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HPLC-ICP-MS speciation of selenium in Se-cultivated Flammulina velutipes
⁎Corresponding author. lbr_simmer@126.com (Huawei Li)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Selenium is an essential micronutrient required at trace levels for human health, and dietary intake is the only source of selenium, which appears mainly in the form of selenocompounds. In this study, Flammulina velutipes was grown for 80 days in standard medium containing selenite, and the level of total selenium in the organism was then determined by inductively-coupled plasma mass spectrometry (ICP-MS). In Se-cultivated F. velutipes, selenium was mainly distributed in the water-soluble form and the content of soluble selenium-containing species in Se-cultivated F. velutipes was 47.10 mg kg−1, accounted for 72.5% of the total selenium content. The water-soluble proteins in F. velutipes were extracted and precipitated by different ammonium sulfate saturation concentrations. Size-exclusion high performance liquid chromatography (SEC-HPLC) analysis of these proteins revealed the presence of at least six selenium-containing protein species, with molecular weights ranging from 9000 to 74,000 Da, Selenium-containing proteins represented about 7.0% of the total soluble selenium. The result of this study suggested that Se-cultivated F. velutipes could potentially be considered as a selenium supplement for human.
Keywords
Flammulina velutipes
Speciation analysis
Selenoproteins
SEC-ICP-MS
1 Introduction
Selenium is a key trace element required in small amounts by humans and animals (Fairweather-Tait et al., 2011; Shamberger and Willis, 1971). It exists in different chemical forms, each of which can determine the bioavailability and toxicity of selenium in the body (Mehdi et al., 2013; Thiry et al., 2012; Umysová et al., 2009; Whanger, 2002). The inorganic forms are mostly selenite and selenate, while the organic forms are mainly selenoproteins, Se-lipids, Se-peptides and Se-amino acids (Hatfield et al., 2014; Kouba et al., 2014; Ly and Wasinger, 2011; Yuan et al., 2012). The toxicity associated with organic selenium is lower than that associated with inorganic selenium, and inorganic selenium is not easily absorbed by the body (Guerrero et al., 2014; Kieliszek and Błażejak, 2013).
The majority of biological and biomedical effects appear to be mediated by proteins that contain selenium. There are two possible mechanisms for selenium incorporation into proteins. The first pathway is the specific incorporation of selenium into proteins in the form of genetically encoded selenocysteine (Thyer et al., 2012; Turanov et al., 2013). The second pathway corresponds to selenium is incorporated nonspecifically into proteins in place of methionine by replacing the sulfur. It is known that selenomethionine can be nonspecifically inserted into proteins by the usual methionine incorporation process. This type of substitution is very common in biological systems; selenomethionine from ingested food can be incorporated randomly into cellular proteins for humans and other animals (Maseko et al., 2013; Knowles and Grace, 2014).
The production of selenium-cultivated plants has been widely studied and the ability of plants to accumulate and transform the inorganic forms of selenium (selenite and selenate) into bioactive organic forms has far-reaching implication in human nutrition and health (Da Silva et al., 2013; Wróbel et al., 2004). Normally, in areas with low selenium concentration in the soils, an effective way to increase the selenium content in plants cultivated in these soils is to use selenium-enriched fertilizers that contain selenium salts. Therefore, it is important to find cultivated plants with the ability to tolerate and transform inorganic selenium into bioactive compounds. Flammulina velutipes is one of most popular fungi in china and japan. Its production and consumption ranks fourth in the edible mushrooms (Lee et al., 2005; Smiderle et al., 2006). Thus, to assess the selenium accumulation capacity of F. velutipes, it is necessary to study the selenium speciation.
Many studies have clarified that hyphenated techniques coupling chromatographic separation with inductively coupled mass spectrometry (ICP-MS) can be a powerful and sensitive technique for selenium speciation (Chen et al., 2013; Kannamkumarath et al., 2002; Mao et al., 2012). In general, chromatographic methods are used to fractionate mixture in separation science. But in protein separation, crude protein extract cannot be directly applied to chromatographic systems due to low protein concentration and interfering compounds. So it is essential to concentrate proteins into a small volume that will minimize the sample loss and remove interfering compounds. In this study, ammonium sulfate was selected as the precipitating agent to concentration; the protein fraction and the distribution of selenium-containing proteins in the extract of F. velutipes were subjected to selenium speciation analysis using size-exclusion chromatography (SEC-HPLC) coupled to ICP-MS. The results obtained suggested that F. velutipes cultivated under selenium-enriched condition could possibly be used as a source of selenium for human consumption.
