichment of peptides from larger volumes of plasma (1 ml) can extend the limits of detection to the low pg/ml range of protein concentration. The method is generally applicable to any protein or biological specimen of interest and holds great promise for analyzing large numbers of biomarker candidates.The current gold standard for quantifying protein biomarkers is the ELISA. A well functioning ELISA can be run at high throughput and has excellent sensitivity; however, the cost associated with development is very high, the lead time is very long, and the failure rate can be high. In addition, sandwich immunoassays are subject to potential interference from endogenous antibodies (1). Unfortunately, there are no quantitative assays available for the majority of biomarker candidates, and a considerable investment is required to generate assays de novo, creating a bottleneck in the biomarker pipeline (2, 3).A technique that has shown potential for bridging the gap between discovery and validation of biomarkers is stable isotope standards with capture by anti-peptide antibodies (SISCAPA)¹ (4) coupled to multiple reaction monitoring (MRM) MS. SISCAPA has several advantages over other immunoassays in that the mass spectrometer provides excellent specificity for the analyte of interest; the sample (including endogenous immunoglobulins) is digested to peptides, avoiding potential interference from endogenous antibodies; and precise, relative quantification is possible via the use of an internal standard. Additionally, although it is very difficult to combine multiple analytes into one assay (i.e. multiplex) using ELISAs, SISCAPA assays can in theory be highly multiplexed as many analytes can be measured from a single enrichment step. To date, individual SISCAPA assays have been successfully configured to a number of analytes (4–9), and up to three peptides have been enriched simultaneously (7, 8). In this study, we sought to advance the utility of SISCAPA for testing large numbers of biomarker candidates in large numbers of patient samples by automating the method to improve throughput and performance, testing the performance of multiplexing analytes, and improving sensitivity.