Novel ¹⁵N Metabolic Labeling-Based Large-Scale Absolute Quantitative Proteomics Method for Corynebacterium glutamicum

Area of Science:

• Synthetic biology
• Proteomics
• Metabolic engineering

Background:

• Synthetic biology relies on precise protein quantification for efficient cell factory development.
• Existing absolute quantitative proteomics methods are often costly due to the need for labeled standards.
• Understanding bacterial host protein regulatory networks is crucial for optimizing bioproduction.

Purpose of the Study:

• To develop a novel, cost-effective absolute quantification method for bacterial proteomes.
• To enable precise protein level measurements for synthetic biology applications.
• To facilitate the engineering of bacterial cell factories for high-value product synthesis.

Main Methods:

• Metabolic labeling of a reference *Corynebacterium glutamicum* strain with 15N.
• Quantification of endogenous anchor proteins using light (14N) peptides.
• Utilizing the quantified proteome as an internal standard (IS) spiked into target (14N) samples.
• Employing SWATH-MS for absolute protein expression level determination.

Main Results:

• A novel metabolic labeling-based absolute quantification approach (nMAQ) was developed.
• The nMAQ method demonstrated robust quantitative performance.
• Estimated cost for nMAQ is less than $10 per sample, significantly reducing expenses.
• The method allows for absolute quantification of protein expression levels in target cells.

Conclusions:

• nMAQ offers a low-cost, efficient solution for absolute protein quantification in bacterial systems.
• This method can enhance the understanding of bacterial regulatory mechanisms in bioengineering.
• nMAQ is expected to accelerate the development of cell factories for synthetic biology applications.