Nano and capillary LC-MS (0-1 – 5 µL/min) is widely recognized as the gold standard for deep-dive proteomics workflows, due to its exceptional sensitivity1,2. However, this comes at the cost of reduced sample throughput and low MS utilization particularly for direct injection (DI) based workflow configurations. 

Here we present a novel tandem direct injection (TDI) workflow which permits offsetting of the gradient runs on the two columns to achieve nearly continuous analyte elution. Coupled with an intelligent instrument method editor (IME) and tailored diagnostic scripts, the workflow also permits comprehensive column washing with no impact on sample throughput. We investigated the capacity of the TDI workflow for both tandem mass tag (TMT) and label-free quantification (LFQ) in both data dependent (DDA) and data independent (DIA) acquisition modes.

_{Thermo Fisher Scientific,} 

_{1 Germany; Thermo Fisher Scientific,} 

_{2 USA; Thermo Fisher Scientific,}

_{3 Bremen; Thermo Fisher Scientific,}

_{4 Sunnyvale; Thermo Fisher Scientific,}

_{5 Lithuania, Thermo Fisher Scientific 6Netherlands, 7Evotec International GmbH, Germany}