Wideband PRM with fully automated dual online reverse-phase liquid chromatography for High-throughput targeted proteomics

Background

Targeted proteomics is a technique used to obtain quantitative information about target proteins, making it widely used for verification and validation of candidate cancer biomarker proteins. Parallel-reaction monitoring (PRM), is favored due to the ease of assay development and the capability to detect all fragment ions. However, PRM has the limitation that the stable isotope labeled (SIL) peptides and endogenous (ENDO) peptides are monitored at different retention time.

Methods

In this study, we developed a wide-band PRM (WBPRM) method to address the limitations of conventional PRM method. The WBPRM utilizes multiplexed single ion monitoring (MSX) scans to increase sensitivity of low-abundance target precursor ions. Furthermore, WBPRM features a wide isolation window that allows for simultaneous isolation and fragmentation of ENDO and SIL precursor ions.

We also developed a fully automated dual online reverse-phase liquid chromatography system for high-throughput analysis. This LC system employs three additional switching valves and two analytical columns that enable mutually independent operation with hidden isocratic elution. As a result, this system eliminates dead time associated with sample injection, column washing and equilibration, thereby increasing experimental throughput by two folds. With this LC system, we optimized 15 minutes gradient with micro flow rate to analyze 96 samples per day (SPD).

Result and conclusions

We integrated the WBPRM method with dual online reverse-phase liquid chromatography system to develop pancreatic ductal adenocarcinoma (PDAC) subtype identification technology using 149 PDAC patient samples. Dual LC system allows short gradient without deadtime, while WBPRM enables the targeting a large number of peptides in highly complex proteome samples within a short LC gradient. Consequently, in a 15-minutes gradient experiment, WBPRM targeted 148 PDAC peptides and detected transition profiles of 106 of 148 SIL peptides, and quantified 93 ENDO peptides on average. This combined system is thus ideal for high-throughput targeted proteomics, involving large cohort sample.