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 b s 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 y s ions as high‐mass quantitation signals in a noise‐free region, facilitating protein quantification in quadrupole ion trap mass spectrometers. Uniquely, b S ions, forming N‐protonated oxazolone, undergo unimolecular dissociation and generate the secondary low‐mass quantitation signals, a S ions. The yield of a S ions derived from b S ions can be used to measure the temperature of b S 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 b S and a S signal ions as well as the isobaric protein quantitation in resonance‐type ion trap using y S (complement of b S ) 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. © 2014 The Authors. Mass Spectrometry Reviews published by Wiley Periodicals, Inc. Mass Spec Rev 34: 209–218, 2015.