Abstract
In recent years, cold-pressed vegetable oils have become very popular on the global market. Therefore, new versatile methods with high sensitivity and specificity are needed to find and combat fraudulent practices. The objective of this study was to identify oilseed species-specific peptide markers, using proteomic techniques, for authentication of 10 cold-pressed oils. In total, over 380 proteins and 1050 peptides were detected in the samples. Among those peptides, 92 were found to be species-specific and unique to coconut, evening primrose, flax, hemp, milk thistle, nigella, pumpkin, rapeseed, sesame, and sunflower oilseed species. Most of the specific peptides were released from major seed storage proteins (11 globulins, 2S albumins), and oleosins. Additionally, the presence of allergenic proteins in the cold-pressed oils, including pumpkin Cuc ma 5, sunflower Hel a 3, and six sesame allergens (Ses i 1, Ses i 2, Ses i 3, Ses i 4, Ses i 6, and Ses i 7) was confirmed in this study. This study provides novel information on specific peptides that will help to monitor and verify the declared composition of cold-pressed oil as well as the presence of food allergens. This study can be useful in the era of widely used unlawful practices.
Subject terms: Plant sciences, Biomarkers
Introduction
Growing consumption of cold-pressed plant oils and a wider range of available oil types have been observed on the market over the last few years. Currently, the most popular are olive oil, palm oil, rapeseed oil, soybean oil, and sunflower seed oil, but less common oils such as evening primrose seed oil, hemp seed oil, flaxseed oil, milk thistle seed oil, nigella seed oil, and pumpkin seed oil are gaining in importance. The global cold-pressed oil market value was $24.62 billion in 2018 and is expected to reach $36.40 billion by 2026, which is a 5.3% CAGR1. The reasons are the growing health awareness of the human population, increasing knowledge of the bioactive compounds detected in various oilseeds and their contribution to diet-related and skin disease prevention and treatment. Cold-pressed oils retain most of their bioactive and nutritional value since the oil’s temperature during the pressing process lies below 50 °C. Therefore, for health benefits, nutritionists advise regular diet supplementation with small amounts (1–3 tablespoons per day) of cold-pressed oils, due to higher yields of pro-healthy compounds over refined oils. They may regulate the blood lipid profile and insulin level, as well as offer anticancer and antioxidant activity2,3. Another trend observed is the modification of food products and nutraceuticals to improve their nutritional composition and positive impact on human health through the addition of edible oils3,4. In particular, in animal food products, the partial replacement of the fat or meat fraction with a plant oil/protein fraction is more frequent4,5.
Oilseeds are rich in n-3 and n-6 essential fatty acids, protein, carbohydrate, fibre, vitamins, minerals, and many small bioactive components belonging to different classes, including phytosterols, tocopherols, phenolic compounds, flavonoids, carotenoids, and bioactive peptides. For instance, hemp oil has a high content of antioxidants, such as phenolic compounds, tocopherols, and phytosterols as well as flavonoids, including flavanones, flavanols, flavonols and isoflavones. Milk thistle oil is a source of silymarin and mixtures of various flavonolignans, i.e. silydianin, silychristin, silybin, and isosilybin, and flaxseed oil is rich in phenols, flavonoids, α- and δ -tocotrienol, and carotenoids, including lutein and β-carotene6–8. The presence of proteins and peptides in cold-pressed oils is less commonly discussed, though they are an important component that may influence the oils’ stability and also cause an allergenic response in sensitive consumers2,3.
The use of cold-pressed oil by the food industry is anticipated to reach the highest growth rate in the next few years. The oils’ online retail distribution channel is also expected to grow considerably1. Since unprocessed oils reach high prices and are a desirable commodity on the market, they are products susceptible to counterfeiting9,10. Therefore, there is an urgent need to implement modern and sophisticated analytical methods to detect adulterations of high-price cold-pressed oils derived from various oleaginous seeds and fruits. Many advanced untargeted approaches have been introduced recently to address authenticity issues of edible oils, based on liquid or gas chromatography mass spectrometry (LC–MS or GC–MS) techniques for measuring various polar, nonpolar, and volatile compounds, as well as spectroscopic techniques including nuclear magnetic resonance (NMR) spectroscopy, near-infrared spectroscopy (NIR), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), or fluorescence, where the result is achieved by analysing the whole spectrum of oils and development of statistical models11–14.
