N‐Terminal Labeling of Peptides by Trypsin‐Catalyzed Ligation for Quantitative Proteomics

Abstract

Labeling tryptic peptides with stable isotopes is one of the most important methods for quantitative proteomics, and many ingenious chemical labeling strategies have been developed. Incorporation of one isotopically labeled tag onto a peptide terminus represents an ideal labeling approach, as it would simplify the interpretation of mass spectra. Moreover, the absence of labels on the side chains would facilitate the quantification of post-translational modifications (PTMs). However, to date all the reported chemical labeling strategies, including dimethyl labeling, iTRAQ (isobaric tags for absolute and relative quantification), and ICAT (isotope-coded affinity tags), result in the modification of side chains. One promising method to achieve the incorporation of a single tag is enzymatic labeling. Proteolytic O labeling can specifically label tryptic peptide termini without modification of side chains, but the small change in mass and O/O back-exchange, i.e. the exchange of O with O after the labeling process hinder its wide application in quantitative proteomics. Herein, we report a novel enzymatic labeling approach, in which trypsin is used as a ligase to specifically incorporate amino acids labeled with stable isotopes onto the N termini of peptides for quantitative analysis. Trypsin is a serine protease that specifically hydrolyzes peptide bonds in proteins after arginine and lysine residues. However, trypsin also catalyses peptide synthesis in organic solvents. Therefore, it is possible to covalently link isotopically labeled amino acids to tryptic peptides by using trypsin as a ligase. In this study, arginine, the prototype substrate for trypsin, was used as an acyl moiety donor. The primary amine group of arginine was protected with a benzoyl group (Bz) to prevent the formation of dipeptides or oligopeptides, and the carboxy group was esterified with ethanol to activate the acyl donor (see the Supporting Information, Figure S1). The final product, Na-benzoyl-l-arginine ethyl ester (Bz–R–OEt), was used as a substrate for the trypsin-catalyzed ligation. The quantitative proteomics workflow based on the trypsincatalyzed N-terminal labeling is shown in Figure 1a. Trypsin was first used as a protease to digest proteins in an aqueous solution (1m urea/50 mm Tris-HCl, pH 8.0). Then, the generated tryptic peptides were lyophilized and transferred to an ethanol solution containing 4% aqueous buffer (0.1m TrisHCl, pH 8.0). Next, trypsin immobilized on magnetic nanoparticles (IM-trypsin) and Bz–R–OEt were added to the ethanol solution for the N-terminal labeling of peptides with Bz–R based on a kinetically controlled mechanism (see the Supporting Information). Finally, quantification of the proteins was achieved by the differential labeling of two samples by using Bz–R–OEt (light label) and Bz–(C6)R–OEt (bearing six C atoms; heavy label), the incorporation of which are indicated by mass shifts of 260 and 266 Da, respectively. We first validated the protease and ligase activities of trypsin by using the synthetic peptide VGKANEELAGVVAEVQK (Figure 1b; m/z= 1740.86), which contains one trypsin cleavage site. In aqueous solution, treatment with IMtrypsin generated a shorter peptide ANEELAGVVAEVQK (Figure 1b, m/z= 1456.82). Treatment of the obtained peptide with an ethanol solution containing Bz–R–OEt and IMtrypsin gave an N-terminus labeled peptide Bz–RANEELAGVVAEVQK (Figure 1b, m/z= 1716.9). Similar results were also obtained for three other synthetic peptides that contained one trypsin cleavage site (Supporting Information, Figure S2). These examples clearly illustrate that trypsin functions as a protease in an aqueous solution whereas it acts as an N-terminus ligase in an ethanol solution. Thus, these results imply that this enzymatic labeling approach can be used to label peptides generated by trypsin digestion of a proteome sample. In this study, free trypsin was used for the digestion of proteins, as is conventional in proteomics analysis, whereas IM-trypsin (trypsin from the same source) was used for ligation because it is more tolerant to organic solvents and it is readily removed using magnetism. We tested whether the labeling of side-chain primary amino groups would also be facilitated by trypsin ligase. A synthetic peptide VIFIEHAKRKG, containing two sidechain amino groups and one terminal primary amino group, was labeled by trypsin. The Arg tag was incorporated only onto the terminal primary amino group and not onto the sidechain amino groups (see the Supporting Information, Figure S3a). These results are markedly different from those of other amine labeling approaches. For example, for labeling [*] Y. Pan, Prof. Dr. M. Ye, Dr. L. Zhao, K. Cheng, M. Dong, C. Song, H. Qin, Dr. F. Wang, Prof. Dr. H. Zou CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences Dalian 116023 (China) E-mail: mingliang@dicp.ac.cn hanfazou@dicp.ac.cn