2 Materials and methods
2.1 Instrumentation
Chromatographic separation was performed using an Agilent 1100 liquid chromatographic system (Agilent Technologies, Palo Alto, CA, USA) equipped with a binary HPLC pump, a temperature-controlled column compartment and a diode array detector (DAD). A TSK-G3000SW column (Japan) was used for size-exclusion chromatography. An Agilent 7500c inductively coupled plasma mass spectrometer (ICP-MS) equipped with Babington nebulizer and Scott-type double-pass spray chamber was used for the determination of total selenium. The coupling between the corresponding column outlet and the sample introduction system of ICP-MS consisted of a 500-mm long PEEK tubing with an inner diameter of 0.17 mm. The instrument-operating conditions are given in Table 1.
| ICP-MS parameters | |
| Forward power | 1490 W |
| Carrier gas (Ar) flow rate | 1.15 L min−1 |
| Dwell time | 0.1 s per isotope |
| Isotopes monitored | 77Se, 82Se |
| SEC-HPLC parameters | |
| Column | TSK-G3000SW (Japan) |
| Mobile phase | 30 mM Tris buffer, pH 7.2 |
| Flow rate | 0.5 mL min−1 |
| Column temperature | 20 °C |
| Injection volume | 50 μL |
2.2 Reagents and standard
All reagents used were of analytical grade. Milli-Q grade water (18.2 MΩ cm at 25 °C) was used throughout the experiment. Sodium selenite was purchased from Sigma. Selenium standard solution (500 mg L−1) was purchased from Sinopharm Chemical Reagent Co., Ltd. Protein standards (bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa)), Coomassie brilliant blue G-250, nitric acid (65%), Tris(hydroxymethyl)aminomethane, and ammonium sulfate were purchased from Sigma.
2.3 Cultivation and preparation of Se-cultivated Flammulina velutipes
Flammulina velutipes was grown under controlled conditions in a glass jar containing corn cob, cornmeal, bran and sodium selenite, and selenium at a fixed concentration of 5 mg kg−1. F. velutipes was aseptically inoculated into the glass jar, and growth was allowed to proceed under a humid condition (Tie et al., 2014). The growth temperature was initially held at 7–23 °C, but once the inside of the jar was covered with mycelium, the temperature was adjusted to 12 °C. The reproductive period of F. velutipes was maintained for several days at 12 °C, but once the fruiting process began, the temperature was reduced to 7 °C until harvest. The harvested F. velutipes was separated into caps and stalks, and then frozen at −85 °C for 24 h, followed by freeze drying. After drying, all parts of F. velutipes were homogenized respectively and stored in a humidity control box at room temperature until analysis.
2.4 Total selenium determination
The digestion procedure and determination method were performed as previous study (Tie et al., 2006). Briefly, approximately 0.5 g of dry F. velutipes powder (caps and stalks) was weighed into a 10-mL tube. Nitric acid (3 mL) was added to the tube and the tube was then incubated on a heating block set at 100 °C for 5 h. After cooling, the final volume of the sample was made to 50 mL with Milli-Q water and the concentration of total selenium in the sample was then measured by ICP-MS. A blank sample which contained no F. velutipes powder was also prepared and measured in the same way. Analysis was performed in triplicate for each sample.
2.5 Extraction of selenium for chromatographic speciation
About 0.5 g dried F. velutipes powder was weighed and transferred into a conical flask. 15 mL of 30 mM Tris-HCl (pH 7.2) was added to the flask and the sample was stirred for 4 h on a magnetic stirrer. In order to identify the soluble selenium-containing molecules and their molecular weight, as well as their distribution and biological stability, the F. velutipes extract was centrifuged at 4000g for 30 min and the supernatant was subjected to SEC-ICP-MS analysis directly (Kápolna et al., 2007; Tie et al., 2015).
To study the distribution of selenium-containing proteins in the water-soluble protein fraction of F. velutipes, the supernatant obtained from the above process was used for ammonium sulfate precipitation. Traditionally, ammonium sulfate concentration is expressed as % of saturation. The amount of ammonium sulfate necessary to reach a % is calculated by the formula g is the amount of ammonium sulfate (in grams) to be added to 1 L of the solution at 20 °C to increase the ammonium sulfate concentration from S1% saturation to S2% saturation (Scopes, 1987).
The concentration of ammonium sulfate was increased from 0% to 30% and followed by centrifugation at 4000g for 30 min. The precipitate was dissolved in 5 mL of 30 mM Tris-HCl (pH 7.2). The supernatant was subjected to a series of further ammonium sulfate precipitation, first at 50%, then at 75%, and finally at 100% saturation. Four precipitates were collected in total, and these were designated as P1, P2, P3 and P4, corresponding to the precipitates of 30%, 50%, 75% and 100% ammonium sulfate saturations, respectively. These precipitates were each dissolved in 5 mL of 30 mM Tris-HCl and then transferred into a dialysis bag and dialyzed against water until no white precipitate was formed when tested with 10% BaCl2. All samples were diluted with 30 mM Tris-HCl (pH 7.2) to a final volume of 15 mL. Speciation of selenium-containing proteins was then carried out for these samples using the SEC-ICP-MS system.
