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

Abstract

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 50% 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.

Quantitative Proteome Analysis Reveals RNA Processing Factors As Modulators of Ionizing Radiation-Induced Apoptosis in the C. elegans Germline.

“…IPTL was performed according to Koehler et al 2011. 16 Briefly, purified and dried Lys-C peptide digests were suspended in 20 mM succinic anhydride (Sigma-Aldrich, Oslo, Norway) for control samples and in tetradeuterated succinic anhydride-d 4 (Larodan Fine Chemicals AB, Malmo, Sweden) for apoptotic samples in 50 mM sodium acetate.…”

“…Protein identification and validation was performed using the IsobariQ software 17 including the force-find algorithm 16 to mine the spectra where Mascot failed to identify opposite sequences. No sequence suggestion below a Mascot ion score of 10 was allowed.…”

Mass Defect-Based Pseudo-Isobaric Dimethyl Labeling for Proteome Quantification

“…Although IPTL improved the quantification accuracy compared to other reported MS/ MS level methods, the side reactions that are inherent in the multistep labeling can adversely affect quantification accuracy and dynamic range. 12 NeuCode SILAC 14 is another strategy based on subtle mass differences resulting from the mass defects of 12 C/ 13 C, …”

Strategies for improving sensitivity and selectivity for the quantitation of biotherapeutics in biological matrix using LC-MS/MS

“…The application of phenyl isocyanate labeling enabled relative quantitation of several styrene oxide adducts of human hemoglobin by LC-MS/MS [32]. Koehler et al [33] established a selective N-terminal peptide modification reaction using succinic anhydride and subsequently applied it to compare the proteome of HeLa cells incubated with S-trityl-l-cysteine to induce mitotic arrest and apoptosis. More recently, Yuan et al [34] reported a selective peptide derivatization approach to improve assay sensitivity for protein bioanalysis.…”