Stable isotope labeled peptides

Peptides labeled with stable, non-radioactive isotopes are increasingly used for convenient detection in research. Stable isotope labeled, or ‘heavy’ amino acids, are derived from natural amino acids by substitution of certain atoms (N, C, H) with their ‘heavy isotope’ variant. The most frequently substitutions are ¹²C by ¹³C (carbon-13), ¹⁴N by ¹⁵N (nitrogen-15), and ¹H by ²H (deuterium).

Stable isotope labeled (SIL) peptides display identical physiochemical properties and chemical reactivity as their non-labeled counterparts (apart from a few exceptions). However, under certain conditions the minute mass difference make labeled and unlabeled peptides behave differently. This constitutes the basis for using SIL peptides in a variety of absolute quantification applications such as quantitative proteomics, the quantification of complex protein mixture at very low concentration, or NMR studies.

CILAt Pepscan we have produced thousands of high quality heavy peptides for proteomics. These SIL peptides are synthesized using only the highly enriched stable amino acids from premium supplier Cambridge Isotope Laboratories (CIL) for the Fmoc-based solid-phase custom peptide synthesis in our laboratory. These uniformly labeled amino acids all have > 99% isotopic purity.

These important attributes have enabled us to successfully respond to the ever changing demand for best quality stable isotope peptides. All SIL peptides undergo mass spectrometric analysis and stringent analytical HPLC to establish the final purity, and to assure that our customers receive only the highest quality peptides for absolute quantitation.

The table below lists the most common SIL amino acids. Because of Pepscan’s supply alliance with Buchem, the Dutch distributor of Cambridge Isotope laboratories, other highly enriched ( >99% isotopic purity) stable amino acids are available upon request for the synthesis of your custom peptides.

The stable isotope labeled peptides are usually supplied with the following specs:

Scale 10 or 40 nmole
Labeling C-terminal, most often R or K (13C, 15N)
Isotopic enrichment >99%
Purity >98%
Formulation fully solubilized and in aliquots (1 nmole each)
Solvent 5% ACN/water; concentration: 5 pmole/µl (200 µl per aliquot)
Concentration precision within 10%
Quality control     liquid chromatography and mass spectrometry
Delivery temperature on dry ice
Aliquots 1 nmole each, in glass tubes
Tube labeling reference peptide number, peptide sequence in one letter code, date of production
Custom specifications upon customer request, supply with other specifications is possible

The most common SIL amino acids are:

Amino Acid Code Mass Difference Isotope Isotopic Enrichment
Alanine A +4Da U- ¹³C₃, ¹⁵N >99%
Arginine R +10Da U- ¹³C₆, ¹⁵N₄ >99%
Isoleucine I +7Da U- ¹³C₆, ¹⁵N >99%
Leucine L +8Da U- ¹³C₆, ¹⁵N >99%
Lysine K +8Da U- ¹³C₆, ¹⁵N₂ >99%
Phenylalanine F +10Da U- ¹³C₉, ¹⁵N >99%
Proline P +6Da U- ¹³C₅, ¹⁵N >99%
Valine V +6Da U- ¹³C₅, ¹⁵N >99%

Selected References for Stable Isotope Labeled Peptides

Mbasu et al: Proteomics (2016) – PMID 27214876.  Advances in quadrupole and time-of-flight mass spectrometry for peptide MRM based translational research analysis

Lalmahomed et al: Am J Cancer Res (2016) – PMID 27186406.  Hydroxylated collagen peptide in urine as biomarker for detecting colorectal liver metastases

Stoop et al: J Pharm Biomed Anal (2016) – PMID 27209449.  A new quantification method for assessing plasma concentrations of pemetrexed and its polyglutamate metabolites

Van Duijn et al: Anal. Chem (2015) – PMID26168337.  Quantitative measurement of immunoglobulins and free light chains using mass spectrometry