Multiplexed complexome profiling using tandem mass tags.

Area of Science:

• Biochemistry
• Proteomics
• Cell Biology

Background:

• Complexome profiling is a key technique for studying protein complexes using mass spectrometry.
• Current methods generate numerous samples, increasing analysis time and cost.
• Characterizing protein complex assembly and interactions is crucial for understanding cellular functions.

Purpose of the Study:

• To develop an improved workflow for complexome profiling using tandem mass tags for multiplexing.
• To reduce the number of samples and mass spectrometry analysis time.
• To maintain or improve the reliability of protein identification and quantification.

Main Methods:

• Implementation of tandem mass tags (TMT) for multiplexing complexome profiling.
• Separation of protein complexes by molecular mass using ultracentrifugation.
• Label-free and TMT-based bottom-up proteomics using mass spectrometry.
• Analysis of mitochondrial fractions from cells recovering from chloramphenicol treatment.

Main Results:

• Tandem mass tags-multiplexed complexome profiling substantially reduced sample numbers and measuring time.
• The method accurately characterized ATP synthase (complex V) migration patterns and assembly intermediates.
• Protein and complex quantification showed comparable or reduced variation versus the label-free approach.
• TMT-multiplexing did not compromise protein identification or quantification reliability.

Conclusions:

• Tandem mass tags provide an efficient and robust method for complexome profiling.
• This multiplexing strategy enhances throughput and may broaden the application of complexome profiling.
• The improved workflow supports advanced protein complex analysis in biomedical research and diagnostics.