Multi-functional MBIT for peptide tandem mass spectrometry.

Isobaric tags have been widely used for the identification and quantification of proteins in mass spectrometry-based proteomics. The mass-balanced, (1) H/(2) H isotope-coded dipeptide tag (MBIT) is a multifunctional isobaric tag based on N-acetyl-Ala-Ala dipeptide containing an amine-reactive linker that conjugates the tag to the primary amines of proteolytic peptides. MBITs provide a pair of isotope-coded quantitation signals separated by 3 Da, which enables 2-plex quantification and identification of proteins in the 15-250 fmol range. Various MBITs diversified at the N-acetyl group or at the side chain of the first alanine provide a pair of bs ions as low-mass quantitation signals in a distinct mass window. Thus, a combination of different MBITs allows multiplex quantification of proteins in a single liquid chromatography-mass spectrometry experiment. Unlike other isobaric tags, MBITs also offer a pair of ys ions as high-mass quantitation signals in a noise-free region, facilitating protein quantification in quadrupole ion trap mass spectrometers. Uniquely, bS ions, forming N-protonated oxazolone, undergo unimolecular dissociation and generate the secondary low-mass quantitation signals, aS ions. The yield of aS ions derived from bS ions can be used to measure the temperature of bS ions, which enables a reproducible acquisition of the peptide tandem mass spectra. Thus, MBITs enable multiplexed quantitation of proteins and the concurrent measurement of ion temperature using bS and aS signal ions as well as the isobaric protein quantitation in resonance-type ion trap using yS (complement of bS ) signal ions. This review provides an overview of MBITs with a focus on the multi-functionality that has been successfully demonstrated in the peptide tandem mass spectrometry.