Introduction

Low-flow UHPLC coupled with mass spectrometry (MS) is the gold standard for deep quantitative analysis of complex proteomes. However, the unmatched sensitivity of low-flow LC-MS typically comes at the cost of low MS utilization and sample throughput. A high proportion of idle MS time results from long sample injection, loading, column washing, and equilibration cycles. Here, we present a novel, facile, and robust workflow for deep dive proteomics that eliminates idle MS time by employing a tandem LC.

Methods

The novel LC-MS configuration, comprising a Vanquish Neo UHPLC system configured for tandem direct injection, was coupled to either an Orbitrap Exploris 240/480 or an Orbitrap Astral mass spectrometer for deep proteome profiling using library-free DIA. Human cell lysates were analyzed to demonstrate the performance of untargeted bottom-up TMT and label-free proteomics on 75 µm (≤ 300 nL/min) and 150 µm ID (1.5 µL/min) columns with throughput up to 180 samples/day. To minimize post-column dispersion the Sonation double-barrel column oven was used for all experiments.

Preliminary results

The tandem LC-MS workflow was tested using gradients from 8 to 120 min (corresponding to 180 to 12 samples/day, respectively) to demonstrate high throughput, run-to-run reproducibility, and close to 100% MS utilization for discovery proteomics. For example, a 65-min gradient with a 75 µm x 75 cm (2 µm particles) column at 300 nL/min enabled the identification of >11,000 protein groups per run in DIA from 200 ng HeLa digest with > 91% MS utilization on the Orbitrap Astral MS. With more than 90% of protein groups overlapped among runs and <5% overhead time, this configuration delivers excellent reproducibility for large-cohort deep-dive proteomics analysis.

(For Research Use/Purposes Only – Not for Diagnostic Procedures)