2.6 Protein assay
Approximately 0.5 g of dry F. velutipes powder was dispensed in a plastic tube and extracted with 15 mL 30 mM Tris-HCl (pH 7.2) for 4 h, then the solution was centrifuged at 4000g for 30 min and after this the supernatant was collected. The concentration of soluble protein was determined by the Bradford assay using BSA as standard.(Jones et al., 1989).
3 Results and discussion
3.1 Selenium content of whole F. Velutipes
The selenium content in the control F. velutipes (cultivated in the absence of added selenium) was determined to be 0.34 mg kg−1 for the dried stalk and 0.54 mg kg−1 for the dried cap. Compared to the control, F. velutipes cultivated in the presence of 5 mg kg−1 selenium showed a significant increase in the selenium content in both stalk and cap. The selenium contents in the stalk and cap were 45.33 and 86.67 mg kg−1, respectively, which corresponded to 133 and 160.5-fold increases. The selenium content in the cap was almost twice the level in the stalk. A similar trend has been observed for selenium-enriched Agaricus bisporus (Maseko et al., 2013). However, selenium distribution in Lentinula edodes shows a higher content in the stalk than in the cap (Alofe et al., 1996). This difference may be due to the different species, which may have different selenium distributions within different tissues.
3.2 Protein and selenium content in F. Velutipes extract
The protein content in the extract of control F. velutipes was 74.9 mg kg−1, whereas the protein content in the extract of Se-cultivated F. velutipes was 241.4 mg kg−1, representing a 3-fold increase. This suggested that the added selenite could stimulate protein deposition in F. velutipes. The content of soluble selenium-containing species in the control and Se-cultivated F. velutipes were 0.37 and 47.10 mg kg−1, respectively, accounting for 42.0% and 72.5% of the total selenium content. Thus, in Se-cultivated F. velutipes, selenium was mainly found in the water-soluble form.
3.3 Determination of the selenium speciation in the F. Velutipes extracts
To study the different forms of water-soluble selenium in F. velutipes, the obtained extract was analyzed by SEC coupled to DAD and SEC coupled to ICP-MS, and their respective chromatograms are shown in Fig. 1a and b. In the case of DAD detection, seven peaks were detected (Fig. 1a), each representing a protein or peptide with or without selenium, while in the case of ICP-MS detection, four selenium-containing species were detected (Fig. 1b). The calibration of the SEC column was accomplished with a standard mixture of bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa) with DAD. The molecular weights and selenium contents of these four species are shown in Table 2. The 79.5-kDa and 60-kDa species could be selenium-containing proteins by virtue of their high molecular weights. These two species accounted for about 8.22% of the total soluble selenium in the extract. Since SEC separates molecules according to their sizes and shapes, and that it is difficult to detect selenium species with a molecular weight of less than 4 kDa, the molecular weights of peak VI and VII therefore could not be detected. The species represented by molecular weights of less than 4 kDa accounted for 81.4% of the total soluble selenium. According to our previous study, the low-molecular weight molecules in the extract of F. velutipes mainly including Se-Cys, Se-Met and peptides composed of these two seleno amino acids, which accounted for 66.7% of the total water-soluble selenium (Tie et al., 2007).
Separation of F. velutipes extract by HPLC. (a) SEC-HPLC-DAD; (b) SEC-HPLC-ICP-MS.
| Se-containing compound | II | III | VI | VII |
|---|---|---|---|---|
| Retention time (min) | 5.59 | 5.82 | 7.15 | 8.76 |
| Molecular weight (kDa) | 79.5 | 60.0 | <4 kDa | <4 kDa |
| Content of selenium (mg/kg) | 1.87 | 2.00 | 32.9 | 5.40 |
To study the stability of selenium-conjugated proteins in F. velutipes, the obtained extract was preserved at 4 °C for 3 and 5 days before subjecting to SEC-HPLC-DAD and SEC-HPLC-ICP-MS analyses. According to the results (Fig. 2), Peak II and III appeared to decrease and peak VI and VII appeared to increase with increase in storage time, suggesting that high-molecular weight selenium species were not stable and could be easily hydrolyzed into selenopepetides and selenoamino acids (Fig. 2). Some studies have observed that selenium can weakly bound to proteins through of the selenodisulfides (RS-Se-SR’) or methylselenyl sulfides (RS-SeCH3), formed during post-translational modification of the proteins, and these forms are easily reduced, releasing low-molecular-weight selenium compounds (Ganther and Kraus, 1987; Kannamkumarath et al., 2002).
