Accurate Label-Free Protein Quantitation on the timsTOF Pro with 4D-Proteomics™

Importance of the Topic

The combination of trapped ion mobility spectrometry and high speed PASEF acquisition in a quadrupole time-of flight system addresses critical challenges in proteomics including sample complexity, dynamic range, sensitivity and throughput.
The ability to perform accurate label free quantitation over five orders of magnitude with minimal sample amounts enables in depth protein profiling in research, quality control and industrial applications.

Objectives and Study Overview

This study evaluates the performance of the timsTOF Pro platform with PASEF technology for label free protein quantitation in a mixed three species proteome.
Key goals include assessing identification depth, quantitative precision and accuracy using defined ratios of human, yeast and E coli peptides.

Methods and Workflow

Sample Preparation

• HeLa cell pellets lysed in trifluoroethanol with reduction and alkylation steps
• Tryptic digestion at enzyme to protein ratio of 1 to 100 overnight
• Peptide cleanup on C18 solid phase cartridges and reconstitution in formic acid
• Hybrid samples HYE A and HYE B containing human, yeast and E coli peptides at defined weight ratios

Chromatography and Separation

• Nanoflow reversed phase C18 column 25 cm length, 1.6 micron particles
• Gradient from 2 to 37 percent organic over 100 minutes at 400 nanoliters per minute
• Column temperature maintained at 50 degrees Celsius

Data Acquisition

• timsTOF Pro instrument combining parallel accumulation with serial fragmentation
• PASEF cycles scheduling ten MS MS scans per cycle with synchronized ion mobility selection
• MS mass range from 100 to 1700 m/z, TIMS ramp time 166 milliseconds
• Collision energies varied between 20 and 59 electronvolts for fragmentation

Data Processing

• PEAKS Studio X plus for identification and label free quantitation
• Search tolerances: 20 parts per million for MS and 0.05 dalton for MS MS
• Variable modifications methionine oxidation and protein N terminus acetylation, fixed carbamidomethylation of cysteine
• Id transfer between runs using four dimensional matching including collisional cross section

Used Instrumentation

The setup includes the Bruker nanoElute nanoflow LC system coupled to the timsTOF Pro quadrupole time of flight mass spectrometer equipped with a dual TIMS funnel for ion mobility separation and time focusing.

Key Results and Discussion

HeLa Digest Performance

• Average of 63 893 peptides identified per triplicate run with 85 percent overlap
• Approximately 6100 protein groups per single 100 minute PASEF analysis

Mixed Proteome Quantitation

• Identification of on average 64 334 peptides and 8410 protein groups in the three species mixture
• Total across six runs: 89 835 peptide sequences and 9467 protein groups
• Excellent chromatographic reproducibility demonstrated by overlayed base peak chromatograms
• Technical replicate correlation coefficients of 0.99 at the protein level
• Accurate fold change measurements for human background proteins centered at log ratio zero
• Yeast and E coli proteins quantified with median ratios close to expected values of 2.0 and 0.25 respectively

High data completeness and reduced missing values attributed to four dimensional id transfer including CCS values.

Benefits and Practical Applications

This approach delivers rapid deep proteome coverage with high quantitative precision making it suitable for:

• Comparative biomarker discovery studies
• Quality control in pharmaceutical and biotech manufacturing
• High throughput screening in academic and industrial laboratories
• Investigations of complex biological mixtures with minimal sample amounts

Future Trends and Potential Applications

Advancements in ion mobility separation and acquisition algorithms are expected to further increase speed and depth of analysis.
Integration with data independent acquisition strategies and machine learning based data processing may enhance quantitation accuracy and proteome coverage.
Expansion of label free methods to single cell and spatially resolved proteomics promises new insights in systems biology and precision medicine.

Conclusion

The timsTOF Pro with PASEF technology enables robust label free protein quantitation across complex proteomes with high sensitivity, precision and throughput.
Combined with intelligent data processing featuring four dimensional matching, more than 8400 proteins can be quantified reproducibly in a single 100 minute run over a five order magnitude dynamic range.

Reference

1 Meier F et al Mol Cell Proteomics 2018 doi 10.1074 mcp TIR118 000900
2 Wang et al J Proteome Res 2005 4 6 2397 2403
3 Navarro P et al Nat Biotechnology 2016 doi 10.1038 nbt 3685