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Case Study
Quantitative Mass Spectrometry using stable isotope labelled peptides
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PEAK™ Peptides
Stable isotope labelled peptides for mass spectrometry and NMR studies
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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.
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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.
Quality
Critical to the accuracy of this technique is the quality of the stable-labelled peptide. CRB’s custom stable–labelled peptides are offered:
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>95% pure by HPLC |
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analysed for net peptide content |
Post-translational modifications
For the quantitation of post-translational modifications, CRB can also offer stable-labelled peptides containing:
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phosphoTyr, phosphoSer, phosphoThr |
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sulfoTyr |
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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. |
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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. |
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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. |
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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
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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
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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.
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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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Should you require a quotation from any of our custom services, we have a clear cut and easy system that enables you to do so. Follow these three simple steps:
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Simply click on the relevant tab and register your details. |
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Once issued with a password you can then log on to our quotation system using your customer account log-in and a quotation will be issued within 24 hours. |
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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.
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T +44 (0)1642 567180 F +44 (0)1642 567181 E enquiries@crbdiscovery.com
17-18 Belasis Court, Belasis Hall Technology Park, Billingham, Cleveland, TS23 4AZ United Kingdom
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