Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal...

Area of Science:

• Proteomics and Mass Spectrometry
• Biochemistry and Molecular Biology

Background:

• Stable isotope-labeled internal standard peptides are essential for accurate mass spectrometry (MS)-based targeted proteomics.
• Existing methods for synthesizing these standards can be time-consuming and limited in scale.

Purpose of the Study:

• To develop a versatile and multiplexed method for absolute protein quantification using MS.
• To improve the MS-based quantification by isotope-labeled cell-free products (MS-QBiC) system for synthesizing multiple peptides in a single reaction.

Main Methods:

• Enhanced the MS-QBiC system to enable one-pot synthesis of multiple isotope-labeled peptides.
• Utilized pluralized quantification tags for simultaneous quantification of synthesized peptides.
• Applied the improved method to determine ribosomal protein stoichiometry in ribosomal subunits.

Main Results:

• Successfully demonstrated the preparation of dozens to hundreds of internal standard peptides within days.
• Enabled absolute quantification of multiple proteins using a single MS analysis run.
• Validated the method's applicability for complex biological samples like ribosomal subunits.

Conclusions:

• The developed multiplexed MS-QBiC method significantly expands the capacity for preparing internal standard peptides.
• This advancement facilitates more practical screening of reliable peptides for absolute quantification.
• The method offers high versatility for proteome analysis across various organisms without requiring cDNA or SIL peptide libraries.