Stable isotope labeled peptides

Peptides labeled with stable, non-radioactive isotopes are increasingly used for convenient detection in research. 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.

At Pepscan we have produced thousands of such custom ‘heavy’ peptides. 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 peptide-synthesis in our laboratory. These uniformly labeled amino acids all have over 99% isotopic purity.

These important attributes have enabled us to successfully respond to the ever changing demand for high quality SIL-compounds. All stable isotope labeled 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.

These 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 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%) stable amino acids are available upon request for the synthesis of your custom peptides.

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) – PMID 26168337. Quantitative measurement of immunoglobulins and free light chains using mass spectrometry.