Mass-Balanced ¹H/²H Isotope Dipeptide Tag for Simultaneous Protein Quantitation and Identification

Abstract

Mass-balanced (1)H/(2)H isotope dipeptide tags (MBITs) are presented for simultaneous protein quantitation and identification. MBIT is derived from N-acetyl-Ala-Ala dipeptide and conjugated to primary amines of target peptides. (1)H/(2)H isotopes are encoded in the methyl groups of N-acetylated dipeptide: one tag deuterated on the N-acetyl group and another on the C-terminal alanine. MBIT-linked peptides comigrate in reversed-phase liquid chromatography without significant (1)H/(2)H isotope effects and provide 2-plex quantitation signals at 114 and 117 Th as well as peptide sequence information upon MS/MS analysis with MALDI TOF/TOF. MBIT shows good quantitation linearity in a concentration range of 20-250 fmol. The performance of MBIT on protein quantitation and identification is further tested with yeast heat-shock protein (Hsp82p) obtained from three different physiological states. MBIT using nanogram-scale samples produces the relative abundance ratios comparable to those obtained from optical imaging of microgram-scale samples visualized with SYPRO Ruby stain. The MBIT strategy is a simple and low-cost alternative for 2-plex quantitation of proteins and offers possibilities of tuning the 2-plex signal mass window by replacing the N-terminal alanine with other amino acid residues.

Quantification of Tryptic Peptides in Quadrupole Ion Trap Using High-Mass Signals Derived from Isotope-Coded N-Acetyl Dipeptide Tags

“…Preparation of the acid form of mass-balanced H/D-isotope tag ( L/H MBIT) is described elsewhere [10]. N-hydroxysuccinimide (NHS), hydroxylamine hydrochloride, N,N'-dimethylformamide (DMF, HPLC grade), trifluoroacetic acid (TFA), formic acid, and 4-hydroxy-α-cyano-cinnamic acid (HCCA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).…”

“…Peptides of interest are differentially labeled with isotopecoded tags and the resulting isotopomeric precursor ions are analyzed by tandem mass spectrometry (MS/MS) to identify the peptide sequence and to simultaneously quantify the amounts of differentially-labeled peptides. All of the isobaric tags available to date are designed to report quantitation signals in the m/z 100-250 region [4][5][6][7][8][9][10][11][12][13]. However, this low-mass region is inaccessible by conventional quadrupole ion trap (QIT) because of the~1/3 mass cut-off problem in ion trapping during resonant excitation of the precursor ion [14].…”

“…However, this low-mass region is inaccessible by conventional quadrupole ion trap (QIT) because of the~1/3 mass cut-off problem in ion trapping during resonant excitation of the precursor ion [14]. Moreover, the m/z 100-250 region is ion-rich due to other small fragment peaks overlapping in this region [10]. Although a couple of novel ion trapping methods have been recently developed to overcome this low-mass cut-off problem [15][16][17][18], they are not directly applicable to most QIT mass spectrometers.…”

“…Recently, we have reported mass-balanced H/D-isotope tags (MBITs) based on Ac-Xxx-Ala dipeptides terminated with an amine-reactive O-succinimidyl (OSu) ester at the Cterminus (Scheme 1) [10,11].…”

“…The variable amino acid Xxx can be chosen either from a natural amino acid [10] or from an artificial amino acid [11] to diversify MBITs (Xxx-tags). The variation of side chain R T shifts quantitation signals and modulates chemical properties of the tagged peptides.…”