Stable Isotope-Labeled Peptides
Genomics research shows that more than a million proteins are encoded by approximately 30,000 human genes. Proteomics, the study of proteins encoded by the genome, includes identification of post-translational modifications, structural analyses, protein localization studies, and protein quantitation. Mass spectroscopy-based techniques have evolved as a powerful tool in proteomics. Stable isotope-labeled peptides (SIL peptides) are chemically and physically indistinguishable from their endogenous counterparts with respect to retention time, ionization efficiency, and fragmentation pathways. Therefore, either are ideal internal standards and template analytes. Peptides can be labeled with one or more isotopes of hydrogen, carbon, nitrogen, or oxygen by incorporating amino acids containing the desired isotopes such as deuterium (D), 13C, 15N, or 18O into the peptide during synthesis.
The Absolute Quantification method (AQUA) enables targeted quantification of protein and post-translational modifications in complex protein mixtures using SIL peptides as internal standards. The SIL peptide is introduced into a biological sample during or after protease digestion. The heavy SIL peptide and its light endogenous peptide fragment are detected by selected reaction monitoring (SRM) in a mass spectrometer. Based on the known amount of SIL peptide added, and the intensity ratio of both peptides, the amount of endogenous protein can be calculated.
Stages of Absolute Quantification of Proteins:
- Proteins of interest identified/selected
- Peptide internal standard sequence selected
- Synthesis of SIL peptide
- Detailed compositional analysis of SIL peptide by LC-MS/MS and method optimization
- SIL peptide added to protein sample prior to enzymatic digestion
- Fragmented peptides are isolated
- AQUA using mass spectrometric analysis
SIL PEPTIDES IN STRUCTURAL ANALYSIS
Nuclear Magnetic Resonance (NMR) is a powerful technique for investigating structural information, dynamics, and molecular interactions of biomolecules. This technique can be used to measure relaxation rates of biomolecules as they dissociate from their bound target. Peptides labeled with D (spin of 1), 13C (spin 1/2), and 15N (spin 1/2) are suitable for NMR studies of proteins. A combination of NMR spectroscopy and segmental isotopic labeling is used to study the mechanism of protein splicing (e.g., the structure of an active protein splicing precursor), a post-translational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein.
HIGH QUALITY SIL PEPTIDES FROM CPC SCIENTIFIC
Absolute quantitation of a complex protein mixture at very low concentrations and structural studies requires high-quality peptides enriched with stable isotopes. The CPC Scientific SIL Peptide Custom Synthesis Service guarantees superior quality and high isotopic enrichment. These stable isotopic peptides are synthesized using the latest Fmoc solid-phase peptide technology in our state-of-the-art peptide laboratory. All heavy isotope-labeled peptides undergo mass spectrometric analysis and stringent analytical HPLC to establish the final purity and assure that our customers receive only the highest quality peptides for absolute quantitation and other studies.
Two major isotopes,15N and 13C, are incorporated into a specific amino acid sequence of peptides. Each atom contains over 99% of an enriched isotope and can be located at multiple positions within the peptide. For example, a Leu amino acid containing one 15N and six 13C, and a peptide containing one such isotope-labeled Leu amino acid, has seven units of molecular weight higher than the corresponding unlabeled peptide.
Wang, Weixun, Nykia D. Walker, Li-Ji Zhu, Weizhen Wu, Lan Ge, David E. Gutstein, Nathan A. Yates et al. "Quantification of circulating D-dimer by peptide immunoaffinity enrichment and tandem mass spectrometry." Analytical chemistry 84, no. 15 (2012): 6891-6898.
Isotope-Labeled Custom Peptide Citations
1. Lee, Anita YH, et al. "Multiplexed quantification of proglucagon-derived peptides by immunoaffinity enrichment and tandem mass spectrometry after a meal tolerance test." Clinical Chemistry 62.1 (2016): 227-235.Learn More »
2. Zhen, Eugene Y., et al. "Circulating FGF21 Proteolytic Processing Mediated by Fibroblast Activation Protein." Biochemical Journal (2015): BJ20151085.Learn More »
3. Dekker, Petrus Jacobus Theodorus, René Marcel De Jong, and Cornelis Marinus Muijlwijk. "Chymosine enzyme variants." U.S. Patent Application No. 14/397,866.Learn More »
4. Harper, Brett, Mahsan Miladi, and Touradj Solouki. "Loss of internal backbone carbonyls: additional evidence for sequence-scrambling in collision-induced dissociation of y-type ions." Journal of The American Society for Mass Spectrometry 25.10 (2014): 1716-1729.Learn More »
5. Zhen, Eugene Y., Richard E. Higgs, and Jesus A. Gutierrez. "Pyroglutamyl apelin-13 identified as the major apelin isoform in human plasma." Analytical Biochemistry 442.1 (2013): 1-9.Learn More »
6. Wang, Weixun, et al. "Quantification of circulating D-dimer by peptide immunoaffinity enrichment and tandem mass spectrometry." Analytical Chemistry 84.15 (2012): 6891-6898.Learn More »
7. Havukainen, Heli, et al. "A vitellogenin polyserine cleavage site: highly disordered conformation protected from proteolysis by phosphorylation." The Journal of Experimental Biology 215.11 (2012): 1837-1846.Learn More »
8. Cox, Jennifer M., et al. "Characterization and quantification of oxyntomodulin in human and rat plasma using high-resolution accurate mass LC-MS." Bioanalysis 8.15 (2016): 1579-1595.Learn More »
9. Qureshi, Tabussom, and Natalie K. Goto. "Impact of Differential Detergent Interactions on Transmembrane Helix Dimerization Affinities." ACS Omega 1.2 (2016): 277-285.Learn More »
|AMINO ACID||ISOTOPE||MASS DIFFERENCE||ISOTOPIC ENRICHMENT|
SIL PEPTIDE APPLICATIONS
- Functional quantitative proteomics
- Quantitation of post translationally-modified proteins
- Protein structure analysis
- Protein expression monitoring
- Biomarker discovery
- Clinical biochemistry for drug and metabolite monitoring
- Anti-doping testing
- Cell signal profiling and pathway validation
- Protein cross-linking analysis
SIL PEPTIDE MODIFICATIONS:
- Phospho-Tyr, Ser, Thr (single or multiple)
- Sulfo-Tyr (single or multiple)
- Methylated Arg, Lys
- Pyroglutamic acid