Pre-Sliced diaPASEF: Narrowband accumulation in the first timsCell for Ultra-Sensitive Plasma Proteomics

Significance of the topic

Mass spectrometry (MS)-based proteomics can quantify thousands of proteins, but measuring very low-abundance proteins in complex biological fluids such as plasma remains difficult because of extreme dynamic range and the dominance of a small set of high-abundance proteins. Improving sensitivity for low-abundance targets (e.g., cytokines) in undepleted plasma is critical for clinical biomarker studies, translational research, and targeted assays where sample pre-depletion or extensive fractionation is undesirable.

Objectives and study overview

This study introduces and evaluates "pre-sliced g-dia-PASEF," a strategy that narrows the ion mobility range injected into the second tims cell of a timsTOF Pro instrument to boost sensitivity for targeted proteomics in plasma. The approach divides a conventional wide ion-mobility g-dia-PASEF acquisition into multiple narrow slices (0.1 1/k0 each) and configures the first TIMS cell to exclude ions outside the selected mobility range (acting as a high-pass filter). The main goals were to determine whether selective accumulation in narrow mobility windows increases peptide signal, preserves quantitative performance, and enables detection of sub-attomolar analytes in undepleted plasma.

Methods

Samples and spike-in design:

• Digested non-depleted human plasma was used as matrix.
• A panel of synthetic peptides representing low-abundance proteins was spiked in a dilution series from 10 fmol down to 0.64 amol (1:5 dilutions).
• Stable-isotope labeled (heavy) peptide standards were present in all samples at 10 fmol to enable targeted quantitation.
• Injection volume reported was 1 µL on column for sensitivity tests.

Acquisition strategy and gradients:

• Analyses were performed on a timsTOF Pro mass spectrometer operating in g-dia-PASEF mode coupled to a nanoElute2 nano-HPLC system.
• Chromatographic gradients evaluated included 60, 40, 20 and 10 min runs.
• The full IM (ion mobility) range 0.7–1.4 1/k0 was compared to pre-sliced narrow windows of 0.1 1/k0 width covering ~0.7–1.25 1/k0; slices were treated as independent dia-PASEF acquisitions for initial testing (Figure depiction indicated six distinct runs to cover the target mobility span).

Data processing:

• MS data were analyzed using Skyline for targeted extraction, quantitation and linearity assessment.

Used instrumentation

• Bruker timsTOF Pro mass spectrometer (tims dual cell architecture).
• nanoElute2 nano-HPLC (Bruker).

Main results and discussion

Principle and effect of pre-slicing:

• By limiting the mobility range that reaches the second TIMS cell and raising the first TIMS inlet potential to exclude unwanted mobilities, the method reduces the population of trapped ions competing for accumulation and MS/MS, effectively enriching ions with the targeted mobility.
• Narrower mobility windows increase ion mobility resolution because more elution time is spent over a smaller mobility span, and they lower background/complexity for the targeted region.

Sensitivity and detectability:

• Peptides that were not identified or quantified using the wide IM range became identifiable and quantifiable in their respective narrow mobility slices.
• Signal intensity for targeted peptides increased when measured in their specific mobility slice, with sensitivity improvements reported up to approximately 4-fold for certain peptides at attomolar/sub-attomolar levels.
• Clinically relevant cytokine peptides (examples shown: IL-18 and IFNA10) were detected and quantified from undepleted plasma at sub-attomolar on-column amounts in this targeted configuration.

Quantitative performance:

• Quantitation maintained linearity across more than four orders of magnitude for the tested peptides, indicating that narrowing the mobility window does not compromise dynamic-range linearity for targeted peptides when appropriate standards are used.

Trade-offs and practical considerations:

• The pre-sliced approach divides the full mobility span into multiple acquisitions; this can increase total acquisition burden if all slices must be measured sequentially, but it can be optimized by selecting only slices relevant to the target list.
• Current implementation treated each slice as an independent run for proof-of-concept; integration into routine workflows may require scheduling, multiplexing, or instrument control adaptations to reduce overhead.

Benefits and practical applications

• Substantially improved sensitivity for low-abundance targets in complex matrices without requiring depletion or extensive front-end fractionation.
• Preservation of quantitative linearity over wide concentration ranges, enabling reliable targeted assays with heavy-labeled standards.
• Applicability to clinical and translational proteomics where detection of low-level biomarkers (e.g., cytokines) in undepleted plasma is desired.
• Can be combined with targeted data analysis tools (Skyline) for high-confidence quantitation.

Future trends and potential applications

• Method optimization: tailoring which mobility slices are acquired based on prior knowledge of target peptide mobilities to minimize instrument time while maximizing sensitivity.
• Integration: developing instrument control that performs rapid sequential slicing or dynamic mobility selection within a single run to avoid multiple injections.
• Multiplexing and scheduling: coupling pre-sliced acquisition with scheduled targeted methods, parallel accumulation, or trapping strategies to improve throughput.
• Broader target panels: expanding the list of clinically relevant low-abundance proteins (e.g., larger cytokine panels, neuropeptides) and validating in clinical cohorts.
• Analytical improvements: combining narrow mobility selection with advanced ion optics, improved accumulation strategies, or optimized chromatography to further enhance limits of detection.

Conclusion

Pre-sliced g-dia-PASEF is a targeted acquisition strategy that narrows ion mobility input to the second TIMS cell, selectively enriching ions within defined mobility windows and increasing sensitivity for low-abundance peptides in undepleted plasma. The approach enabled detection and quantitation of cytokine peptides at sub-attomolar levels with preserved quantitative linearity and up to ~4x sensitivity gains for certain analytes. While the method increases acquisition granularity (potentially requiring more runs), it offers a practical path to higher sensitivity targeted proteomics applicable to clinical and translational studies. Further development should focus on reducing acquisition overhead and expanding validated target lists.

References

• Study authors and affiliations as reported: Marta L. M. Mendes (Luxembourg Institute of Health), Ryan Marisco (Bruker Daltonics Inc.), Pierre-Olivier Schmit (Bruker Datonique S.A.), Gunnar Dittmar (Luxembourg Institute of Health).
• Instrument/manufacturer information: timsTOF Pro and nanoElute2 (Bruker).