Multiple forms of Selenoprotein P in a Candidate Human Plasma Standard Reference Material
Guillaume Ballihaut, Lisa E. Kilpatrick, Eric L. Kilpatrick and W. Clay Davis
Human selenoprotein P (SePP) is responsible for the transport and distribution of the essential and beneficial trace element selenium (Se) through the human body. The concentration of SePP in human blood represents the most useful marker of Se nutritional status. Although SePP has been extensively studied, the structure of SePP in human plasma remains unresolved and must be determined for its use as a Se biomarker. For this reason, a human plasma Standard Reference Material (SRM 1950, NIST) was used to develop mass spectrometry-based methods for the specific characterization of SePP in a pooled sample representative of a healthy US population. Following immunoprecipitation, three SePP isoforms were unequivocally identified at 45, 49 and 57 kDa (termed as SePP45, SePP49 and SePP57) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Se was detected by gel electrophoresis laser ablation inductively coupled plasma mass spectrometry in SePP49 and SePP57, which was confirmed by the identification of three selenopeptides by LC-MS/MS analyses. Conversely, neither Se nor peptides covering SePP sequence after residue 306 was identified in SePP45 that suggests that SePP45 is a truncated isoform transcriptionally terminated at the 2nd in-frame UGA codon. An additional band at 23 kDa was found to contain Se but no peptides of SePP. Instead, glutathione peroxidase 3 (GPx3) was identified within the band presumably being co-immunoprecipitated with the SePP providing preliminary evidence that SePP and GPx3 interaction may take place in vivo. Therefore, this study has brought new insights into selenoprotein isoform studies as well as a more thorough understanding of Se metabolism.