Protocol to identify human subcellular alternative protein interactions using cross-linking mass spectrometry.

Area of Science:

• Proteomics
• Cell Biology
• Biochemistry

Background:

• Alternative proteins (AltProts) from non-referenced open reading frames are often overlooked in mass-spectrometry-based proteomics.
• Understanding AltProt functions is crucial for a comprehensive view of cellular processes.

Purpose of the Study:

• To present a protocol for identifying human subcellular AltProts.
• To decipher AltProt interactions using cross-linking mass spectrometry.
• To enable non-targeted identification of signaling pathways involving AltProts.

Main Methods:

• The protocol involves cell culture and in cellulo cross-linking.
• Subcellular extraction and sequential digestion are key steps.
• Liquid chromatography-tandem mass spectrometry and cross-link data analysis are employed.

Main Results:

• The workflow allows for the non-targeted identification of AltProts.
• It facilitates the deciphering of interactions involving AltProts.
• Signaling pathways involving AltProts can be uncovered.

Conclusions:

• The developed protocol offers a comprehensive approach to study AltProts.
• This method expands the scope of proteomic analysis beyond canonical proteins.
• It opens new avenues for understanding cellular signaling and function.