Limited Proteolysis Mass Spectrometry to Identify Protein Structural Differences in Brain Tissue

Area of Science:

• Proteomics
• Structural Biology
• Biochemistry

Background:

• Structural proteomics enables the study of protein structural differences between conditions.
• Limited proteolysis mass spectrometry (LiP-MS) uses protease cleavage to report on protein structure.
• Previous LiP-MS implementations require specific protocols for proteome-wide analysis.

Purpose of the Study:

• To present a detailed protocol for a Limited Proteolysis Mass Spectrometry (LiP-MS) workflow.
• To enable the identification of proteome-wide protein structural changes between two experimental conditions.
• To provide a reliable and accessible method for structural proteomics research.

Main Methods:

• The protocol involves homogenizing tissue under native conditions, followed by limited proteolysis with proteinase K (PK).
• Samples are prepared for mass spectrometry (MS) and analyzed using data-dependent acquisition (DDA) or data-independent acquisition (DIA).
• Data processing utilizes FragPipe and FragPipe Limited-Proteolysis Processor (FLiPPR), including permutation analyses for reliability.

Main Results:

• The protocol successfully identifies proteins exhibiting structural changes between different experimental conditions in a proteome-wide manner.
• The workflow is applicable to brain tissue, providing insights into condition-specific structural alterations.
• Key features include the use of free and open-source software (FragPipe and FLiPPR).

Conclusions:

• This LiP-MS protocol offers a robust method for investigating proteome-wide protein structural changes.
• The described workflow is efficient, with sample preparation achievable in two days.
• The protocol facilitates reliable structural proteomics studies using accessible tools.