A reliable method of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry for determining selenoamino acids in selenoproteins from Lactococcus lactis

• In this study, we addressed a topical issue “A reliable HPLC-ICP-MS method for determining selenoamino acids, especially Selenocysteine (Sec)” after optimizations of proteolysis conditions, chromatographic conditions and determination conditions. The innovation and breakthrough of our research are reflected as the following aspects: • The physiological health function of selenium is realized through selenoprotein composed of selenocysteine (SeCys) mostly[1], so we planned to detect whether the selenoproteins expressed by lactic acid bacteria have this active selenoamino acid. And it was finally realized through rigorous experimental design and certification. • Compared to the studies in Table 1, our method can successfully detect SeCys, SeCys2, SeMeCys and SeMet simultaneously within 10 min, especially to separate SeCys from SeCys2 after optimization of operation conditions for HPLC-ICP-MS and chromatographic conditions (see Table S1 and S2); And the reagents used in our study are milder and can only be extracted with water and enzyme. • Although SeCys is reductive and can be oxidized, it could be detected from Se-enriched yeast[2, 3]. However, the derivatization method used in those studies is more time-consuming and cumbersome, since the reduction with DTT and other reducing reagents and the derivatization with IAM reagents are easy to produce by-products, which needs extra operation steps to eliminate their effects[2]. So the method developed here is more dominant and can be easily conducted. • In contrast to other molecular identification methods, especially the popular NMR and ESI-MS/MS, HPLC-ICP-MS is more suitable for identifying amino acids containing selenium (see Table 1). Being of the same class of mass spectrometry technique as ESI-MS/MS, HPLC-ICP-MS can more directly separate and identify qualitatively and quantitatively selenoamino acid mixtures through external standard method, and give more intuitive results. NMR is widely used as a "Gold Standard" method to determine the structure of molecular substance, but usually only single pure compound. In our study, we used the commercially available NMR-confirmed standard selenium amino acids to establish and verified the HPLC-ICP-MS method. • The results also indirectly indicated the random incorporation of selenium into LlGPx. This work throws new light on the identification and biosynthesis of organic selenium species in selenoproteins and selenium-riched organisms. • All in all, we believe this paper is suitable for “Journal of Chromatography A”. • [1] D.E. Fomenko, W. Xing, B.M. Adair, D.J. Thomas, V.N. Gladyshev, High-throughput identification of catalytic redox-active cysteine residues, Science 315 (2007) 387-389. doi: 10.1126/science.1133114 • [2] K. Bierla, J. Bianga, L. Ouerdane, J. Szpunar, A. Yiannikouris, R. Lobinski, A comparative study of the Se/S substitution in methionine and cysteine in Se-enriched yeast using an inductively coupled plasma mass spectrometry (ICP MS)-assisted proteomics approach, Journal of proteomics 87 (2013) 26-39. • [3] K. Bierla, R. Lobinski, J. Szpunar, Determination of proteinaceous selenocysteine in selenized yeast, International Journal of Molecular Sciences 19 (2018) 543. doi: 10.3390/ijms19020543 A reliable method for simultaneous determination of four organic selenium species by HPLC-ICP-MS was developed and implemented in determining organic selenoamino acids (Se-AAs) in selenoproteins from Lactococcus lactis ( L. lactis ) NZ9000. The method consisted of liberating Se-AAs from selenoproteins using ultrasound-assisted protease hydrolysis, and quantitatively detecting Se-AA speciations by HPLC-ICP-MS. After optimizations of proteolysis conditions, chromatographic conditions and determination conditions, the established method could efficiently separate the four Se-AAs, including SeCys, SeCys2, SeMeCys and SeMet within 10 min. It presented high sensitivity with the limits of detection and quantitation in the range of 0.197∼0.240 μg∙L −1 and 0.788∼0.960 μg∙L −1 , respectively, good repeatability with a relative standard deviation (RSD) of less than 5%, and good recovery in the desired floating range of 90%∼105%, verifying the good accuracy. The method successfully detected four selenium species in the purified glutathione peroxidase (LlGPx) overexpressed in L. lactis NZ9000, SeCys (0.9716∼1.6784 μg∙g −1 ), SeCys2 (1.0695∼1.2124 μg∙g −1 ), SeMeCys (0.7288∼0.7984 μg∙g −1 ) and SeMet (1.0058∼1.9571 μg∙g −1 ), accounting for up to 80.14% of total selenium. There was no difference of order of magnitude in the four Se-AAs, indirectly indicating the random incorporation of selenium into selenoprotein LlGPx in L. lactis NZ9000. This work throws new light on the identification and biosynthesis of organic selenium species in selenoproteins and selenium-riched organisms like L. lactis .