Exploring the proteomics capabilities of a new Trapped Ion Mobility Q-TOF designed for enhanced metabolomics performances

Significance of the topic

This study evaluates whether a newly developed Trapped Ion Mobility Q-TOF platform (timsMetabo), engineered primarily for enhanced metabolomics and lipidomics performance, maintains high-quality proteomics capabilities. Demonstrating robust proteomics on a metabolomics‑optimized instrument is important for multi-omics workflows where a single benchtop system must deliver high sensitivity, broad dynamic range and high throughput across analyte classes.

Goals and overview of the study

The principal aim was to assess depth, throughput, dynamic range and targeted quantitation performance of the timsMetabo using standard proteomics samples (K562 cell digest) and complex biofluid (neat human plasma) at low sample loads. The study compared data-dependent PASEF (DDA/PASEF) and data-independent dia-PASEF acquisition modes, and evaluated a targeted prm-PASEF approach for limit of quantitation (LOQ) assessment using isotopologue peptide standards.

Methods and experimental design

The experimental workflow used 100 ng injections on a 25 cm x 75 µm IonOpticks column with nanoElute2 nano‑HPLC at 250 nL/min and gradients of 22, 44 and 60 minutes (5%–35% ACN). Acquisition modes included dia-PASEF (24 × 25 Th windows from 400–1000 m/z distributed over 8 PASEF ramps) and PASEF DDA (5 PASEF ramps per cycle; 300 Hz acquisition); a reduced-duty-cycle (≈30%) setting was chosen for prm-PASEF to match sensitivity conditions. For targeted experiments, a Promega peptide reference mix (isotopologs) was spiked into a neat plasma background. Data analysis: dia‑PASEF processed with DIA‑NN (library‑free mode) and PASEF DDA processed with FragPipe; prm-PASEF analyzed in Skyline. Analyses were performed without match-between-runs (MBR) to report conservative identification numbers.

Used instrumentation

• timsMetabo Trapped Ion Mobility Q‑TOF (Bruker) with TIMS‑MX cartridge, Athena Ion Processor (AIP) and Mobility Range Extension (MoRE) scan mode.
• nanoElute2 nano‑HPLC system (Bruker) and 25 cm × 75 µm IonOpticks column.
• Promega Peptide Reference Mix (V7491) for prm-PASEF LOQ assessment.
• Software: DIA‑NN v2.0.1 (library‑free), FragPipe v22.0, Skyline‑daily v26.1.

Main results and discussion

- Discovery proteomics (dia‑PASEF): Using a 22‑min gradient and a reduced duty cycle, up to ~69,000 precursors and ~7,200 protein groups were identified from K562 digest (100 ng load). Extending the gradient to 60 minutes increased identifications to ~103,000 precursors and ~8,200 protein groups, showing clear gains with longer LC separation.

- DDA PASEF: For comparison, PASEF DDA yielded ~28,000 peptides and ~4,700 protein groups with a 22‑min gradient and ~62,000 peptides and ~6,500 protein groups with a 60‑min gradient, indicating the platform’s value for projects requiring high selectivity and de novo sequencing (e.g., immunopeptidomics, metaproteomics, cross‑linking studies).

- Neat plasma proteome: With a 100 ng neat plasma injection and no special dia‑PASEF optimization, the system delivered on average ~412 protein groups and ~5,050 precursors. The measured plasma dynamic range spanned roughly five orders of magnitude, consistent with expectations for plasma proteomics at this sample load.

- Targeted quantitation (prm‑PASEF): Using isotopolog peptide standards in a plasma background, the timsMetabo achieved a limit of quantitation down to ~25 amol on‑column with linearity R2 > 0.99 across evaluated peptides, demonstrating excellent sensitivity and quantitative precision even with short gradients and complex matrix.

- Data quality: Spectral quality at LOQ remained high for the evaluated peptides. The timsMetabo’s MoRE and AIP elements preserved ion transmission and MS/MS quality across the measured m/z and mobility ranges despite the platform’s low‑m/z optimization.

Key contributions and practical implications

• The timsMetabo, though optimized for metabolomics, retains proteomics performance comparable to predecessor timsTOF instruments for discovery workflows, offering a versatile solution for multi‑omics laboratories.
• High throughput is achievable (e.g., deep proteome coverage with 22–60 min gradients), enabling balance between speed and depth depending on study goals.
• Targeted workflows are supported with excellent LOQ and linearity in complex matrices, making the platform suitable for quantitative proteomics assays in translational and clinical research contexts (research use).
• Operation without MBR and usage of library‑free DIA analysis show the instrument can produce robust identifications with conservative processing choices.

Future trends and potential applications

• Integration in multi‑omics pipelines: The ability to handle metabolites, lipids and intact proteomes makes timsMetabo attractive for single‑platform multi‑omics studies where sample throughput and consistency across omics layers are critical.
• Method optimization: Further tuning of dia‑PASEF windows and duty‑cycle tradeoffs could increase identifications at short gradients, enabling even faster proteomics assays for large cohorts.
• Targeted clinical assays: Demonstrated LOQ suggests potential for development of high‑sensitivity targeted proteomics assays for biomarker verification, pending regulatory and validation work.
• Advanced applications: The preserved TIMS‑PASEF capabilities support specialized proteomics such as immunopeptidomics, metaproteomics and cross‑linking experiments, benefiting from enhanced selectivity and de novo sequencing capacity.

Conclusions

The timsMetabo platform, while engineered primarily for improved low m/z transmission for metabolomics and lipidomics, preserves high‑performance proteomics capabilities. It provides competitive depth, dynamic range and sensitivity for both discovery and targeted proteomics at low sample loads and short gradients. These attributes support its deployment as a flexible benchtop instrument for multi‑omics laboratories that require robust, high‑throughput analyses across analyte classes.

Reference

Exploring the proteomics capabilities of a new Trapped Ion Mobility Q‑TOF designed for enhanced metabolomics performances. Benoit Fatou et al., Bruker (2026).