Session: Parallel session 5 - New topics in OMICs

Trap-Refocusing-Elute nano liquid chromatography: how to take advantage of both Direct Injection and Trap-and-Elute mode?

Christopher KUNE², Maximilien FLERON¹, Dominique BAIWIR¹, Gauthier EPPE², Gabriel MAZZUCCHELLI^1,2

¹University of Liege - GIGA Proteomics Facility, Liège, Belgium
²University of Liege - MS Lab - Molsys, Liège, Belgium

Liquid chromatography coupled with mass spectrometry (LC-MS) has emerged as a tool of choice for identifying and quantifying an organism's proteome, particularly in bottom-up proteomics where sample complexity necessitates high resolution and sensitivity. The two primary LC methodologies include Direct Injection (DI), where samples are introduced directly onto the analytical LC column, and Trap-and-Elute (TE), where samples are initially trapped on a column before elution onto the analytical LC column. DI typically achieves superior chromatographic resolution compared to TE but is limited to injection of small volumes (~ 1 µL) and does not allow online sample desalting.

Here, we introduce a novel LC method termed "Trap-Refocusing-Elute" (TRE), which integrates the strengths of trap-elute techniques with enhanced chromatographic resolution capabilities. TRE involves a post-trap column dilution step by water increasing water content before the analytical column, thanks to an additional LC pump. This enables a secondary sample trapping at the head of the analytical column.

This new approach has been evaluated, in comparison to DI and TE with a standard solution of HeLa cells tryptic digest by monitoring peptide peak widths as an indicator of LC resolution. The dilution factor (trap column flow / analytical column flow) has been optimized to reach the DI resolution minimizing the impact of the dilution. Preliminary results indicate that TRE exhibits comparable peak widths to direct injection methods. TRE represents a promising advancement in LC-MS-based proteomics, offering researchers a practical solution for achieving high LC resolution, online desalting, and efficient sample handling within their analytical workflows. This methodological innovation holds potential for enhancing the sensitivity and robustness required in high throughput complex proteomic analyses.