Peptides Labeling with Stable Isotope


Peptides are organic compounds formed by the dehydration of amino acids and intermediate products of protein hydrolysis. Peptides have strong biological activity, which can regulate the functional activities of cells in living organisms, and play a variety of roles in medicine, cosmetics, food, and other fields. With the more and more extensive and in-depth applications of peptides, the demand for labeled and modified peptides is growing, and the quality requirement is also higher and higher. To obtain peptides with higher biological activity, fluorescent modification, isotope labeling, and other peptide modification techniques have been applied in biomedical and life science research fields.

 

Isotope labeling is a technique to track isotopes through reactions, metabolic pathways, or cellular channels. Stable isotope labeling can realize the study of the metabolic pathway of peptides and can sensitively and accurately track changes according to the location and quantity of peptides containing isotope-labeled in vivo or in vitro at any time. The most common stable isotopes are 2H, 13C, and 15N. Stable isotope-labeled tracers have the advantages of high sensitivity, simple location, and accurate quantification.

 

A stable isotope-labeled (SIL) peptide is a chemically synthesized peptide with the native sequence, but some constituent amino acids are replaced by stable isotope-labeled amino acids containing 2H, 13C, and/or 15N atoms. The result of this labeling is to change the mass of a peptide from as small as a few Da to as large as tens of Da compared to the mass of the parent peptide. SIL peptides exhibit identical physicochemical properties and chemical reactivity as unlabeled peptides (with a few exceptions), and under certain conditions, the minute mass differences cause labeled and unlabeled peptides to behave differently. In addition, peptides can be labeled with one or more isotopes of hydrogen, carbon, nitrogen, or oxygen during synthesis by incorporating amino acids containing the desired isotopes, such as deuterium, 13C, 15N, or 18O, into the peptide.

 

These unique features constitute the basis for using stable isotope labeling peptides in a variety of absolute quantification applications such as quantitative proteomics and the quantification of complex protein mixtures at very low concentrations. To be more specific, absolute quantitation of a complex protein mixture at very low concentrations and structural studies requires high-quality peptides enriched with stable isotopes. Besides, stable isotope-labeled peptides are routinely used as internal standards for the absolute quantitation of proteins in targeted proteomics. These peptides can either be synthesized chemically on solid supports or expressed biologically by concatenating multiple peptides together to a large protein. With rapid advances in the discovery of optimal peptides for targeted proteomics, there is an urgent need to generate isotope-labeled peptides for each protein in the human proteome, especially for low-abundance proteins.

 

Stable isotope-labeled peptides have also been widely used in (NMR) spectroscopy and mass spectrometry (MS) as well as reference materials for pharmacokinetic analysis and metabolite identification. 13C and 15N labeled peptides are suitable for NMR to study the protein structure, kinetics, and molecular interaction of biomolecules. Other applications include protein expression monitoring, protein cross-linking analysis, biomarker discovery, pharmacokinetics, clinical biochemistry for drug and metabolite monitoring, anti-doping testing, and cell signal profiling and pathway validation.

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