Optimized DDA+ and HR-DIA workflows for standardized, reproducible, precise and robust label-free quantitation of proteomes

Significance of the Topic

The accurate and reproducible quantitation of proteins in complex samples is essential for biomarker discovery and comparative proteomics. Traditional label free workflows often suffer from missing values and variability in nano flow chromatography and data dependent acquisition methods. Developing robust and high throughput label free quantitation workflows addresses key challenges in large scale studies and quality controlled environments

Objectives and Study Overview

This work aimed to establish two complementary label free precursor level quantitation workflows that deliver standardized operation, reproducible performance, comprehensive proteome coverage and minimal missing values. The data dependent acquisition plus workflow known as DDA plus and a high resolution data independent acquisition workflow referred to as HR DIA were evaluated for robustness over more than one thousand injections of HeLa digest and for precision in replicate analyses of complex samples

Methodology and Instrumentation

A unified capillary flow based platform was configured using a Thermo Scientific UltiMate 3000 RSLCnano system coupled to a 150 micrometer EASY Spray column operating at 1.2 microliters per minute. Peptides were separated on a 45 to 40 percent acetonitrile gradient over 45 minutes. The Q Exactive HF X hybrid quadrupole Orbitrap mass spectrometer provided high resolving power and mass accuracy for both MS1 and MS2 scans. Data analysis for DDA plus employed Proteome Discoverer 2.2 software with the Minora Feature Detector and Precursor Ions Quantifier nodes. HR DIA data were processed with Spectronaut software using a 1 percent false discovery rate filter at the peptide level

Instrumentation Used

• UltiMate 3000 RSLCnano HPLC system with capillary flow pump and trap cartridge
• 150 micrometer EASY Spray column in back flush configuration
• Q Exactive HF X hybrid quadrupole Orbitrap mass spectrometer
• Proteome Discoverer 2.2 software and Spectronaut DIA software

Main Results and Discussion

DDA plus showed durable performance over 1000 injections with less than five percent relative standard deviation in quantified proteins and over 4500 protein groups consistently identified. The enhanced Minora algorithm boosted peptide quantitation reproducibility to 97 percent across triplicates compared to about 50 percent in conventional DDA. HR DIA achieved over 4200 protein groups quantified per run with coefficients of variation below twenty percent for the majority of measurements. Both workflows effectively eliminated traditional missing value issues and maintained high sensitivity and throughput

Benefits and Practical Applications of the Method

• Highly reproducible quantitation enabling large cohort studies with minimal data gaps
• Robust capillary flow chromatography reduces downtime and maintenance
• Flexible choice between data dependent and data independent acquisition based on study needs
• User friendly software workflows yield standardized and automated data processing

Future Trends and Potential Applications

Ongoing developments in faster scanning Orbitrap analyzers and advanced data analysis algorithms will further improve throughput and depth of coverage. Integration with machine learning based spectral prediction and cloud based data management can facilitate real time quality control and remote laboratory standardization. Expansion to post translational modification analysis and single cell proteomics applications represents emerging areas for these workflows

Conclusion

The optimized DDA plus and HR DIA label free quantitation workflows provide robust, precise and high throughput proteome analysis with minimal missing values. Standardized instrumentation and software tools ensure reproducible results across extended injection series and replicate analyses, making these methods well suited for large scale biomarker discovery and industrial quality control

References

1. A Gajadhar Pushing the leading edge in protein quantitation Integrated precise and reproducible proteomic workflows PlanetOrbitrap com
2. Top reasons to upgrade to a Q Exactive HF X hybrid quadrupole Orbitrap mass spectrometer Technical Guide Grant Application Resource PlanetOrbitrap com
3. X Zhang A Gajadhar X Sun M Baynham A highly sensitive and robust 150 micrometer column to enable high throughput proteomics Application Note 21744 PlanetOrbitrap com