A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data-Independent Acquisition Modes.

Area of Science:

• Proteomics
• Mass Spectrometry
• Quantitative Biology

Background:

• Multiplexed quantitative proteomics using reporter ions faces challenges like ratio distortion from peptide cofragmentation.
• Data-independent acquisition (DIA) offers superior reproducibility compared to data-dependent acquisition (DDA), but multiplexed labeling is less common in DIA due to spectral complexity and quantification issues.
• Existing methods struggle to balance multiplexing capabilities with accuracy and precision in DIA.

Purpose of the Study:

• To develop a versatile labeling strategy compatible with both DDA and DIA for accurate multiplexed protein quantification.
• To introduce a novel acetyl-alanine-glycine (Ac-AG) tag that conceals and reveals quantitative information via peptide-coupled reporter ions.
• To demonstrate the Ac-AG tag's ability to overcome ratio distortion and maintain quantification accuracy in complex proteomic samples.

Main Methods:

• Development of a novel acetyl-alanine-glycine (Ac-AG) tag for isobaric labeling of peptides.
• Utilizing peptide-coupled reporter ions that are precursor-specific, enabling compatibility with DDA and DIA.
• Applying the Ac-AG tag to triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) across a 10-fold dynamic range.

Main Results:

• The Ac-AG tag effectively quantifies proteins in both DDA and DIA modes, mitigating ratio distortion.
• In DDA, BSA spiked at 1:5:10 ratios was quantified as 1.00:4.87:10.13.
• In DIA, the same BSA ratios were quantified as 1.16:5.20:9.64, demonstrating high accuracy and precision.

Conclusions:

• The Ac-AG tag is a versatile tool for multiplexed quantitative proteomics, applicable to both DDA and DIA workflows.
• This method overcomes limitations of previous reporter-ion strategies, offering improved accuracy and precision.
• The Ac-AG tag facilitates robust protein quantification in complex biological samples, enhancing proteomic analysis capabilities.