Changes in the distribution of selenium-containing compounds in F. velutipes extract as detected by HPLC. (a) SEC-HPLC-DAD; (b) SEC-HPLC-ICP-MS.
3.4 Selenium content of precipitated protein by ammonium sulfate
The water-soluble proteins in the F. velutipes extract were divided into four groups (P1–P4), based on their likelihood to precipitate in the presence of different ammonium sulfate concentrations. The total protein from the four groups accounted for 51.7% of the soluble protein. Selenium was found in P2, P3 and P4 only, with the highest level in P3, and represented 63.69% of the total soluble selenium (four groups), compared to 62.6% in the case of total soluble protein content (four groups). The orders of decreasing selenium content and protein content were the same; P3 > P2 > P4 > P1 (Fig. 3).
Selenium and protein content in the water-soluble extract of F. velutipes.
3.5 Distribution of selenium-containing proteins in the water fraction of F. velutipes
The chromatograms of selenium-containing proteins in the water-soluble protein fraction are shown in Fig. 4. Non-selenium-conjugated proteins were precipitated by ammonium sulfate at 30% saturation. As for selenium-conjugated proteins, two species were detected from the proteins precipitated between 30% and 50% ammonium saturation (Fig. 4F), whereas three species were detected from the proteins precipitated between 50% and 75% ammonium saturation, while only one species was detected from the proteins precipitated between 75% and 100% ammonium saturation. According this result, it is evident that a certain amount of proteins which do not contain selenium, can be removed by increasing the concentration of ammonium sulfate in the crude extract up to 30% of saturation and removing the precipitation after centrifugation. A range of molecular weights were obtained for these selenium-conjugated protein species (Table 3). According to Table 3 and Table 4, at least six selenium-containing proteins existed in the Se-cultivated F. velutipes extract, ranging from 9000 to 74,000 Da. Selenium-containing proteins represented about 7% of the total soluble selenium, and P3 consisted of the highest level, covering three species having molecular weights of 34000 Da (P3a), 25000 Da (P3b) and 12000 Da (P3c). These findings were similar to those reported in some other studies. Jayasinghe et al. have observed that most of selenium-containing proteins in Brazil nuts can be precipitated during 50% to 80% ammonium sulfate saturation, and distributed in high molecular weight region as well as in low molecular weight region. Hammmel et al. performed the extraction of Lecythis ollaria at pH 4.5 and pH 7.5 and the extracts were analyzed by SDS-PAGE method. The results showed that the protein-bound selenium of the two extracts account for 9% and 29% in total selenium, and more than 90% of the firmly bound selenium protein was present in the molecular mass range below 20 kDa (Hammel et al., 1996).
Analysis of soluble proteins in F. velutipes extract. Soluble proteins in F. velutipes extract (P1, P2, P3, and P4) fractionated by ammonium sulfate were either subjected to SEC-HPLC with DAD (A, B, C, and D) or SEC-HPLC-ICP-MS analysis (E, F, G, and H).
| Water-soluble protein | Molecular weight of selenoprotein/kDa | Contents of selenium in selenoproteins/(μg kg−1) | Relative content of selenium in selenoproteins (%) |
|---|---|---|---|
| P1 | – | – | – |
| P2a | 74 | 28.57 | 10.75 |
| P2b | 10 | 25.38 | 9.55 |
| P3a | 34 | 37.67 | 26.21 |
| P3b | 25 | 26.29 | 18.29 |
| P3c | 12 | 26.00 | 18.10 |
| P4 | 9 | 49.34 | 17.10 |
| Component | Consistence of selenium/(mg kg−1) |
|---|---|
| Total | 65.00 |
| Soluble selenium-containing compound | 47.10 |
| Soluble selenoprotein | 3.29 |
| P2a | 0.354 |
| P2b | 0.314 |
| P3a | 0.862 |
| P3b | 0.602 |
| P3c | 0.595 |
| P4 | 0.563 |
4 Conclusion
In this study, water-soluble proteins of F. velutipes were fractionated by ammonium sulfate precipitation, followed by size exclusion chromatography (SEC) with DAD or ICP-MS detection to investigate the distribution of different molecular weights of soluble selenium-containing proteins in F. velutipes. At least six selenium-containing proteins were found in Se-cultivated F. velutipes, and their molecular weights ranged from 9000 to 74000 Da and accounted for 7.0% of soluble organic selenium. Se-cultivated F. velutipes could be considered as a valuable dietary of selenium for human. Further study is in progress to identify the unknown selenium-containing species.
Acknowledgements
This work was supported by Liaoning Scientific Research Fund 2011205001. Special thank is given to Dr Alan K Chang (Liaoning University, China) for his valuable discussion and for revising the language of the manuscript.
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