Maximizing proteome coverage through improved on-line Orbitrap peak determination

Significance of the topic

Modern proteomic analyses depend on efficient on line peak determination to detect and select peptide precursors for fragmentation. Traditional algorithms impose limits on proteome coverage and reproducibility. An improved peak determination method can overcome these bottlenecks, enabling deeper and more reliable peptide and protein identification workflows.

Objectives and study overview

This study introduces an advanced on line peak determination algorithm implemented on the Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. The aim is to increase unique peptide identifications and reproducibility in data dependent LC MS/MS experiments and to demonstrate enhanced performance for top down protein analysis. Comparative runs alternating between the conventional THRASH algorithm and the new method were performed on a HeLa tryptic digest and on intact protein standards.

Methodology and instrumentation

• Sample types: Thermo Pierce HeLa digest protein standard and Pierce Intact Protein Standard mix
• Liquid chromatography: Thermo Scientific Easy n LC 1000 or Dionex Ulti Mate 3000 UHPLC
• Mass spectrometry: Orbitrap Fusion Lumos Tribrid in data dependent mode
• Acquisition parameters: rapid scan rate, automatic MS range selection, dynamic exclusion 20 seconds; precursor charge filter 2–6 for peptides and ≥7 for proteins
• ITMS2 optimization: scan rate turbo, auto mass range, investigation of injection times (20–35 ms)
• Data processing: Proteome Discoverer 2.1 with UniProt human database, peptide spectral matches filtered at 1% false discovery rate using Percolator

Main results and discussion

• The advanced algorithm resolves overlapping isotopic envelopes and improves charge state assignment across entire precursor envelopes
• Ion trap MS2 capacity utilization increased from ~60% with the conventional algorithm to ~95% with the advanced method
• Optimized ITMS2 settings (20 ms injection time) enabled >40 Hz MS2 acquisition, generating ~250 000 MS2 spectra in a two hour LC run
• Unique peptide identifications increased by more than 35%, rising from ~33 000 to ~45 000 per two hour analysis
• Run-to-run reproducibility improved by 2–3 fold, lowering the threshold for reproducible detection
• Top down experiments benefited from accurate detection of highly charged protein envelopes and enabled targeted MS/MS fragmentation strategies

Benefits and practical applications

• Substantially enhanced proteome coverage with additional unique peptide identifications
• Improved reproducibility across technical replicates
• More efficient use of instrument cycle time for faster sequencing without sacrificing spectral quality
• Support for advanced top down workflows and large biomolecule analysis
• Applicability to high throughput academic, clinical, and industrial proteomics laboratories

Future trends and potential applications

As proteomics moves toward deeper coverage and characterization of intact protein forms, on line peak determination algorithms will integrate real time decision making and machine learning based feature recognition. Future instrumentation with higher field orbitraps and improved chromatography will further leverage these algorithms to enhance sensitivity, throughput, and quantitative accuracy across diverse applications.

Conclusion

The advanced on line peak determination algorithm significantly boosts the performance of Orbitrap based LC MS/MS by increasing proteome coverage, reproducibility, and enabling complex top down analyses. Optimized acquisition parameters in conjunction with the new algorithm provide a practical solution for maximizing the capabilities of modern mass spectrometers in deep proteomic studies.

References

• Hoopmann MR Finney GL MacCoss MJ High speed data reduction feature selection and MS/MS spectrum quality assessment of shotgun proteomics datasets using high resolution mass spectrometry Anal Chem 2007 79 5630 5632
• Senko MW Beu SC McLafferty FW Automated assignment of charge states from resolved isotopic peaks for multiply charged ions J Am Soc Mass Spectrom 1995 6 52 56
• Senko MW Beu SC McLafferty FW Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions J Am Soc Mass Spectrom 1995 6 229 233
• Horn DM Zubarev RA McLafferty FW Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules J Am Soc Mass Spectrom 2000 11 320 332