These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Dimethyl isotope-coded affinity selection for the analysis of free and blocked N-termini of proteins using LC-MS/MS.
    Author: Shen PT, Hsu JL, Chen SH.
    Journal: Anal Chem; 2007 Dec 15; 79(24):9520-30. PubMed ID: 18001127.
    Abstract:
    Because of enhanced a(1) ion signals, dimethyl labeling is useful for identifying the N-termini of proteins or peptides. Herein, we describe a novel strategy that uses dimethyl isotope-coded affinity selection (DICAS) to isolate peptides that contain either the dimethylated or in vivo blocked N-termini of proteins for comprehensive sequence analyses by LC-MS/MS. In this method, dimethyl labeling at the protein level was first performed using formaldehyde-d(2) to label all unblocked protein N-termini and lysine residues, followed by trypsin digestion. The free N-terminal amines of internal peptides generated by digestion were captured by solid supports with aldehyde functionalities through reductive amination. The flow-through fractions, which contained either in vivo or in vitro blocked N-terminal peptides, were subjected to sequence analyses by LC-MS/MS. Owing to the unique feature of a1 signal enhancement associated with dimethylated peptides and the use of the deuterium reagent, the in vitro blocked (or in vivo free) N-termini of proteins could be easily differentiated from the in vivo blocked N-termini. Thus, their sequences and N-terminal modifications could be assigned unambiguously from MS/MS spectra. In this study, the completeness of the labeling and the efficiency of the isolation method were first confirmed by the use of a mixture of model proteins composed of hemoglobin, myoglobin, and alpha-lactalbumin. The N-termini of all three proteins, including both alpha and beta chains of hemoglobin as well as a signal sequence located in the N-termini of alpha-lactalbumin, were successfully identified. The protocol was then applied to the analysis of an unfractionated lysate of MCF-7 cells. Results indicate that more than 80% of the isolated peptides contained the N-termini of unique proteins, and many of them were consistent with known or inferred N-terminal processing such as methionine removal and acetylation. In addition, using the DICAS approach, we identified a novel N-terminal formylation for the Ig kappa chain V-III region SIE protein and a novel N-terminal signal sequence (1th-32th amino acid) for profilin.
    [Abstract] [Full Text] [Related] [New Search]