Exploring Depth and Breadth of a Protein Complex Mixture with Top-Down Data-Independent Acquisition Using an Orbitrap Fusion Tribrid Mass Spectrometer

Importance of Topic

Top-down proteomics provides direct identification and characterization of intact proteins and their proteoforms, complementing bottom-up approaches. By analyzing full-length proteoforms, researchers can assess sequence variants, post-translational modifications, and microheterogeneity. Extending the dynamic range of detectable proteins in complex mixtures is critical for comprehensive profiling in discovery and quality-control settings.

Objectives and Overview of the Study

This study systematically compares four top-down workflows on a standard protein mixture (UPS2) using a Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer. Two MS1 detection modes (high-resolution Orbitrap and high-sensitivity ion trap) are combined with two MS2 acquisition strategies: data-dependent (DDA) and data-independent (DIA) with broad isolation windows (100 or 250 m/z). The goal is to evaluate how each configuration impacts proteoform detection across size, concentration, and complexity ranges.

Methods and Instrumentation

Sample Preparation and Chromatography:

• UPS2 standard (48 proteins, 6–83 kDa, 0.05–5000 fmol) reconstituted in 10% acetonitrile/0.1% TFA.
• 1 µg injections onto a 200 µm×25 cm EASY-Spray Pepswift monolith column.
• 60 min gradient from 10% to 60% acetonitrile/0.1% formic acid at 4 µL/min on an nLC1000 HPLC.

Mass Spectrometry:

• Thermo Scientific Orbitrap Fusion Tribrid instrument.
• MS1 detection: Orbitrap (R=240 K, 150–2000 m/z) or ion trap (normal mode).
• MS2 detection: Orbitrap (R=240 K), HCD fragmentation at 20% collision energy.
• DDA settings: 5 m/z isolation, 30 s exclusion, charge state filter z>6.
• DIA settings: serial windows of 100 or 250 m/z covering 500–1500 m/z.

Data Analysis:

• Top-down data processing via TopDown cRAWler (ReSpect for ion trap, Xtract for Orbitrap) and AbsoluteMass search within ProSightPD node of Proteome Discoverer 2.0.

Main Results and Discussion

• Ion trap MS1 with DDA excels in sensitivity but struggles to resolve charge states, identifying only highest-abundance proteins by DDA.
• Ion trap MS1 with DIA extends detection two orders of magnitude for small proteins and improves MS2 confidence scores by >2×.
• Orbitrap MS1 with DDA captures low-abundance small proteins (e.g., IGF II at 5 fmol) but underperforms on larger, complex proteoforms.
• Orbitrap MS1 with DIA (especially 250 m/z windows) uncovers mid-range proteins and low-level species invisible to DDA, albeit with diminishing returns for very large proteoforms (e.g., serum albumin).
• Microheterogeneity within protein groups can dilute ion current and impede DDA triggering; DIA co-isolates multiple charge states to boost total fragment ion current, facilitating proteoform identification.
• Combining all four workflows yielded identification of >160 proteoforms across 13 protein groups spanning three orders of magnitude in concentration.

Benefits and Practical Applications of the Method

By selecting appropriate MS1 detector and acquisition mode, laboratories can tailor top-down workflows to sample characteristics:

• DDA workflows provide high confidence for selected proteoforms, ideal for targeted characterization.
• DIA workflows improve discovery of low-abundance and mid-range proteoforms in complex samples.
• Automated ProSightPD processing streamlines data analysis and supports high-throughput studies.

Future Trends and Potential Applications

• Optimization of DIA window schemes and dynamic window placement for improved proteoform coverage.
• Advanced deconvolution algorithms to separate co-isolated proteoforms and resolve near-isobaric species.
• Hybrid DDA-targeted acquisition strategies to combine discovery and quantitation in a single run.
• Integration with bioinformatic pipelines for in-depth proteoform mapping in clinical and biopharmaceutical research.
• Instrument enhancements for faster scan rates and increased sensitivity on large proteoforms.

Conclusion

This comparison highlights that no single top-down configuration suffices for all proteoform classes. Ion trap and Orbitrap MS1 each bring complementary strengths, and DDA vs. DIA trade sensitivity, selectivity, and dynamic range. A combined approach leveraging both detectors and acquisition modes maximizes proteoform coverage across diverse size and abundance regimes.

Instrumentation Used

• Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer
• Thermo Scientific EASY-Spray Pepswift monolith column
• Thermo Scientific nLC1000 HPLC

References

• Ivanov AR, Colangelo CM, Dufresne CP, Friedman DB, Lilley KS, Mechtler K, Phinney BS, Rose KL, Rudnick PA, Searle BC, Shaffer SA, Weintraub ST. Interlaboratory studies and initiatives developing standards for proteomics. Proteomics. 2013 Mar;13(6):904–909.