When food fraud is considered in terms of food safety issues, and related, for example, to food allergenicity, a targeted approach based on identifying specific proteins and peptide markers could be a method of choice. From this aspect, MS-based proteomic analysis has the advantage of detecting the adulteration of unrefined oils and tracing potential allergenic proteins present in the sample, simultaneously during one MS run. Depending on the type of oil, the method of protein extraction and assessment methodology, the protein content of edible oils varies from 0.01 to 14.8 ppm2,15. Allergenic proteins, such as cruciferin (11S globulin) and napin (2S albumin) have been identified in rapeseeds (Brassica napus) but also in unrefined rapeseed oil using a gel-based LC–MS method; however, these allergens have not been shown in refined rapeseed oil16. Moneret-Vautrin et al.17 found that the residual allergenic proteins, i.e. Ara h1, in peanut oil can cause an allergic reaction in infants with atopic dermatitis sensitive to peanut, whereas, Blom et al.18 estimated the risk of allergenic reactions to refined peanut oil as extremely low.
This study aimed to identify the unique peptide markers specific to oilseed species and proteins present in cold-pressed oils using proteomic techniques. An additional goal was to assess the presence of allergens. The protein and peptide composition of oils produced from 10 oilseeds, namely coconut, evening primrose, hemp, flax, milk thistle, nigella (also known as black cumin), pumpkin, rapeseed, sesame, and sunflower seeds, was analysed using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF–MS/MS). The markers identified could be useful for the detection of adulteration.
Results and discussion
Protein profiles
A previous study, comparing five methods using various solvents to separate proteins from unrefined and refined oils, showed that the protein content differs significantly depending on the extraction method15. Therefore, the two most efficient methods were tested on cold-pressed sunflower oil, i.e. extraction with acetone-hexane and extraction with acetone, at the beginning of this work. Since no considerable differences were observed in the extracted protein profiles, extraction with acetone was applied to all oil samples. Acetone extraction has been used successfully to identify allergenic proteins from commercial cold-pressed rapeseed oils16. Proteins were present in all the examined oils but the proteomic profiles differed considerably in the distribution and intensity of protein bands (Fig. 1). Visible differences reflect the species diversity of the major storage proteins. Under reducing SDS-PAGE conditions, the most abundant bands belonged to 11S globulin α and β subunits (MW 30–45 and 20–30 kDa, respectively). 11S globulin monomers (MW about 45–56 kDa) were less intense. Lower molecular weight bands (MW 10–21 kDa) were less abundant but showed species specificity.
Figure 1.
Protein profiles of 10 acetone-extracted cold-pressed oils. Lanes: S—sunflower; Se—sesame; P—pumpkin; C—coconut; E—evening primrose; H—hemp; N—nigella; L—flax (linen); R—rapeseed; M—milk thistle.
Globulin, albumin and oleosin subunits have been reported in electrophoretic bands obtained from oilseed meals or isolates of various species, such as sesame protein isolate19, hemp protein isolate20 and sunflower seed and kernel proteins21. Regarding cold-pressed oils, proteins are minor components present in very low amounts as a result of gentle processing by pressing the oilseeds with a screw press without heating, and the resulting oil is purified only by sedimentation, filtration, or centrifugation. Therefore, identification of the proteins in vegetable oils has not been common in the literature. The fruit and seed proteins, mainly storage proteins and different oleosins have been found in olive oil and palm oil22,23, whereas knowledge about nigella, milk thistle, and evening primrose proteins is negligible. Major rapeseed allergens, such as napin and cruciferin (2S albumin and 11S globulin, respectively), have been extracted and identified from commercial cold-pressed rapeseed oil16. Accordingly, there is a need to conduct research to determine species-specific proteins and peptides found in cold-pressed vegetable oils.
