An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers

Abstract

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 microl 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.

Fig. 1.

Response curves for multiplexed SISCAPA assay. The ion signals for varying amounts of heavy stable isotope-labeled peptide were measured relative to the signal of the light peptide channel. a, the peptide concentrations are calculated from the response curve and plotted versus the expected peptide concentration based on the level of peptide added to the sample. b, protein concentrations are plotted based on the molar equivalents of the peptides determined in a and hence do not adjust for trypsin digestion efficiency, which could be an issue if the peptide were being released from the endogenous protein versus spiked-in as in this experiment. Each point was measured in triplicate on three separate days; error bars are ±1 S.D.

Fig. 2.

Improving overall sensitivity through enrichment from large volume of plasma. Response curves for the multiplexed SISCAPA assay measured in 1 ml of plasma are shown. The ion signals for varying amounts of heavy stable isotope-labeled peptide were measured relative to the signal of the light peptide channel. a, the concentration of each peptide is calculated based on the response curve and plotted versus the expected peptide concentration based on the level of peptide added to the sample. b, protein concentrations are plotted based on the molar equivalents of the peptides determined in a and hence do not adjust for trypsin digestion efficiency, which could be an issue if the peptide were being released from the endogenous protein versus spiked-in as in this experiment. Each point was measured in duplicate; error bars are the range of measured values.