PNNL

Abstract

Sensitive, multiplexed protein quantification remains challenging despite recent advancements in LC-MS assays for targeted protein biomarker quantification. High-sensitivity protein biomarker measurements usually require immuno-affinity enrichment of target protein; a process which is highly dependent on capture reagent and limited in capability to measure multiple analytes. Herein, we report a novel antibody-free platform, which measures multiple biomarkers from complex matrices employing a strategically optimized solid-phase extraction cleanup and orthogonal multidimensional LC-MS. Eight human protein biomarkers with different specifications were spiked into dog plasma as a model investigation system. The developed analytical strategy achieved the desired sensitivity, robustness, and throughput via the following steps: (1) post digestion mixed-mode cation exchange-reverse phase SPE enrichment to remove the majority of non-peptide components; (2) rapid, high-pH peptide fractionation to eliminate background peptide components efficiently while selectively enriching signature peptides (SP) to provide sufficient sensitivity for multiple targets; and (3) trapping-micro-LC-MS analysis, which achieves high sensitivity comparable to a nano-LC-MS method but with much better robustness and throughput for the final analysis. Compared with a conventional LC-MS assay with direct protein digestion and limited clean-up, analysis with this antibody-free platform improved the LLOQ by 1-2 orders of magnitude for the eight protein biomarkers, reaching as low as 5 ng/mL in plasma. In parallel comparison with previously PRISM method, at least comparable sensitivity was demonstrated with 5 times faster throughput using our platform. This platform was applied for the quantification of biomarkers of respiratory conditions in subjects with various lung diseases, demonstrating real-world applicability.