A novel method for identification and relative quantification of N-terminal peptides using metal-element-chelated tags coupled with mass spectrometry

Abstract

By means of the reaction between a DOTA-NHS-ester bifunctional reagent and N-terminal peptides of proteins, and then chelation of lanthanide metal ions as tags, we established a novel method for the identification of N-terminal peptides of proteins and their relative quantification using metal-element-chelated tags coupled with mass spectrometry. The experimental results indicate that metal elements are able to completely label N-terminal peptides at the protein level. The N-terminal peptides are enriched as the peptides digested with trypsin are selectively eliminated by isothiocyanate-coupled silica beads. We successfully identified the N-terminal peptides of 158 proteins of Thermoanaerobacter tengcongensis incubated at 55 and 75 °C, among which N-terminal peptides of 24 proteins are partially acetylated. Moreover, metal-element tags with high molecule weights make it convenient for N-terminal peptides consisting of less than 6 amino acids to be identified; these make up 55 percent of the identified proteins. Finally, we developed a general approach for the relative quantification of proteins based on N-terminal peptides. We adopted lysozyme and ribonuclease B as model proteins; the correlation coefficients (R 2) of the standard curves for the quantitative method were 0.9994 and 0.9997, respectively, with each concentration ratio ranging from 0.1 to 10 and both relative standard derivations (RSD) measured at less than 5%. In T. tengcongensis at two incubation temperatures, 80 proteins possess quantitative information. In addition, compared with the proteins of T. tengcongensis incubated at 55 °C, in T. tengcongensis incubated at 75 °C, 7 proteins upregulate whereas 16 proteins downregulate, and most differential proteins are related to protein synthesis.