ROSQuant Kit

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Quantification of Reactive Oxygen Species


US$ 800.00

Reactive Oxygen Species Identification and Quantification Kit (ROSQunt Kit)

 
ROSQuant Demo Experiment:
To demonstrate ROSQuant can be used for the quantification of thiol containing proteins extracted from case and control sample.
 
Demo Experiment Background
Bovine Serum Albumin (BSA) is protein that contain 35 cysteine amino acids and 34 of which are in oxidized form. In other word, BSA contain many internal disulfide bond (14 pairs in total). After trypsin digestion, 43% of the peptides will contain cysteine. Therefore, BSA is a good model protein for experimenting the possibility of combining ROS and dimethyl labeling together. 
 
Methodology:
Two aliquots of BSA are prepared in ammonium bicarbonate buffer. One aliquot is completely reduced by TCEP to prepare reduced BSA while the second aliquot is set aside as oxidized BSA. After reduction and acetone precipitation, the two aliquots are mixed together so that oxidized and reduced BSA is in 1:1 ratio. This serve to mimic a biological situation in which half of a thiol protein is in oxidized/reduced state. MPB and BIAM was used sequentially to labeled reduced and oxidized BSA. The labeled BSA proteins are digested with trypisn overnight. The tryptic peptides are then splited into two aliquots and labeled as "case" and "control" sample respectively. Case and control sample are lightly and heavily labeled using dimethyl labeling technique. These sample are mixed and desalted for LC-MS/MS analysis.
 
Result:
(1) 127 BSA peptides are observed
(2) 97/127 BSA peptides have an ion score >=20
(3) 19/97 high score peptides are labeled with (BIAM/MPB) and Dimethyl
(4) 7/19 labeled peptides are unique and correspond to 30% of the computationally predicted cysteine containing BSA peptides
(5) 1/7 unique peptide has all of the following peptide forms
  • Lightly-labeled and oxidized peptide form
  • Heavily-labeled and oxidized peptide form 
  • Lightly-labeled and reduced peptide form 
  • Heavily-labeled and reduced peptide form 
Their spectra are shown below.
 
(6) The light:heavy ratio for oxidized or reduced peptides in (3) matched with the 1:1 theoretical ratio (data not shown)
 
Conclusion:
ROSQuant can be used for the quantification of thiol containing proteins extracted from case and control sample. As thiol peptides have been successfully labeled BIAM/MPB, which contains biotin tag, thiol containing peptides can be enriched by streptavidin to further enhance the percentage of thiol peptides in the total number of peptides identified.
 
 
Oxidized Form
oxidized form
oxidized form
 
 
​​Reduced
 Form
reduced form
 
 
 
reduced form
 
 
 
 
 
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