Isobaric Peptide Termini Labeling Utilizing Site-Specific N-Terminal Succinylation

摘要

Recently, we introduced a novel approach for protein quantification based on isobaric peptide termini labeling (IPTL). In IPTL, both peptide termini are dervatized in two separate chemical reactions with complementary isotopically labeled reagents to generate isobaric peptide pairs. Here, we describe a novel procedure for the two chemical reactions to enable a cost-effective and rapid method. We established a selective N-terminal peptide modification reaction using succinic anhydride. Dimethylation was used as second chemical reaction to derivatize lysine residues. Both reactions can be performed within 15 min in one pot, and micropurification of the peptides between the two reactions was not necessary. For data analysis, we developed the force-find algorithm in IsobariQ which searches for corresponding peaks to build up peak pairs in tandem mass spectrometry (MS/MS) spectra where Mascot could not identify opposite sequences. Utilizing force-find, the number of quantified proteins was improved by more than 50percent in comparison to the standard data analysis in IsobariQ. This was applied to compare the proteome of HeLa cells incubated with S-trityl-L-cysteine (STLC) to induce mitotic arrest and apoptosis. More than 50 proteins were found to be quantitatively changed, and most of them were previously reported in other proteome analyses of apoptotic cells. Furthermore, we showed that the two complementary isotopic labels coelute during liquid chromatography (LC) separation and that the linearity of relative IPTL quantification is not affected by a complex protein background. Combining the optimized reactions for IPTL with the open source data analysis software IsobariQ including force-find, we present a straightforward and rapid approach for quantitative proteomics.