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.
Different Stages of Absolute Quantification of Proteins (AQUA) using SIL Peptides:
Step 1: Proteins of interest identified/selected
Step 2: Peptide internal standard sequence selected
Step 3: Synthesis of SIL peptide
Step 4: Detailed compositional analysis of SIL peptide by LC-MS/MS and method optimization
Step 5: SIL peptide added to protein sample prior to enzymatic digestion
Step 6: Fragmented peptides are isolated
Step 7: 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.
|Amino Acid||3-Letter Code||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