Stable-isotope dimethyl labeling for quantitative proteomics.
In this paper, we report a novel, stable-isotope labeling strategy for quantitative proteomics that uses a simple reagent, formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys through reductive amination. This labeling strategy produces peaks differing by 28 mass units for each derivatized site relative to its nonderivatized counterpart and 4 mass units for each derivatized isotopic pair. This labeling reaction is fast (less than 5 min) and complete without any detectable byproducts based on the analysis of MALDI and LC/ESI-MS/MS spectra of both derivatized and nonderivatized peptide standards and tryptic peptides of hemoglobin molecules. The intensity of the a(1) and y(n-1) ions produced, which were not detectable from most of the nonderivatized fragments, was substantially enhanced upon labeling. We further tested the method based on the analysis of an isotopic pair of peptide standards and a pair of defined protein mixtures with known H/D ratios. Using LC/MS for quantification and LC/MS/MS for peptide sequencing, the results show a negligible isotopic effect, a good mass resolution between the isotopic pair, and a good correlation between the experimental and theoretical data (errors 0-4%). The relative standard deviation of H/D values calculated from peptides deduced from the same protein are less than 13%. The applicability of the method for quantitative protein profiling was also explored by analyzing changes in nuclear protein abundance in an immortalized E7 cell with and without arsenic treatment.
Hsu
Jue-Liang
JL
Department of Chemistry, National Cheng Kung University, No.1 Ta-Hsueh Road, Tainan, 701, Taiwan.
Huang
Sheng-Yu
SY
Chow
Nan-Haw
NH
Chen
Shu-Hui
SH
D000586:Amination; D002460:Cell Line; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D020543:Proteome; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D010455:Peptides; D011506:Proteins; D020543:Proteome