Protein identification
To identify specific proteins, an in-solution tryptic digest of acetone-extracted proteins was analysed using the UHPLC-Q-TOF–MS/MS method. Figure 2 presents the 3D chromatograms obtained from the 10 examined cold-pressed oils. The National Center for Biotechnology Information (NCBI, U.S. National Library of Medicine) protein database was searched for protein and peptide identification across green plant taxonomy entries or appropriate genera (i.e. Brassica, Cannabis, Cocos, Cucurbita, Helianthus, Nigella, Linum, Oenothera, Sesamum, and Silybum). The number of identified proteins and tryptic peptides obtained from cold-pressed oils is shown in Fig. 3. In total, over 380 proteins and 1050 peptides were identified in the samples analysed. The highest number of proteins and peptides was found in the pumpkin and sunflower oils (106 and 105 proteins, respectively). The smallest number of proteins and peptides was determined in evening primrose, milk thistle, and nigella oils (only four, two, and three proteins, respectively). For these species, only a small fraction of the proteins have been sequenced, and thus, databases are highly incomplete. SDS-PAGE protein profiles obtained from these three oils confirmed the presence of a storage protein bands of species-specific distribution and intensity (Fig. 1).
Figure 2.
3D LC-Q-TOF–MS/MS chromatograms of tryptic digested proteins extracted from cold-pressed oils obtained from 10 species of oilseeds: coconut, evening primrose, flax/linen, hemp, milk thistle, nigella, pumpkin, rapeseed, sesame, and sunflower. The mass-to-charge ratio (m/z), the retention time, and the intensity of the signal are present on the X-, Y-, and Z-axis, respectively.
Figure 3.
Number of identified proteins and tryptic peptides obtained from cold-pressed oils.
All proteins which were identified with high confidence scores using the Spectrum Mill MS Proteomics Workbench with > 70% score peak intensity and 10 ppm precursor mass tolerance are gathered in Supplementary material 1 for protein datasets. The matches and Spectrum Mill scores were evaluated at 1% of the false discovery rate (FDR), for identity and homology threshold. The identified proteins belong, among others, to 11S and 7S globulins, 2S albumins, and oleosin families. Some of those derived from hemp oil and sesame oil have been identified previously in hempseed and sesame seed defatted powders using LC–MS/MS24,25. For the remaining eight oilseed species studied, there is a lack of comprehensive proteomic MS-based analysis. Recently, only the key proteins of oil palm mesocarp (Elaeis guineensis) have been identified by 2-DE and MALDI-TOF/TOF methods23. None of them coincided with the proteins detected in coconut oil, despite the close relationship between these two species as they belong to the same family of Arecaceae.
Species-specific peptide markers
The peptides identified and selected, based on the Spectrum Mill output scores, were searched against the NCBInr database using the protein Basic Local Alignment Search Tool (BLAST) and blastp algorithm (U.S. National Library of Medicine, Bethesda, MD), for species and protein specificity. In total, 92 species-specific peptides, that were released from 42 proteins (including different subunits), were identified in the examined oils. The largest amount of unique peptides was found in sesame, sunflower and pumpkin oils (21, 18, and 17 peptides, respectively). Table 1 presents the unique, species-specific peptide markers identified in coconut, evening primrose, flax, hemp, milk thistle, nigella, pumpkin, rapeseed, sesame, and sunflower cold-pressed oils. The peptides’ total intensities were in the range of 105–108 and scored peak intensities (SPI) were > 60%. All peptides detected in the examined cold-pressed oils are shown in Supplementary material 2 for peptide datasets.
Table 1.
Unique peptide markers identified in coconut, evening primrose, flax, hemp, milk thistle, nigella, pumpkin, rapeseed, sesame, and sunflower cold-pressed oils.
