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April Newsletter:

Peak Peptides: Internal standards for protein quantification
Proteomics: Directly quantify protein levels via mass spectrometry
Chromis Dyes: New dye labelling kits for 2D Gels

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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.

Quality

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
  • sulfoTyr
  • 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.

The stable-labelled amino acids most commonly used in this technique are Leucine, Phenylalanine, Proline and Valine. These amino acids provide the greatest shift in the molecular weight for the least cost; however, heavy atom versions of other amino acids are available upon request.

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.

Peak Peptides

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

References

C. Barton, R. G. Kay, W. Gentzer, F. Vitzthum & S. Pleasance. Quotient Bioresearch Ltd. & Siemens Healthcare Diagnostics Products GmbH.
Development of High-Throughput Chemical Extraction Techniques and Quantitative HPLC-MS/MS (SRM) Assays for Clinically Relevant Plasma Proteins.
J. Proteome Res., 2010, 9, 333-340

C. Barton, R. G. Kay, W. Gentzer, F. M. Quaglia, C. Pritchard, Z. Hall & G. O'Connor. LGC Ltd.
Amine-reactive isobaric tagging reagents: Requirements for absolute quantification of proteins and peptides.
Anal. Biochem., 2008, 379, 164-169

C. Pritchard, M. Quaglia, C. Mussell, W. L. Burkitt, H. Parkes & G. O'Connor. LGC Ltd.
Fully Traceable Absolute Protein Quantification of Somatropin That Allows Independent Comparison of Somatropin Standards.
Clin. Chem., 2009, 55, 1984-1990.

H. Neubert, I. James., Pfizer Ltd.
Online capillary weak cation exchange enrichment hyphenated to nanospray mass spectrometry for quantitation of a basic pegvisomant derived peptide.
J. Chromatography A, 1216, 2009, 6151-6154.

  Proteomics -
Application Notes

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.

Chromis Diagram

To assist with the selection of the most appropriate stable-labelled peptide standard, we have prepared simple to follow guidelines.

Stable-labelled amino acids most commonly used in this technique. These amino acids provide the greatest shift in the molecular weight for the least cost; however, heavy atom versions of other amino acids are available upon request.

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.

Chromis Dyes for protein protection

2-D Fluorescence Difference Gel Electrophoresis is a method that labels protein samples with fluorescent dyes before 2-D electrophoresis, enabling accurate analysis of differences in protein abundance between samples.

CRB offers the Chromis DGE minimal labelling kits for this purpose bringing the performance of these new fluorescent labels to the technique.

The kits include Chromis 490, 550, 645 dyes which are size and charge-matched, as well as spectrally distinct. The Chromis dyes give bright and intense colours with narrow excitation and emission bands. Co-separation of different labelled samples in the same gel is therefore possible and all samples are subject to exactly the same first and second dimension electrophoresis running conditions. This limits experimental variation and ensures accuracy within gel matching.

Chromis Diagram

More about Chromis dyes

Take a look at our Chromis DGE kits

 View our full range of products

Visit our website to view our full range of products, we offer products for the following:

TARGET Antibodies INFINITY Antibodies Peptide Immunisation Protein Immunisation

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