Comprehensive comparison of sample preparation workflows for proteomics

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Mass spectrometry-based proteomics experiments often suffer from high variability, hindering accurate and deep protein identification.
• Optimizing sample preparation is essential for overcoming these limitations in human tissue analysis.

Purpose of the Study:

• To systematically compare key sample preparation steps for in-depth proteome identification in human tissues.
• To establish an optimal workflow for highly efficient and unbiased global proteomic analysis.

Main Methods:

• Comparison of lysis buffers (SDS vs. urea/thiourea), precipitation methods (acetone), proteolytic digestion techniques (in-solution vs. FASP), and pre-fractionation strategies (SDS-PAGE vs. hi-pH RPLC).
• Evaluation of combined methods for identifying low molecular weight (LMW) proteins.

Main Results:

• The workflow combining urea/thiourea lysis, in-solution digestion, and hi-pH RPLC significantly increased proteome coverage (+15%) and matched peptides (+42.4%).
• This optimized workflow also identified 3 previously classified missing proteins (MPs) according to Human Proteome Project (HPP) guidelines.
• Performance varied across different protein groups, highlighting the importance of workflow selection.

Conclusions:

• The urea/thiourea lysis, in-solution digestion, and hi-pH RPLC method provides an optimal sample preparation workflow for human tissues.
• This workflow enhances proteome coverage, protein identification rates, and the discovery of low molecular weight and missing proteins.
• The findings contribute to more efficient and unbiased global proteomic analysis.