NEW Strategy for the Detection, Identification and Quantification of Selenoproteins in Living Organisms: Application to selenoproteins in human plasma.

 

Guillaume Ballihauta, W. Clay Davisa, Colleen Bryana, Eric L. Kilpatricka, Lisa E. Kilpatrickb, Stephen E. Longc, Stephen A. Wisec

 

aNational Institute of Standards and Technology, Analytical Chemistry Division, Hollings Marine Laboratory, Charleston, SC 29412;

 bNational Institute of Standards and Technology, Physical and Chemical Properties Division, Hollings Marine Laboratory, Charleston, SC 29412;

 cNational Institute of Standards and Technology, Analytical Chemistry Division, Gaithersburg, MD  20899;

Guillaume.Ballihaut@noaa.gov

 

Selenium is recognized as an essential micronutrient for human health and its deficiency is associated with a wide variety of human diseases including cancer and cardiovascular disease.  Its biological functions are primarily mediated by selenoproteins, which are an essential part of antioxidant mechanisms in cells and critical players in a variety of essential biological processes. Therefore, accurate and precise quantification of selenoproteins in human fluids is crucial to assess the nutritional needs of selenium and to better understand its biological role.  In human blood, selenium is mainly distributed among two selenoproteins: glutathione peroxidase and selenoprotein P, whose detection and identification by using classical proteomic approaches are challenged by the presence of 15 highly-abundant proteins (HAP).  Therefore, to target more specifically these proteins, new analytical strategies need to be developed. 

 

In this work, a multidisciplinary approach is proposed for the specific removal of the most abundant HAP followed by the isolation, identification, and quantification of selenoproteins in fresh-frozen human plasma (candidate SRM 1950) standard reference material.  A particular emphasis was placed on the development of a method for the specific detection and identification of selenoproteins after gel electrophoresis.  Selenoproteins were detected by laser ablation coupled to inductively coupled plasma mass spectrometry (ICP-MS) and identified after tryptic digestion by liquid chromatography tandem mass spectrometry (LC/MS/MS).  Future work will focus on selenoprotein quantification by on-line isotope dilution LC/ICP-MS strategies, and the potential of ICP-MS will be investigated for metalloproteins detection and quantification.