| Protein | Protein score | Parent ion (m/z) | Mr (exp) | Exp z | Total intensity | RT (min) | SPI (%) | Delta (ppm) | Peptide marker |
|---|---|---|---|---|---|---|---|---|---|
| Coconut | |||||||||
| 11S globulin isoform 2 (AKS26849.1) | 234.02 | 635.3332 | 1269.6474 | 2 | 2.28E+07 | 20.45 | 94.1 | 9.3 | AGSEGFQFVSIK |
| 991.5129 | 1982.0052 | 2 | 3.44E+06 | 40.52 | 91.7 | 6.8 | GFGTELLAAAFGIDMELAR | ||
| 714.3443 | 2140.9977 | 3 | 5.77E+06 | 27.08 | 83.8 | 9.7 | GMVGLVMPGCPETFQSFQR | ||
| Cocosin (ASQ40963.1) | 202.8 | 929.0134 | 929.0134 | 2 | 1.06E+07 | 29.65 | 94.1 | 7.7 | QGQLLIVPQNFAMLER |
| 1011.4302 | 2021.8359 | 2 | 1.13E+07 | 19.43 | 88.0 | 8.5 | SEAGVTDYFDEDNEQFR | ||
| Oleosin isoform OLE500c partial (ACH91013.1) | 16.77 | 599.3204 | 1197.6222 | 2 | 3.04E+06 | 10.67 | 78.7 | 9.4 | VPGAEQLEQAR |
| Evening primrose | |||||||||
| Putative LOV domain-containing protein (AML79398.1) | 2.42 | 575.2869 | 1723.8511 | 3 | 1.03E+06 | 15.48 | 41.5 | − 2.8 | DPGSRLGSNNGSXFFR |
| Flax | |||||||||
| Conlinin (CAC94011.1) | 133.74 | 658.3132 | 1315.6059 | 2 | 5.62E+06 | 6.47 | 96.3 | 10.0 | DLPGQCGTQPSR |
| 745.3305 | 1489.6448 | 2 | 3.53E+06 | 1.40 | 79.9 | 6.0 | GGQGGQGQQQQCEK | ||
| 857.4170 | 1713.8151 | 2 | 4.60E+07 | 5.05 | 92.4 | 6.8 | QDIQQQGQQQEVER | ||
| Conlinin (CAC94010.1) | 101.34 | 1331.6097 | 2662.2023 | 2 | 2.19E+07 | 14.87 | 73.1 | 3.7 | GGGQGGQGQQQSCEQQIQQQDFLR |
| 863.9330 | 1726.8467 | 2 | 5.02E+06 | 4.45 | 93.4 | 6.9 | QEIQQQGQQQEVQR | ||
| Oleosin high molecular weight isoform (ABB01624.1) | 50.01 | 600.2677 | 1199.5184 | 2 | 1.29E+06 | 8.17 | 80.4 | 8.2 | MQDAAGYMGQK |
| 1082.5430 | 2164.0669 | 2 | 1.35E+07 | 19.22 | 83.9 | 5.4 | YLQQAGQGVGVGVPDSFDQAK | ||
| Oleosin high molecular weight isoform (ABB01616.1) | 51.29 | 1089.5514 | 2178.0826 | 2 | 7.14E+06 | 19.38 | 94.4 | 6.0 | YLQQAGQGVGVGVPDSFEQAK |
| Oleosin low molecular weight isoform, partial (ABB01620.1) | 24.57 | 795.8659 | 1590.7143 | 2 | 3.35E+06 | 6.60 | 62.5 | 6.5 | ASDFGQQHVTGQQTS |
| Oleosin low molecular weight isoform (ABB01617.1) | 15.34 | 838.3924 | 1675.7671 | 2 | 8.05E+06 | 7.17 | 75.4 | 6.2 | ASEFAQQHVTGGQQTS |
| Oleosin low molecular weight isoform (ABB01618.1) | 14.42 | 831.3859 | 1661.7514 | 2 | 1.54E+07 | 6.78 | 66.3 | 7.8 | ASEFGQQHVTGGQQTS |
| Hemp | |||||||||
| Edestin 3 (SNQ45160.1) | 453.22 | 847.4206 | 1693.8214 | 2 | 6.73E+07 | 19.87 | 94.8 | 7.4 | AMPEDVIANSYQISR |
