Enhancing sample throughput for deep dive proteomics with Tandem LC

Importance of the Topic

High-throughput and deep-dive proteomics demand both exceptional sensitivity and efficient use of mass spectrometer time. Traditional nano-LC-MS workflows achieve deep coverage but suffer from low sample throughput and extended idle periods of the MS. The Tandem Direct Injection (TDI) approach addresses this challenge by overlapping column loading, washing and equilibration with active gradient elution, maximizing MS utilization while maintaining or enhancing proteome depth.

Objectives and Study Overview

The study evaluates a novel intelligent tandem nano-LC workflow on a Vanquish Neo UHPLC system coupled to Orbitrap Exploris MS platforms. It aims to assess:

• MS utilization and sample throughput gains
• Proteome coverage for label-free and TMTpro 18-plex HeLa samples
• Quantitative performance in DDA and DIA modes
• Carryover elimination under aggressive wash cycles

Methodology and Workflow Configuration

The TDI setup uses two binary pumps and two columns in a double-barrel oven. One pump drives the analytical separation while the second performs parallel sample loading, column reconditioning and washing. An intelligent Method Editor (IME) on the Vanquish System Controller automates configuration and scheduling. Key steps run in parallel:

• Gradient elution on Column A
• Sample aspiration, loading and wash cycles on Column B
• Column equilibration for the next injection

Sample types include TMTpro 18-plex labeled and label-free HeLa digests. Columns ranged from 15 cm to 75 cm length with 50 µm or 75 µm inner diameter, flow rates from 0.1 to 1 µL/min, delivering throughputs from 4 to 180 samples per day (SPD). Data were processed with Proteome Discoverer 3.1 (SEQUEST HT, INFERYS, CHIMERYS) and Spectronaut 19, maintaining FDR <1%.

Used Instrumentation

• Vanquish Neo UHPLC with dual binary pumps
• Vanquish Column Compartment N and Sonation Double Barrel Oven
• Low-dispersion switching valves and Tandem Workflow Kit
• Nanospray Flex ion source, Orbitrap Exploris 480/Astral MS
• FOSSILionTECH microtight unions, various PepMap columns

Main Results and Discussion

• On a 75 µm × 75 cm column with 90 min gradient, TDI matched DI proteome coverage but raised MS utilization from 70% to 93% and throughput to 18 SPD.
• In 100 SPD trap-and-elute comparisons, TDI delivered 20% more protein groups and, at 500 nL/min, boosted sensitivity by 62%, identifying >4,300 proteins from 40 ng digest in DIA mode.
• TMTpro 18-plex DDA analysis achieved >5,700 proteins in 360 min; employing CHIMERYS increased identifications to >6,700.
• Transitioning a 72 SPD method on 15 cm × 50 µm columns raised MS utilization from 55% to 74% and quantified up to 1,564 proteins from 10 ng input.
• A 96 SPD cell lysate study on Orbitrap Astral MS quantified ~10,000 protein groups with robust discrimination of treatment effects.
• Extended wash protocols virtually eliminated carryover (<1–2%) without throughput penalty for both TMT-DDA and LFQ-DIA.

Benefits and Practical Applications

• Significantly improved MS duty cycle across gradients from 7 to 357 min.
• Flexible deployment for both deep proteome profiling and high-throughput screening.
• Automated diagnostics and method setup enhance robustness and reproducibility.
• Minimal carryover supports large cohort and multiplexed TMT studies.

Future Trends and Potential Applications

Integration of TDI with emerging MS architectures and higher levels of multiplexing (e.g. TMTpro-35plex) may further accelerate large-scale studies. Software advances in AI-driven identification and quantification will enhance throughput and depth. Miniaturized column designs and faster gradients could enable real-time clinical proteomics and automated quality control in bioprocessing.

Conclusion

The Tandem Direct Injection workflow on the Vanquish Neo platform dramatically improves MS utilization and throughput without compromising sensitivity or data quality. It enables both deep proteome coverage and rapid screening workflows, offering a versatile solution for modern proteomics challenges.

Reference

1. Zheng R. et al. Deep single-shot NanoLC-MS proteome profiling with a 1500 Bar UHPLC system, long fully porous columns and HRAM MS. J. Proteome Res. 2022;21(10):2545–2551.
2. Stewart H. et al. Parallelized acquisition of Orbitrap and Astral analyzers enables high-throughput quantitative analysis. Anal. Chem. 2023;95(42):15656–15664.
3. Sonation GmbH. Double Barrel Oven for Thermo Scientific NanoSpray Flex Ion Source ES071 and ES072. 2024.
4. Zheng R. et al. Fast, sensitive and reproducible nano- and capillary-flow LC-MS methods for high-throughput proteome profiling using the Vanquish Neo UHPLC system hyphenated with the Orbitrap Exploris 480 MS. Thermo Fisher Scientific Technical Note 000138, 2021.