This article is a U.S. Government work and is in the public domain in the U.S.A.
In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics†
Article first published online: 24 JUL 2006
This article is a U.S. Government work and is in the public domain in the U.S.A. Published in 2006 by John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Volume 20, Issue 16, pages 2463–2477, 30 August 2006
How to Cite
Simons, B. L., Wang, G., Shen, R.-F. and Knepper, M. A. (2006), In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics. Rapid Commun. Mass Spectrom., 20: 2463–2477. doi: 10.1002/rcm.2615
- Issue published online: 24 JUL 2006
- Article first published online: 24 JUL 2006
- Manuscript Accepted: 15 JUN 2006
- Manuscript Revised: 6 JUN 2006
- Manuscript Received: 17 MAR 2006
- NHLBI intramural budget. Grant Number: ZO1-HL001285
We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100 mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the ε-amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b-ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9- to D9-trimethyl-labeled peptides of β-casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes. Published in 2006 by John Wiley & Sons, Ltd.