title: Insights into the binding sites of sulforaphane on insulin studied by electrospray ionization mass spectrometry.
abstract: BACKGROUND
  Sulforaphane (SFN) is a natural isothiocyanate, known to reduce the risk of cancer and also aortic damage and diabetic cardiomyopathy induced by type 2 diabetes, etc. A more detailed knowledge on the direct interaction of SFN with insulin and its binding sites is necessary for better understanding the role of SFN on diabetes.
  
  
  METHODS
  Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and in-source fragmentation experiments were performed on a Thermo Exactive orbitrap mass spectrometer. The solution of insulin and SFN was incubated and analyzed by mass spectrometry. Isotopic distribution pattern, accurate mass values and theoretical product ions were used to analyze the mass spectrometry data. The nature of binding of SFN and its binding sites with insulin were evaluated by LC/MS data.
  
  
  RESULTS
  ESI-MS analysis of the incubated solution of insulin and SFN showed 1:1 and 1:2 complexes of [Insulin-SFN]. LC/MS analysis revealed that the [Insulin+SFN] complexes were due to covalent binding of SFN at two different sites. The in-source fragmentation experiments revealed that the SFN is binding to the NH2 groups of N-terminal amino acids of A and B chains of insulin. Further study of SFN with insulin reduced with dithiothreitol (DTT) showed exclusive modification of cysteines with SFN.
  
  
  CONCLUSIONS
  The interaction of SFN was studied with insulin using ESI-MS. SFN is found to bind covalently with the free NH2 group of the N-terminal of the A and B chains of insulin. However, when insulin is reduced SFN preferably binds to SH groups of cysteines. Hence, the present study helps in the understanding of the binding sites of SFN on insulin.
authors.affiliation: National Centre for Mass Spectrometry, CSIR - Indian Institute of Chemical Technology, Hyderabad, 500 007, Telangana, India.
authors.forename: V
authors.initials: V
authors.lastname: Nagaveni
authors.affiliation: National Centre for Mass Spectrometry, CSIR - Indian Institute of Chemical Technology, Hyderabad, 500 007, Telangana, India.
authors.forename: S
authors.initials: S
authors.lastname: Prabhakar
mesh_terms: D001665:Binding Sites; D002853:Chromatography, Liquid; D004229:Dithiothreitol / Q000737:chemistry; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism*; D017879:Isothiocyanates / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013454:Sulfoxides
chemical_list: D007328:Insulin; D017879:Isothiocyanates; D013454:Sulfoxides; C016766:sulforaphane; D004229:Dithiothreitol