Low-Input Proteomics Protocols for Laser-Captured FFPE Samples with Orbitrap Astral DIA-MS.

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Laser microdissection (LMD) coupled with mass spectrometry (MS) is vital for analyzing tissue heterogeneity.
• Analyzing low-input tissue samples presents significant challenges in proteomic studies.
• Existing protocols may not be optimized for minimal sample loss or manual processing.

Purpose of the Study:

• To present two optimized protocols for low-input sample preparation for LMD-MS analysis.
• To enable sensitive, high-resolution proteomic analysis of microdissected tissue regions.
• To provide guidance for reproducible proteomic profiling of clinical samples.

Main Methods:

• Development and optimization of two low-input sample preparation workflows: Solid-Phase Enhanced Preparation (SP3) and Detergent/Organic Solvent (DDM/ACN).
• Application of protocols to small tissue areas (down to 1500 μm² of 10 μm thick FFPE tissue).
• Integration with high-resolution mass spectrometry (Orbitrap Astral) using Data-Independent Acquisition (DIA).

Main Results:

• Protocols minimize sample loss, enabling analysis of very small tissue regions.
• Achieved reproducible proteomic profiling with substantial proteome depth from low-input samples.
• Demonstrated sensitive and high-resolution proteomic analysis of microdissected FFPE tissue.

Conclusions:

• The developed SP3 and DDM/ACN protocols offer robust solutions for low-input LMD-MS.
• These methods facilitate the investigation of spatial heterogeneity in clinical samples with high precision.
• The protocols are suitable for manual processing, increasing accessibility for researchers without automation platforms.