Accurate Quantification of Selenoprotein P (SEPP₁) in Plasma Using Isotopically Enriched Seleno-peptides and Species-Specific Isotope Dilution with HPLC Coupled to ICP-MS/MS

Abstract

A novel strategy for the absolute quantification of selenium (Se) included in selenoprotein P (SEPP1), an important biomarker for human nutrition and disease, including diabetes and cancer, is presented here for the first time. It is based on the use of species-specific double isotope dilution mass spectrometry (SSIDA) in combination with HPLC-ICP-MS/MS for the determination of protein bound Se down to the peptide level in a complex plasma matrix with a total content of Se of 105.5 μg kg(-1). The method enabled the selective Se speciation analysis of human plasma samples without the need of extensive cleanup or preconcentration steps as required for traditional protein mass spectrometric approaches. To assess the method accuracy, two plasma reference materials, namely, BCR-637 and SRM1950, for which literature data and a reference value for SEPP1 have been reported, were analyzed using complementary hyphenated methods and the species-specific approach developed in this work. The Se mass fractions obtained via the isotopic ratios (78)Se/(76)Se and (82)Se/(76)Se for each of the Se-peptides, namely, ENLPSLCSUQGLR (ENL) and AEENITESCQUR (AEE) (where U is SeCys), were found to agree within 2.4%. A relative expanded combined uncertainty (k = 2) of 5.4% was achieved for a Se (as SEPP1) mass fraction of approximately 60 μg kg(-1). This work represents a systematic approach to the accurate quantitation of plasma SEPP1 at clinical levels using SSIDA quantification. Such methodology will be invaluable for the certification of reference materials and the provision of reference values to clinical measurements and clinical trials.

Determination of Selenium Species in Muscle, Heart, and Liver Tissues of Lambs Using Mass Spectrometry Methods

“…Hydride generation atomic absorption spectrometry (HG-AAS) [12,13], electrothermal atomic absorption spectrometry (ET-AAS) [14,15], radiochemical and instrumental neutron activation analysis (RNAA and INAA) [16], and inductively coupled plasma mass spectrometry (ICP-MS) [17,18] are frequently used for this purpose. In order to perform speciation, it is necessary to use separate techniques coupled to sensitive detection methods, i.e., elemental or molecular mass spectrometry [19][20][21][22][23][24][25][26][27][28][29][30]. Among the mentioned techniques ICP-MS is the most powerful element-specific method for total Se determination in biological samples.…”

Standardization approaches in absolute quantitative proteomics with mass spectrometry

“…This approach can be used in quantitative proteomics measuring isotope ratios in elements either naturally present in the peptides/proteins, or tagged to them, usually with reagent molecules. Hence, absolute quantification has been reported for either amino acids, peptides, and even full‐length proteins …”

“…Such elemental concentrations accurately computed can be translated into biomolecule concentration given the known stoichiometry heteroatom/ biomolecule. This approach can be used in quantitative proteomics measuring isotope ratios in elements either naturally present in the peptides/proteins, or tagged to them, usually with reagent molecules.Hence, absolute quantification has been reported for either amino acids,157,158 peptides,159 and even full-length proteins 160. 161 3.2.2 | Species-unspecific isotope dilution approaches On the other hand, quantitative proteomics with species-unspecific isotope dilution using ICP-MS detection is based on use of an online continuous addition, commonly after the on-column separation, of a generic species isotopically enriched in the detected heteroelement, in a known concentration and isotopic composition.…”

Accurate quantification of selenoproteins in human plasma/serum by isotope dilution ICP-MS: focus on selenoprotein P

“…However, in this case, due to a higher sample complexity, the measured isotope ratios in the sample blend and in the calibration standard accounted for about 75% and 10% of the total uncertainty budget, respectively. (Figure 1.4a) 25 . Se mass fractions for SEPP1 of (56.2 ± 1.7) ng g -1 Se and (60.6 ± 3.2) ng g -1 Se (k=2) in BCR-637 and SRM 1950, respectively, were obtained at the peptide level, which agree well with those obtained for the whole protein using single SS IDA-ICP-…”