A Versatile Isobaric Tag Enables Proteome Quantification in Data Dependent and Data Independent Acquisition Mode

Quantifying proteins based on peptide-coupled reporter-ions is a leading multiplexed quantitative strategy in proteomics that significantly alleviates the problem of ratio distortion caused by peptide co-fragmentation, as commonly observed in other reporter-ion based approaches, such as TMT and iTRAQ. Fueled by improvements in mass spectrometry and data processing, data-independent acquisition (DIA) is an attractive alternative to data dependent acquisition (DDA) due to its better reproducibility. While multiplexed labeling is widely used in DDA, it is rarely used in DIA, presumably because current approaches lead to more complex MS2 spectra or to a reduction in quantification accuracy and precision. Herein, we present a versatile acetyl-alanine-glycine (Ac-AG) tag which conceals quantitative information in isobarically labeled peptides and reveals it upon tandem MS in the form of peptide-coupled reporter-ions. Since the peptide-coupled reporter-ion is precursor-specific while fragment ions of the peptide backbone originating from different labeling channels are same, the Ac-AG tag is compatible with both the widely adopted DDA as well as with the DIA mode. By isolating the monoisotopic peak of the precursor ion in DDA, intensities of the peptide-coupled reporter-ions simply represent the relative ratios between constituent samples. While in DIA, the ratio can be inferred after deconvoluting peptide-coupled reporter-ions. The proteome quantification capability of the Ac-AG tag was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. Within a complex proteomics background, the BSA spiked at 1:5:10 ratios was detected at ratios of 1.00 : 4.87 : 10.13 in DDA and 1.16 : 5.20 : 9.64 in DIA.