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CRB Discovery

Case Study

Quantitative Mass Spectrometry using stable isotope labelled peptides

PEAK™ Peptides

Stable isotope labelled peptides for mass spectrometry and NMR studies

Protein Quantitation

Direct quantification of protein levels via mass spectrometry

PEAK Peptides
CRB specialises in the rapid synthesis of peptides containing stable isotope labelled amino acids.
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Pep Talk
Case Study
Quantitative Mass Spectrometry Using Stable Isotope Labelled Peptides
N-terminal peptide chosen from hGH: FPTIP-[13C6, 15N-L]-SRLFDNANL-acid
M+7 Mass shift of 7 from stable labelled Leucine at position 6

Using a stable isotopically labelled peptide synthesised at Cambridge Research Biochemicals, Dr Hendrik Neubert at Pfizer UK utilised quantitative mass spectrometry to assess the immunogenicity of a biological protein therapeutic. Biological protein therapeutics raising an off-target immune response can lead to possible serious clinical effects due to the generation of anti-drug antibodies. Existing assay techniques can be subject to poor reliability due to high interference caused by high concentrations of protein therapeutics. In this work a magnetic bead based immunoprecipitation method has been developed to determine anti-drug antibodies despite the high background of protein therapeutics in serum. Anti-drug binding sites are saturated by addition of excess therapeutic protein followed by isolation of IgG antibodies and bound antigens. LC/MS is then used to quantify peptides of the target therapeutic proteins using stable isotopically labelled peptides as internal standards. A typical mass shift of the isotopic envelope is shown in the MALDI trace above.

With thanks to Dr Hendrik Neubert at Pfizer Global Research and Development, Sandwich U.K. for granting permission to highlight his work. For further information please refer to:
Anal Chem 2008, 80, 6907-6914
Assessing Immunogenicity in the Presence of Excess Protein Therapeutic Using Immunoprecipitation and Quantitative Mass Spectrometry
Hendrik Neubert, Christopher Grace, Klaus Rumpel and Ian James

Pfizer Global Research and Development , Sandwich, U.K.

Read more Find out more on stable labelled peptides
PEAK Peptides PEAK Peptides
PEAK Peptides

Stable Isotope Labelled Tryptic Peptides:

CRB specialises in the rapid synthesis of peptides containing stable isotope labelled amino acids.

By using stable-labelled versions of tryptic peptides as internal standards, it is possible to directly quantify protein levels in a digested sample via mass spectrometry.


Critical to the accuracy of this technique is the quality of the stable-labelled peptide. CRB’s custom stable–labelled peptides are offered:


>95% pure by HPLC


analysed for net peptide content

Post-translational modifications

For the quantitation of post-translational modifications, CRB can also offer stable-labelled peptides containing:


phosphoTyr, phosphoSer, phosphoThr




methylated Lys and Arg

Other proteolysis standards

Isotopically labelled versions of peptides derived from proteolysis by other enzymes, such as Lys-C, Glu-C, can also be produced.

Amino Acid Range

Typically for peptides digested via Trypsin, Arginine and Lysine are commonly used. Other common stable-labelled amino acids used in this technique are Leucine, Phenylalanine, Proline and Valine. Heavy atom versions of other amino acids are also available upon request.

Rapid Delivery

Streamlined operation allows delivery in 2 - 3 weeks. Most common stable-labelled amino acid building blocks are kept in stock to ensure rapid turn-around.

Technical support

CRB are happy to advise on the design of stable-labelled peptides. CRB can also assist with pre-concentration techniques to significantly reduce the limit of detection via the production of antibody pre-concentration columns.

For more information about applications using CRB stable labelled peptides see references section, or contact us by visiting our website.

Download Peptide Buyer’s Guide
More about PEAK Peptides
Protein Quantitation Using Peptides Take a look at our Peak Peptides section on our website to find out more

The need for absolute quantitation of protein expression levels in biological samples is of major importance in proteomics.

The simplest and most cost effective method of quantitation involves the use of peptides containing amino acids incorporating heavy stable isotopes. Typically, the biological sample is spiked with a known amount of a heavy version of a tryptic peptide related to the protein of interest prior to enzyme digest. Since the stable-labelled and natural peptide have identical HPLC and electrophoretic properties but different molecular masses, the levels of target protein can be directly quantified via mass spectrometry from the peak ratios of the extracted ion chromatograms.

To assist with the selection of the most appropriate stable-labelled peptide standard, please see our Peptide Selection Rules.

Quantitate protein concentration from peak ratios of extracted mass chromatograms
More about Proteomics
For further advice on designing your stable-labelled peptide or for help in choosing which amino acid to label within your peptide, please contact us on our website


Internal peptide standard GPETLCGAELVDALQF-[U-13C5,15N-Val]-CGDR
Development of High-Throughput Chemical Extraction Techniques and Quantitative HPLC-MS/MS (SRM) Assays for Clinically Relevant Plasma Proteins.
C. Barton, R. G. Kay, W. Gentzer, F. Vitzthum & S. Pleasance. Quotient Bioresearch Ltd. & Siemens Healthcare Diagnostics Products GmbH.
J. Proteome Res., 2010, 9, 333-340

Reference dash

Internal peptide standards [U-13C6,15N-Leu]-LFDNAMLR & TGQI-[U-13C9,15N-Phe]-K
An immunoaffinity liquid chromatography-tandem mass spectometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum.
M. Fernandez Ocana, H. Neubert. Pfizer Ltd.
Analytical Biochemistry 399 (2010) 202-210

Reference dash

Internal peptide standard KFLNHRGSPLQG[U-13C5,15N-Pro]-F-[U-13C6,15N-Leu]-TARTWPALPK
Fully Traceable Absolute Protein Quantification of Somatropin That Allows Independent Comparison of Somatropin Standards.
C. Pritchard, M. Quaglia, C. Mussell, W. L. Burkitt, H. Parkes & G. O’Connor. LGC Ltd.
Clin. Chem., 2009, 55, 1984-1990.

Reference dash

Multiple phosphorylated internal peptide standards
Stoichiometric Quantification of Akt Phosphorylation using LC-MS/MS
A. Atrih, D. Turnock, G. Sellar, A. thompson, G. Feurstein, M. A. J. Ferguson & J. T.J. Huang. University of Dundee & Pfizer Inc.
J. Proteome Research 2010, 9, 743-751

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At Cambridge Research Biochemicals we have a fast-track quotation system that tailors your requirements, gives you all the information you need and a specific price within 24 hours. You are also given a personal contact so that if you have any further questions you can directly contact one of our approachable representatives who has the technical knowledge to assist you.

Meet a CRB representitive

If you would like any support, help or technical advice we would like to hear from you.

CRB is delighted to be sponsoring the Cambridge Cell Biology Seminar Series and the next event is being held on the 27th April 2011.
Professor Maxence Nachury, Stanford University.
Adhesive control of cell polarity, organelle positioning and cell division axis in growing cells and of primary ciliogenesis in cell-cycle arrested cells.
The Sackler Lecture Theatre, Level 7, Wellcome Trust/MRC Building, Addenbrooke's Site. Wednesday 27 April 2011, 4pm Durham University Logo
Click here to find out more
CRB will be exhibiting and attending the following exhibitions:
BioTrinity 2011
Signalling Meeting
Proteomics Method Forum

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