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Mass-balanced 1H/2H-isotope dipeptide tag (MBIT) is diversified as aliphatic tags for multiplexed protein quantification. Aliphatic MBITs are based on the N-acetyl-Xxx-Ala dipeptide, where Xxx is an artificial amino acid with a linear alkyl side chain from C2H5to C8H17(C2–C8tags). 1H/2H isotopes are encoded in the methyl groups of N-acetyl and Ala to yield a pair of isobaric tags with 2-plex quantitation signals separated by 3 Da. C2–C5tags are prepared by solid-phase synthesis, while C6–C8tags are synthesized by olefin metathesis in solution. These aliphatic tags are made reactive toward the primary amines of peptides, and the relative abundances of quantitation signals are characterized using both matrix-assisted laser desorption ionization and electrospray ionization tandem mass spectrometry. MBIT-linked peptides co-migrate in reverse-phase liquid chromatography (LC), and their tandem mass spectra exhibit 2-plex quantitation signals as well as sequence ions in similar abundances. As the length of alkyl side chain increases, C2–C8tags show a stepwise increase in both the LC retention time and the relative abundance of quantitation signals. In addition, the quantitation linearity is well-maintained in a 15–250 fmol range. The multiplexing capability of aliphatic MBITs is demonstrated by applying three different tags (C6–C8tags) to the quantification of yeast heat shock proteins expressed under four different physiological conditions. [ABSTRACT FROM AUTHOR]