Trap-Refocusing-Elute NanoLC-MS: A Robust Strategy Combining Trap-and-Elute Configuration with Direct-Injection Chromatographic Performance

Liquid chromatography coupled with mass spectrometry (LC−MS) is essential for proteome identification and quantification, and chromatographic resolution strongly affects their performance. Direct injection (DI) typically provides superior resolution but is restricted to low injection volumes, particularly in nano-flow setups, and does not allow online desalting. Trap-and-elute (TE) workflows enable larger sample loading and efficient desalting but generally produce broader chromatographic peaks. Here, we introduce trap-refocusing-elute (TRE), a nanoLC strategy, evaluated here in forward-flush mode, that combines the benefits of DI and TE by introducing an auxiliary water flow between the trap and analytical columns. This flow reduces the organic solvent strength and restores peptide retention at the column head, minimizing peak dispersion during trap-to-column transfer. Using HeLa tryptic digest and data-independent acquisition (DIA), we compared DI, TE, and TRE at matched injection volume. At the strongest dilution tested (2-fold), TRE achieved a median full width at half maximum (FWHM) of 3.1 s, versus 4.7 s for TE and 3.6 s for DI, over a 28 min gradient. Gains were largest for early-eluting peptides, consistent with reduced broadening during trap-to-column transfer, the main source of dispersion in TE. When the injection volume was increased ninefold at a matched total injected quantity, TRE retained most of this resolution advantage. By enhancing resolution while preserving the injection volume flexibility of trap-based workflows, TRE offers an easily implementable solution for clinical, single-cell, post-translational modification and high-throughput proteomics, addressing the long-standing trade-off between resolution and robustness in nanoLC-MS.