Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC–MS/MS View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2010-08-26

AUTHORS

Richard D Unwin, John R Griffiths, Anthony D Whetton

ABSTRACT

In this paper, we describe the use of iTRAQ (isobaric Tags for Relative and Absolute Quantitation) tags for comparison of protein expression levels between multiple samples. These tags label all peptides in a protein digest before labeled samples are pooled, fractionated and analyzed using mass spectrometry (MS). As the tags are isobaric, the intensity of each peak is the sum of the intensity of this peptide from all samples, providing a moderate enhancement in sensitivity. On peptide fragmentation, amino-acid sequence ions also show this summed intensity, providing a sensitivity enhancement. However, the distinct distribution of isotopes in the tags is such that, on further fragmentation, a tag-specific reporter ion is released. The relative intensities of these ions represent the relative amount of peptide in the analytes. Integration of the relative quantification data for the peptides allows relative quantification of the protein. This protocol discusses the rationale behind design, optimization and performance of experiments, comparing protein samples using iTRAQ chemistries combined with strong cation exchange chromatographic fractionation and MS. More... »

PAGES

1574-1582

References to SciGraph publications

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nprot.2010.123

DOI

http://dx.doi.org/10.1038/nprot.2010.123

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1008894697

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/21085123


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