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Quantitative Proteome Analysis Using Differential Stable Isotopic Labeling and Microbore LC−MALDI MS and MS/MS
journal contribution
posted on 2005-06-13, 00:00 authored by Chengjie Ji, Liang LiWe demonstrate an approach for global quantitative analysis of protein mixtures using differential
stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC
provides higher sample loading, compared to capillary LC, which facilitates the quantification of low
abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a
heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification
to carboxylic groups and dimethylation to amine groups of peptides) with this LC−MALDI technique
are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in
individual sample spots are obtained to determine the abundance ratio among pairs of differential
isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing
significant abundance differences to determine the sequences of selected peptides for protein
identification. The peptide sequences determined from MS/MS database search are confirmed by using
the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The
effectiveness of this microbore LC−MALDI approach is demonstrated in the quantification and
identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of
known relative concentrations. It is shown that this approach provides a facile and economical means
of comparing relative protein abundances from two proteome samples.
Keywords: quantitative proteomics • isotope labeling • microbore LC • LC−MALDI