| 889.4256 | 1777.8293 | 2 | 1.81E+07 | 15.58 | 94.4 | 8.2 | FYIAGNPHEDFPQSR | ||
| 1049.0236 | 2097.0247 | 2 | 1.54E+08 | 30.92 | 82.7 | 7.3 | GFSVNLIQEAFNVDSETAR | ||
| 846.7562 | 2538.2293 | 3 | 1.04E+08 | 29.63 | 93.6 | 9.8 | GVLGTLFPGCAETFEEAQVSVGGGR | ||
| 1166.5912 | 2332.1655 | 2 | 4.89E+06 | 21.75 | 66.8 | 4.2 | NAMYAPHYNINAHSIIYAIR | ||
| 834.3816 | 1667.7442 | 2 | 2.15E+07 | 8.68 | 92.9 | 7.0 | TAVYGDQNECQLNR | ||
| 1044.4852 | 3131.4132 | 3 | 2.41E+07 | 25.43 | 95.7 | 8.9 | VECEGGMIESWNPNHEQFQCAGVALLR | ||
| Edestin 3 (SNQ45158.1) | 390.94 | 823.0533 | 2467.1282 | 3 | 6.42E+06 | 20.03 | 88.8 | 7.0 | FYIAGNPHQEFPQSMMTQQGR |
| 563.7565 | 1126.4946 | 2 | 2.08E+06 | 3.43 | 75.7 | 9.9 | LEACEPDHR | ||
| 800.4523 | 1599.8853 | 2 | 1.34E+07 | 20.57 | 83.6 | 7.5 | QGQALTVPQNFAVVK | ||
| 7S vicilin-like protein (SNQ45153.2) | 65.5 | 1016.029 | 2031.0393 | 2 | 1.53E+06 | 22.47 | 74.8 | 5.6 | EILSSQQEGPIVYIPDSR |
| Milk thistle | |||||||||
| Preprosilpepsin 2 (AGE15495.1) | 38.35 | 764.4145 | 1527.8094 | 2 | 6.24E+06 | 28.93 | 94.9 | 8.1 | IFELTPEQYIFK |
| 772.3684 | 2315.0687 | 3 | 1.67E+07 | 21.68 | 78.4 | 9.5 | NVNEEEGGELVFGGVDPNHFR | ||
| Nigella | |||||||||
| Chain A, nigellin-1.1 (pdb|2NB2|A) | 51.07 | 775.3724 | 1549.7284 | 2 | 1.08E+06 | 18.88 | 85.7 | 5.9 | ACIGLCAPACLTSR |
| 673.8030 | 1346.5868 | 2 | 2.54E+06 | 17.22 | 94.1 | 8.8 | CTYIPDYAGMR | ||
| 716.2902 | 1431.5627 | 2 | 2.23E+06 | 6.40 | 72.0 | 7.2 | YQDCLSECNSR | ||
| Thionin NsW1 (C0HJH9.1) | 11.29 | 521.7133 | 1042.4114 | 2 | 1.55E+06 | 3.47 | 53.8 | 7.5 | TCSGLCGCK |
| Pumpkin | |||||||||
| 11S globulin subunit beta-like (XP_023553731.1) | 552.27 | 1039.4212 | 2077.8217 | 2 | 5.40E+06 | 9.22 | 95.5 | 6.5 | GDEQQWEEEQEEEQER |
| 1215.0627 | 2429.1038 | 2 | 9.60E+06 | 17.10 | 95.0 | 5.9 | GVVLSGCPETYQESQQSAGEFR | ||
| 805.1246 | 2413.335 | 3 | 1.77E+07 | 31.4 | 94.0 | 10.0 | LIIVVLLDVSNHANQLDFHPR | ||
| 699.8968 | 1398.7740 | 2 | 4.62E+07 | 14.53 | 90.0 | 8.8 | VEGQFEVIKPPR | ||
| 786.9318 | 1572.8414 | 2 | 6.18E+07 | 23.20 | 98.1 | 9.5 | VLAEVLNINTEMAR | ||
| Vicilin-like (XP_023527143.1) | 408.19 | 885.8216 | 2655.4287 | 3 | 5.85E+07 | 26.48 | 96.6 | 8.2 | LSEGGVLVIPAGHPIAIMASPNENLR |
| 709.3693 | 1417.7183 | 2 | 1.09E+07 | 16.30 | 93.0 | 9.3 | LVGFGINAENNNR | ||
| 671.3671 | 1341.7161 | 2 | 1.82E+06 | 13.52 | 90.0 | 8.1 | LVQPVNNPGEFK | ||
