1000 Proteins Per Hour [pph] Maximizing Protein ID From Complex Mixtures

Significance of the Topic

The ever-increasing complexity of proteomic samples demands advanced mass spectrometry solutions that maximize protein identification rates and sequence coverage. Recent developments in hybrid linear ion trap–Orbitrap instruments offer enhanced scan speeds, sensitivity, and resolution, enabling the identification of over 1,000 proteins per hour from complex mixtures, such as E. coli digests.

Objectives and Study Overview

This study evaluates the performance of a new Thermo Scientific LTQ Orbitrap Velos hybrid mass spectrometer. By applying two data-dependent acquisition strategies—ddTOP20 CID and ddTOP10 HCD—across 60- and 90-minute nanoLC gradients, the work aims to compare protein and peptide identification rates, sequence coverage, and confidence levels at different sample loads (1 µg, 100 ng, and 20 ng of E. coli digest).

Applied Methodology and Instrumentation

Sample Preparation and Chromatography:

• E. coli whole-cell lysates were reduced, alkylated, and enzymatically digested using a K/R-specific protease.
• Peptides were separated on a reversed-phase trap (100 µm×2 cm) and analytical column (75 µm×10 cm, 3 µm particles) with gradients of 2–30 % acetonitrile at 300 nL/min.

Data Acquisition Methods:

• ddTOP20 CID: One full MS scan at 30,000 resolution followed by 20 collision-induced dissociation MS/MS events in the linear ion trap.
• ddTOP10 HCD: One full MS scan at 30,000 resolution followed by 10 higher-energy collisional dissociation events detected in the Orbitrap.

Instrument Features:

• Stacked-ring ion guide (S-Lens) for increased ion transmission.
• Dual-pressure ion trap for optimized trapping and scan speed.
• Predictive automatic gain control (AGC) and improved HCD cell with axial field.
• Enhanced vacuum in the Orbitrap chamber for better resolution.

Main Results and Discussion

• At 1 µg load with ddTOP20 CID (60 min), ~1,050 proteins and 6,582 peptides were identified at <1 % FDR.
• Using ddTOP10 HCD (90 min), >1,000 proteins per hour were consistently identified, demonstrating the instrument's high throughput capability.
• HCD provided 81 % overlap at the protein level and 68 % overlap at the peptide level when compared to CID, with reduced fill times accelerating cycle times.
• Sensitivity gains enabled reliable identification down to 100 ng and 20 ng loads, maintaining robust sequence coverage and confidence.

Benefits and Practical Applications

The enhanced scan speed, improved ion transmission, and high mass accuracy of the LTQ Orbitrap Velos allow laboratories to

• Increase proteome depth in limited sample contexts.
• Perform rapid quality control in biopharmaceutical workflows.
• Enable de novo sequencing and quantitation of labeled samples.

Future Trends and Potential Applications

• Integration with ion mobility for multidimensional separations.
• Implementation of real-time database searching to prioritize low-abundance species.
• Adoption in clinical proteomics for biomarker discovery and personalized medicine.
• Enhanced fragmentation schemes (e.g., electron transfer dissociation) for labile post-translational modifications.

Conclusion

The hybrid linear ion trap–Orbitrap mass spectrometer substantially advances proteomic analysis by delivering rapid, high-confidence protein identifications from complex mixtures. The instrument’s innovative design features drive deeper proteome coverage, faster throughput, and greater sensitivity, positioning it as a cornerstone technology for both fundamental research and industrial applications.

References

• Wouters ER, Splendore M, Senko MW, Syka JEP, Dunyach E, Zabrouskov V. Ion Transmission at High Pressure. ASMS 2008.
• Olsen JV, Schwartz JC, Griep-Raming J, Nielsen ML, Damoc E, Denisov E, Lange O, Remes P, Taylor D, Splendore M, Wouters ER, Senko MM, Makarov A, Mann M, Horning S. Dual-Pressure Linear Ion Trap–Orbitrap Instrument with Very High Sequencing Speed. Mol Cell Proteomics. 2009 Dec;8(12):2759-69.
• Pekar T, Blethrow JD, Schwartz C, Merrihew J, MacCoss MJ, Swaney DL, Russell JD, Coon JJ. Improving the Analysis of Complex Protein Mixtures. Anal Chem. 2009;81(20):7757-65.