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

Abstract

Isotope-labeled N-acetyl dipeptides (Ac-Xxx-Ala) are coupled to the primary amines of tryptic peptides and then analyzed by tandem mass spectrometry. Amide bond cleavage between Xxx and Ala provides both low-and high-mass isotope-coded signals for quantification of peptides. Especially, facile cleavage at the modified lysine side chain yields very strong high-mass quantitation signals in a noise-free region. Tagging tryptic peptides with isobaric N-acetyl dipeptides is a viable strategy for accurate quantification of proteins, which can be used with most quadrupole ion trap mass spectrometers carrying the 1/3 mass cut-off problem.

Multi‐functional MBIT for peptide tandem mass spectrometry

“…Furthermore, the b S ion (N‐protonated oxazolone) enables a precise measurement of ion temperature from the reaction yield of its subsequent unimolecular dissociation to the a S ion (Seo et al, ), because the dissociation rate depends on the internal energy of the b S ion which varies with the temperature of the peptide ion. Most importantly, the isotope‐coded high‐mass H/L y S ions (protonated Ala‐peptides), unlike their complementary low‐mass L/H b S ions, afford the isobaric quantification of peptides in the quadrupole ion trap (QIT) where a 1/3 mass cutoff problem hinders the use of other isobaric tags based on low‐mass signals (Seo, Yoon, & Shin, ).…”

“…In addition, the m / z 100–220 region is ion‐rich due to other fragment peaks overlapping with the signal ions. MBITs can circumvent these problems by providing high‐mass signals in a noise‐free region for the accurate protein quantification (Seo, Yoon, & Shin, ).…”

“…To examine high‐mass quantitation signals for peptide quantification, we conjugated the Gln‐tag (Ac‐QA‐OSu) to two model peptides, LISFYAGR ( 1 ) and LISFYAGK ( 2 ), which have the same sequence except for the C ‐terminal amino acid (Seo, Yoon, & Shin, ). The amine‐reactive coupling via the OSu‐linker resulted in Ac‐QA‐LISFYAGR ( 1 1 ) with one Gln‐tag conjugated at the N ‐terminus and Ac‐QA‐LISFYAGK‐AQ‐Ac ( 2 2 ) with two Gln‐tags conjugated at the N ‐terminus and lysine.…”

“…Lastly, the performance of MBITs on protein quantification was demonstrated with a protein mixture containing horse myoglobin, bovine serum albumin (BSA), and human ubiquitin by using a linear QIT mass spectrometer connected to a nano‐LC system (Seo, Yoon, & Shin, ). These proteins were mixed in two different ratios (samples A and B) and digested with trypsin.…”

“…They offer a pair of 3‐Da gap quantitation signals in both low‐ and high‐mass regions with no or little overlapping peptide fragments. Thus, MBITs can be employed in ion trap for the isobaric protein quantification (Seo, Yoon, & Shin, ). The signal mass window of MBITs can be varied by diversifying the dipeptide structure (Seo et al, ; Suh et al, ).…”