| 1030.9556 | 2060.8944 | 2 | 6.23E+06 | 11.92 | 84.9 | 4.6 | TEQEQSNNPYYFQEQR | ||
| 11-beta-hydroxysteroid dehydrogenase 1B-like (XP_022923933.1) | 133.1 | 878.9664 | 1756.9150 | 2 | 1.17E+06 | 25.58 | 83.0 | 6.0 | DALIGLVPVETAEACAK |
| 823.4332 | 1645.8465 | 2 | 1.91E+06 | 18.43 | 92.8 | 7.7 | ILAMPAAGASESDALTK | ||
| 788.9426 | 1576.8668 | 2 | 2.94E+06 | 26.02 | 88.7 | 7.0 | IVALSAPPAWMPAPR | ||
| 2S albumin-like (XP_023520423.1) | 74.43 | 669.3239 | 2005.9396 | 3 | 5.74E+05 | 15.18 | 67.2 | 8.8 | AHEEIGSCVQYLTQQSR |
| 11 kDa late embryogenesis abundant protein-like (XP_022940345.1) | 63.21 | 823.9135 | 1646.8093 | 2 | 4.18E+06 | 4.53 | 71.4 | 6.4 | TGAHTGLTTGTGTGTGTR |
| oleosin 18.2 kDa-like (XP_022928435.1) | 62.5 | 632.8148 | 1264.6128 | 2 | 2.82E+06 | 2.40 | 73.5 | 7.6 | TTTTTAAQEQGR |
| 17.1 kDa class II heat shock protein-like (XP_022982419.1) | 21.0 | 800.4250 | 1599.8337 | 2 | 6.42E+05 | 15.97 | 94.0 | 5.7 | VQIEDNQLVVTGER |
| 17.4 kDa class I heat shock protein-like (XP_022939316.1) | 14.44 | 766.3943 | 1531.7711 | 2 | 2.40E+06 | 8.95 | 64.0 | 6.7 | VPSSGAGETTAIANTR |
| Rapeseed | |||||||||
|
Napin small chain S2 (pir||S70337) |
14.45 | 537.2426 | 1073.4680 | 2 | 3.14E+06 | 4.98 | 86.1 | 9.2 | QAMQPGGGSGPS |
| Sesame | |||||||||
| Legumin B-like (XP_020549903.1) | 421.56 | 1398.1465 | 2795.2689 | 2 | 5.79E+07 | 29.35 | 97.2 | 6.0 | AGEQGCEWVEFNTNDNALINTLSGR |
| 1113.6036 | 2226.1852 | 2 | 1.09E+08 | 26.83 | 87.0 | 6.6 | ALMLPAYHNAPILAYVQQGR | ||
| 764.7779 | 2292.2962 | 3 | 7.37E+07 | 44.67 | 95.1 | 10.0 | FSTINSLTLPILSFLQLSAAR | ||
| 691.9919 | 2073.9407 | 3 | 7.24E+07 | 14.87 | 85.1 | 9.9 | GESDMQIVNHNGQAVFDGR | ||
| 1055.5338 | 2110.0451 | 2 | 3.88E+07 | 34.67 | 96.6 | 7.2 | GFDVQILSEVFGVDEQTAR | ||
| 794.4355 | 1587.8489 | 2 | 9.07E+07 | 23.93 | 97.2 | 9.3 | GLPADVIANAYQISR | ||
| 952.0832 | 2854.2117 | 3 | 2.71E+07 | 23.70 | 94.1 | 8.2 | GMYGVMISGCPETFESSQQQFEEGR | ||
| 752.3698 | 1503.7186 | 2 | 3.14E+07 | 10.37 | 91.7 | 9.1 | GQEQQEYAPQLGR | ||
| 595.3016 | 1189.5861 | 2 | 2.32E+07 | 10.82 | 64.3 | 8.3 | GQHQFGNVFR | ||
| 604.2730 | 1207.5273 | 2 | 7.92E+06 | 4.22 | 66.6 | 9.4 | GSTWQQGQCR | ||
| 1025.8021 | 3075.3650 | 3 | 1.58E+07 | 21.77 | 89.4 | 8.7 | IQAEGGVSEFWDHNSDEFQCAGVSIHR | ||
| 7S globulin (AAK15089.1) | 154.07 | 614.7641 | 1228.5124 | 2 | 6.43E+05 | 1.18 | 60.1 | 7.0 | GCEQQHGEQR |
