Babu Purkayastha
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iTRAQ Reagents in Action

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The world of proteomics continues to evolve. Although many techniques and workflows have been developed over the years for high throughput identification and quantitation of proteins by mass spectrometry, there are some strategies that seem to excel beyond others for consistently producing high-quality data that moves research forward. iTRAQ® Reagents are one of those strategies.

First developed over 10 years ago1, iTRAQ Reagents enable identification and quantitation of complex proteome samples from LC-MS/MS data of up to 8 samples simultaneously. Their high accuracy in quantitation and sensitivity for identification, combined with their simple sample preparation protocol, and versatility for use with any sample type, has proven to be a winning formula for iTRAQ reagent adoption around the world. Since their introduction, the number of iTRAQ reagent publications has steadily increased with now over 2000 publications as of October 2016.2

iTRAQ Reagents have been applied to the study of plants, bacteria, humans, and animals. In the world of human health and disease, the applications are far reaching, with cancer the number one disease being studied to date.2

And did we mention the versatility of iTRAQ Reagents for sample types? The wide range of samples that can be studied using iTRAQ reagents is impressive. Due to their versatility—just one extra step in your normal sample preparation protocol—iTRAQ reagents can be used with virtually any sort of human, animal, or plant fluid, extract, or tissue.2

iTRAQ Reagents are continuing to help promote understandings in human, animal, and plant health and disease around the world. Here are three recent applications from across the globe.

England:  Using a combination of analytical techniques including proteomics experiments and iTRAQ Reagents, researchers at the University of Manchester and Glasgow found a way to kill leukemia stem cells (LSCs) which could lead to an actual cure of chronic myeloid leukemia (CML) instead of simply managing the disease.3 Through proteomics, transcriptomics, and network analysis, researchers were able to achieve a deeper level of understanding on how the BCR–ABL signaling network functions in CML. From this revelation, they showed that dual targeting of both p53 and c-MYC provided a much-improved kill rate for the LSCs compared to current treatments.

Australia: Recently researchers at the Australian Proteome Analysis Facility (APAF) investigated IκB kinase inhibition in breast cancer cells using iTRAQ Reagents.4 An assessment of the effects of the IKK inhibitor IMD0354 on the breast cancer cell line T47D following 30 minute drug treatment showed that in total, 36 and 65 proteins were significantly up- or down-regulated, respectively following IMD0354 treatment. iTRAQ Reagents were then used for time course studies to further reveal the dynamic effects of the inhibitor on protein expression. Among other observations, 60s ribosomal proteins significantly increased over time, while histones and related proteins were seen to significantly decrease and then recover again over time. Pathway analysis revealed that nine broad pathways were shown to be significantly regulated by IKK inhibition.

Czech Republic: Researchers in the Czech Republic at the Institute of Molecular Pathology and the University Hospital Hradec Kralove, in collaboration with SCIEX researchers looked at the combination of isobaric tagging reagents and cysteinyl-peptide enrichment (CPE) for in-depth quantification.5 By combining the enrichment benefits of cysteine capture with the high multiplexing properties of isobaric iTRAQ reagents, a powerful new quantitative workflow was created that allowed quantitative analysis of both cysteinyl and non-cysteinyl peptide fractions. Using an intersection analysis between the various sample preparation fractions and the Protein Alignment Template, the researchers found that one injection of each non-cysteinyl and cysteinyl fractions of an amniotic fluid sample detected 38% more unique proteins than two injections of the unfractionated sample, showing that the CPE enrichment clearly led to increased proteome coverage. Ultimately, the combined evaluation of three injections of both cysteinyl and non-cysteinyl peptides fractions resulted in the identification of 60% more proteins compared to three injections of the unfractionated amniotic fluid sample.

To learn more about iTRAQ Reagents, visit the iTRAQ Reagent website, and read our first blog on the history of isotope labeling for mass spectrometry.

References:

  1. P. L. Ross, et. al., (2004). “Multiplexed Protein Quantitation in Saccharomyces Cerevisiae Using Amine-Reactive Isobaric Tagging Reagents.” Molecular & Cellular Proteomics, 3(12), 1154-1169.
  2. PubMed Search: https://www.ncbi.nlm.nih.gov/pubmed/
  3. Abraham SA et al., Dual targeting of p53 and c-MYC selectively eliminates leukemic stem cells (2016) Nature 534, 341–346. cells
  4. Tech Note: Investigating IκB Kinase Inhibition in Breast Cancer Cells
  5. Tech Note: Combination of Isobaric Tagging Reagents and Cysteinyl Peptide Enrichment for In-Depth Quantification

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