Analyzing Various Biological Fluids with a Single Automated Proteomic Workflow for Biomarker Discovery

Area of Science:

• Proteomics
• Biomarker Discovery
• Clinical Mass Spectrometry

Background:

• Protein biomarker discovery in biological fluids is crucial for understanding human diseases.
• Technological advancements have improved proteome coverage and sample throughput in recent years.
• While blood is common, other fluids like CSF, urine, and milk offer unique insights for specific pathologies.

Purpose of the Study:

• To adapt and validate a scalable automated proteomic pipeline (ASAP²) for diverse biofluid samples.
• To demonstrate the workflow's applicability beyond blood and CSF, including milk and urine.
• To maintain high-throughput and robustness for large-scale clinical proteomic studies.

Main Methods:

• Adaptation of the ASAP² workflow for milk (defatting) and urine (acetone precipitation) samples.
• Automated sample preparation including reduction, alkylation, digestion, and peptide labeling (isobaric).
• Liquid chromatography-mass spectrometry (LC-MS) analysis and subsequent data processing.

Main Results:

• The ASAP² workflow was successfully modified for milk and urine sample preparation.
• Key automated steps (reduction, alkylation, digestion, labeling, pooling, purification) are consistent across biofluids.
• The pipeline supports high-throughput analysis essential for biomarker discovery in clinical studies.

Conclusions:

• The adapted ASAP² workflow provides a robust and scalable solution for proteomic analysis of diverse human biofluids.
• This standardized approach facilitates large-scale protein biomarker discovery across various sample types.
• The methodology supports efficient sample preparation for LC-MS-based clinical research.