Data Processing Workflows for DDA and DIA LC-MS data using Symphony and MSConvert

Significance of the Topic

Open source mass spectrometry data formats such as mzML and mgf have become essential for enabling flexible and transparent analysis of LC MS datasets across multiple platforms. The ability to convert proprietary raw files into community standards allows researchers to apply advanced analytics including molecular networking, library searches, and multivariate statistics using third party tools. This interoperability enhances data reuse and accelerates discoveries in metabolomics and proteomics.

Objectives and Study Overview

This brief demonstrates a streamlined workflow that converts Waters MassLynx high resolution DDA and DIA raw data into open source formats using Symphony Data Pipeline in combination with ProteoWizard MSConvert. A catechin standard mixture is used to illustrate conversion steps, compatibility with the GNPS molecular networking platform, and the benefits of automated processing immediately after acquisition.

Methodology and Instrumentation

A three tiered processing scheme is presented:

• Raw data access via Application Programming Interface for direct third party analysis
• Format conversion of continuum data to mzML or mzXML using MSConvert
• Peak detection, centroiding, and deconvolution to generate precursor product lists for complex DIA modes such as MSE, HDMSE and SONAR

Symphony pipelines orchestrate command line modules for three dimensional peak detection, deisotoping, conversion to mgf, and final transformation to mzML. Processing is triggered automatically from the sample list in MassLynx upon completion of each run.

Used Instrumentation

• Waters Xevo or Synapt series LC MS system with MassLynx software
• Symphony Data Pipeline for workflow automation
• ProteoWizard MSConvert for format translation
• GNPS platform for molecular networking and library searching

Main Results and Discussion

The catechin dataset was processed through Symphony to yield mgf and mzML files without user intervention. Submitting these files to GNPS produced high confidence library matches for individual spectra and constructed a molecular network of the mixture. The in line conversion enabled immediate availability of open source files for downstream analysis, reducing manual handling and improving throughput.

Benefits and Practical Applications

• Automated conversion ensures consistent data handling and reduces risk of manual errors
• Immediate file availability after acquisition supports high throughput studies
• Open formats foster use of diverse informatics tools for metabolomics flux analysis, compound screening, and statistical modeling
• Compatibility with both DDA and advanced DIA acquisition modes expands application scope

Future Trends and Applications

Advances are expected in cloud based processing pipelines and integration of machine learning for automated feature detection. Expansion of open spectral libraries and community curation will enhance compound annotation. Emerging acquisition strategies may require further development of vendor agnostic deconvolution algorithms to maintain seamless interoperability.

Conclusion

Symphony Data Pipeline combined with MSConvert provides a robust and efficient workflow to convert Waters LC MS raw files into open source formats. This approach supports diverse third party applications, accelerates data analysis, and promotes open science practices in high resolution DDA and DIA mass spectrometry.

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