Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Mass spectrometry (MS)-based proteomics has advanced significantly, particularly with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling.
• Accurate quantitation of whole proteomes in complex biological samples remains a challenge.

Purpose of the Study:

• To introduce a deep-proteomics profiling protocol for accurate whole proteome quantitation.
• To combine 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform and computational interference correction.

Main Methods:

• Protein extraction, digestion, and 10-plex TMT labeling.
• Two-dimensional (2D) liquid chromatography (LC) coupled to high-resolution tandem mass spectrometry (LC-MS/MS).
• Post-MS computational interference correction for accurate quantitation and quality control.

Main Results:

• The protocol enables confident quantitation of over 10,000 proteins in mammalian samples.
• Demonstrated robustness and applicability to various complex samples like cell cultures, tissues, and clinical specimens.
• Potential for adaptation to quantitate post-translational modifications.

Conclusions:

• This multiplexed and robust method offers a powerful tool for deep proteome profiling.
• The protocol enhances the accuracy and capacity of quantitative proteomics.
• Facilitates comprehensive proteomic analysis across diverse biological contexts.