Multiplexing Cysteine Redox Proteomics

Dr. Kay-Hooi Khoo from the Institute of Biological Chemistry at Taiwan’s Academia Sinica uses Thermo Scientific tandem mass tags (TMTs) in his work elucidating the effect of redox conditions on cysteine residues. In this short video, presented at the American Society for Mass Spectrometry conference in June 2014, he explains why the Thermo Scientific reagent, a vital part in his research program, is ideal for the job.

Khoo’s work looks into the effect of hypoxia (low tissue oxygen levels) on the post-translational modification (PTM) of proteins. He is interested in the mechanisms of reperfusion injury, whereby reversal of tissue ischemia—low or absent blood supply to an area that decreases tissue oxygenation—causes cellular damage. This damage arises through the release of toxic metabolites and development of free radicals and reactive oxygen species.

Focusing on alterations in susceptible cysteine residues brought about by the physiological change, Khoo quantifies these PTMs, characterizing their nature and how they occur. With Thermo Scientific iodoTMT (iodoacetyl tandem mass tags), he can irreversibly label cysteine residues and then quantify them using mass spectrometric proteomic analysis.

Replacing the previous cysTMT kit, the iodoTMT reagents work by irreversibly labeling only the sulfhydryl groups on reduced, unmodified cysteine residues. Important for Khoo’s work, this tagging is not susceptible to reducing agents. In addition, each iodoTMT kit enables up to 6-plex multiplexing, thus extending the experimental range and sample handling capabilities.

Another useful feature is the ability to immunoenrich tagged analytes by using an immobilized anti-TMT antibody resin prior to mass spectrometry. In this way, low-abundance peptide modifications can be concentrated prior to assay and quantified accurately.

Using these features, Khoo is able to label susceptible cysteines to monitor PTMs induced with hypoxic stress in cell culture. Through differential labeling followed by treatment with ascorbate or other reducing agents, Khoo can follow the pattern of cysteine PTMs sequentially. In conjunction with reporter ion quantitation, he can determine the different ratios of modifications occurring with each experiment, probing the process as it occurs. From this information, Khoo is able to determine the effect of potential protective agents, such as oxygen donors, on regulation of each PTM.

Through the understanding of cellular stresses and redox activation/inactivation, Khoo hopes to develop better therapeutics that could prevent irreversible tissue damage.

 

Further Reading

Read about iodoTMT kits at http://www.piercenet.com/product/cysteine-reactive-tandem-mass-tag-reagents.

Bomgarde, R.D., et al. (2012) “Iodoacetyl Tandem Mass Tags for Cysteine Peptide Modification, Enrichment and Quantitation.”

More on Dr. Khoo’s work may be found at http://www.ibc.sinica.edu.tw/PI_DetailE.asp?Auto=23.

 

 

Post Author: Amanda Maxwell. Mixed media artist; blogger and social media communicator; clinical scientist and writer.

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