Dimethyl isotope-coded affinity selection for the analysis of free and blocked N-termini of proteins using LC-MS/MS.
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.
Shen
Po-Tsun
PT
Department of Chemistry, National Cheng Kung University, No. 1 College Road, Tainan, 701, Taiwan.
Hsu
Jue-Liang
JL
Chen
Shu-Hui
SH
D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D003903:Deuterium; D005557:Formaldehyde; D006801:Humans; D007553:Isotope Labeling; D008745:Methylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D011506:Proteins; D005557:Formaldehyde; D003903:Deuterium