Enhancing sample throughput and proteome coverage with a novel tandem-LC MS/MS approach

Enhancing sample throughput and proteome coverage with a tandem direct-injection LC–MS/MS workflow — concise expert summary

Significance of the topic

High-sensitivity nano-flow LC–MS/MS is central to modern bottom-up proteomics but is often limited by instrument overheads and low mass-spectrometer utilization when operated as a single direct-injection (DI) column workflow. For large-scale screening and industrial proteomics (thousands of samples), balancing throughput, depth of proteome coverage, and quantitative precision is critical. The tandem direct injection (TDI) approach evaluated here increases effective gradient time and reduces overhead, enabling deeper, more precise proteome measurements at higher sample throughput while reducing carryover and preserving quantitative accuracy across columns.

Goals and study overview

• Evaluate a tandem direct-injection (TDI) workflow implemented on the Thermo Scientific Vanquish Neo UHPLC with a Sonation double-barrel ion source, paired with an Orbitrap Astral MS.
• Compare TDI versus single-column DI across multiple sample throughputs (30, 48, 60, 96 samples/day, SPD) using mixed proteome standards (human/yeast/E. coli) and label-free / SILAC cell-line experiments for targeted protein degradation (TPD) screening.
• Quantify gains in identification, quantification precision (CV), carryover, and practical throughput scaling via multiplexing (SILAC).

Methodology and instrumentation used

• Sample types: HeLa (human), S. cerevisiae, and E. coli digests mixed in defined ratios; cell-line samples treated with BRD4 degrader MZ1 vs DMSO and prepared by automated workflows.
• Columns: Self-packed pulled-tip C18 columns (75 μm i.d. × 20 cm, 1.5 μm beads), maintained at 60 °C in a Sonation double-barrel oven.
• Flow and gradient: Nano-flow regimes (0.4–0.7 μL/min) with method durations adjusted per throughput (effective gradient times from ~13 to 44 min depending on SPD).
• Mass spectrometry: Orbitrap Astral MS operated in DIA (directDIA+) mode; acquisition parameters held constant between DI and TDI comparisons.
• Software and processing: Spectronaut (Biognosys) directDIA+ for identification and MaxLFQ-style quantification; combined FASTA databases for HYE analyses.
• Key hardware enabling TDI: Vanquish Neo tandem UHPLC, Vanquish switching valves and binary pumps, nanoViper capillary/tandem workflow kit, Sonation double-barrel oven and tandem source kit, NanoSpray Flex ion source with automated spray-voltage switching.

Main results and discussion

• TDI increases effective gradient time: By preparing (wash/equilibrate/load) the second column while the first is acquiring, TDI preserves more gradient time per run versus a single-column DI workflow at identical SPD. The effect is especially pronounced at high throughputs (e.g., 96 SPD).
• Higher proteome depth: At 30 SPD nearly 11,000 protein groups were identified from cell-line samples; even at 96 SPD the workflow yielded ~10,000 proteins. For mixed HYE standards, TDI outperformed DI in identifications due to improved peak capacity.
• Improved quantification precision: At 96 SPD the number of proteins quantified with CV <10% was substantially higher with TDI (nearly doubled compared with DI in HYE experiments); the Orbitrap Astral high scanning speed plus longer effective gradients preserves points-per-peak and quantitative quality.
• Robustness across columns: Quantitative comparisons made across the two tandem columns show that intercolumn variation is small relative to sample-driven differences. PCA plots separated biological conditions more strongly than column identity.
• Substantially reduced carryover: The extended washing achievable in TDI decreased peptide-level carryover—up to ~4-fold reduction at 96 SPD versus DI—reducing the risk of contamination of subsequent runs and extending column lifetime.
• Application to targeted protein degradation screening: In MZ1-treated vs DMSO controls, TDI resolved expected down-regulation of BRD4 (and related BRD proteins) across throughputs, demonstrating sensitivity to small, biologically relevant changes even at high SPD.
• Throughput scaling with multiplexing: Introducing multiplexing (double or triple SILAC) converts gradient throughput (injections/day, IPD) into higher effective sample throughput (SPD). For example, double SILAC at 60 IPD corresponds to 120 SPD; triple SILAC at 60 IPD gave ~180 SPD while maintaining robust quantification (median CV ~5.7% for triple SILAC 60 IPD with ~7,500 proteins at CV <10%).

Benefits and practical applications

• Higher sample throughput without sacrificing proteome depth or precision—critical for industrial screening, CRO/CDMO pipelines, and large-cohort studies.
• Lower carryover and improved column cleaning reduces instrument downtime, maintenance needs, and false-positive identifications from contaminants.
• Flexible experimental designs: TDI supports label-free and multiplexed strategies; multiplexing enables large gains in samples-per-day while retaining per-channel quantification.
• Compatibility with existing nano-LC and Orbitrap Astral DIA workflows and bioinformatic processing (Spectronaut), facilitating integration into established pipelines like Evotec’s ScreenPep.

Future trends and potential applications

• Further scaling via multiplexing: Combining TDI with higher-order chemical or metabolic labeling can multiply effective throughput (SPD) without proportionally reducing identification rates; however, careful design is required to avoid ratio compression and channel interference in quantitative readouts.
• Optimization of gradient length vs. data quality: Shorter gradients will increase SPD but risk deteriorating quantification due to insufficient points-per-peak; TDI mitigates this trade-off by maximizing gradient time. Continued enhancements in MS scan speed and ion transmission will push this boundary further.
• Automation and integration with sample-prep: Higher throughput will benefit from end-to-end automation (plate-based prep, automated desalting) to keep sample handling aligned with LC–MS capacity.
• Industrial screening and phenotypic assays: TDI is well-suited for large-scale compound screens (e.g., targeted protein degradation, molecular-glue discovery) where thousands of samples and subtle proteome changes must be detected reliably.
• Column and emitter technology evolution: Advances in column packing, emitter designs, and lower-dead-volume microfluidics will further increase peak capacity, reduce carryover, and improve robustness for tandem workflows.

Conclusion

The tandem direct-injection LC–MS/MS workflow on the Vanquish Neo platform with a double-barrel nanoelectrospray source and Orbitrap Astral MS demonstrably increases effective gradient time, proteome depth, and quantitative precision at high sample throughputs. TDI reduces carryover and enables robust cross-column quantification, while multiplexing strategies (SILAC) further escalate practical sample throughput. This approach offers a pragmatic balance of sensitivity, precision, and throughput for large-scale proteomics and screening operations, enabling more efficient discovery and profiling in industrial and academic settings.

References

1. Nagaraj N., Boychenko A., Valenta A. Thermo Fisher Scientific Webinar: A tandem LC workflow for maximizing mass spectrometer utilization in proteomics (September 19, 2024).
2. Zheng R., Rendl M., Valenta A., et al. Thermo Fisher Scientific Technical Note 003335: Maximizing sample throughput and sensitivities in nano and capillary LC-MS: harnessing the advantage of a tandem direct injection workflow and a double barrel ion source (2024).
3. Sonation GmbH. Double barrel oven for Thermo Scientific NanoSpray Flex ion source ES071 and ES072 (2024).