Comprehensive Prostate Fluid-Based Spectral Libraries for Enhanced Protein Detection in Urine

Area of Science:

• Proteomics
• Biomarker Discovery
• Mass Spectrometry

Background:

• Biofluid analysis offers insights into disease states.
• Standardized data-independent acquisition mass spectrometry (DIA-MS) workflows are needed for large-scale biofluid proteome characterization.
• Prostate cancer biomarker discovery requires robust analytical methods.

Purpose of the Study:

• To evaluate various DIA-MS data analysis workflows for biofluid proteomic analysis.
• To optimize DIA-MS for enhanced prostate cancer biomarker discovery using urine samples.
• To develop and provide high-quality urine spectral libraries for DIA-MS.

Main Methods:

• Collected urine from prostate cancer patients.
• Acquired data using data-dependent acquisition (DDA) and DIA scan modes.
• Searched DIA data against various urine spectral libraries (sample-specific, library-free, pan-human, pooled/fractionated).
• Incorporated urine subproteomes into spectral libraries.

Main Results:

• DIA-MS doubled sample throughput compared to DDA-MS with minimal peptide detection loss.
• Sample-specific spectral libraries maximized peptide detection compared to other library approaches.
• Adding urine subproteomes improved prostate protein detection in unfractionated urine.

Conclusions:

• An optimized DIA-MS workflow for prostate cancer urine analysis was established.
• Sample-specific spectral libraries are superior for maximizing peptide detection in DIA-MS.
• Provided comprehensive urine spectral libraries will facilitate future biomarker discovery studies.