| 934.9765 | 1868.9330 | 2 | 3.40E+06 | 26.42 | 71.1 | 6.8 | IPYVFEDQHFITGFR | ||
| 982.1603 | 2944.4404 | 3 | 1.49E+07 | 37.47 | 100.0 | 8.8 | LLQPVSTPGEFELFFGAGGENPESFFK | ||
| 1003.8700 | 3009.5720 | 3 | 2.31E+06 | 31.63 | 84.5 | 7.8 | VAILEAEPQTFIVPNHWDAESVVFVAK | ||
| Steroleosin (AAL09328.1)/11-beta-hydroxysteroid dehydrogenase 1B-like (NP_001291322.1) | 82.77 | 578.3271 | 1155.6368 | 2 | 6.91E+05 | 17.05 | 87.6 | 8.8 | DLGSPDVVVVR |
| 614.8542 | 1228.6896 | 2 | 1.31E+06 | 16.68 | 75.7 | 9.5 | DVQVSTTPILR | ||
| 800.4400 | 1599.8629 | 2 | 1.47E+06 | 21.57 | 94.7 | 6.2 | SLLYPETVQVPEPK | ||
| 2S albumin precursor isoform 3 (ABB60053.1) | 68.82 | 655.7865 | 1310.5538 | 2 | 1.96E+07 | 9.13 | 96.1 | 9.1 | EQEMQQMMQK |
| 695.7788 | 1390.5371 | 2 | 6.28E+06 | 10.52 | 79.1 | 9.5 | MCGMSYPTQCR | ||
| Caleosin (AAF13743.1)/Peroxygenase (NP_001291323.1) | 55.71 | 697.0523 | 2089.1223 | 3 | 1.41E+06 | 20.25 | 87.5 | 9.6 | NAALAPDAPLAPVTMERPVR |
| 751.8763 | 1502.7348 | 2 | 1.82E+06 | 30.18 | 87.0 | 7.0 | YLPMNFENLFSK | ||
| Sunflower | |||||||||
| 11S globulin seed storage protein G3-like (XP_021988017.1) | 234.0 | 973.9860 | 1946.9566 | 2 | 1.24E+07 | 19.9 | 94.3 | 4.1 | VQIVDNQGNSVFDNELR |
| 11S globulin seed storage protein G3-like (XP_021973262.1) | 172.37 | 704.4060 | 2111.1832 | 3 | 2.76E+06 | 19.38 | 82.2 | 9.6 | GHIVNVGQDLQIIRPPQAR |
| 973.4946 | 1945.9726 | 2 | 1.89E+06 | 19.23 | 93.9 | 4.8 | VQIVNNQGNSVFDNELR | ||
| Putative 11-S seed storage protein (OTG28570.1) | 209.52 | 581.7473 | 1162.4793 | 2 | 1.35E+06 | 1.53 | 75.8 | 6.9 | GMDSSADSHQK |
| 885.4581 | 2654.3420 | 3 | 3.28E+06 | 29.82 | 80.9 | 6.7 | NQEVVAIIVDDVNNPANQLDFQAK | ||
| 610.8050 | 1220.5906 | 2 | 8.31E+06 | 12.27 | 91.1 | 9.9 | SPFGGQEELTR | ||
| 2S seed storage albumin 1 (XP_021993221.1) | 168.15 | 655.7993 | 1310.5794 | 2 | 4.32E+06 | 8.80 | 87.1 | 9.2 | GQFGGQEMETAR |
| 2S seed storage albumin 2 precursor (XP_021993622.1) | 153.25 | 654.8089 | 1308.6001 | 2 | 1.37E+07 | 13.68 | 90.9 | 8.0 | GQFGGQEMDIAR |
| 11S globulin subunit beta-like (XP_021998813.1) | 123.79 | 700.0163 | 2098.0173 | 3 | 9.00E+06 | 5.32 | 52.0 | 8.1 | TGQSQRPGWETGRPEQQR |
| Oleosin 16.4 kDa-like (XP_022005568.1) | 72.45 | 736.8887 | 1472.7591 | 2 | 6.82E+06 | 10.92 | 76.8 | 7.5 | QITGTVPEQVDSAK |
| Vicilin-like seed storage protein At2g28490 (XP_022001204.1) | 60.64 | 1050.9588 | 2100.9033 | 2 | 1.49E+06 | 23.27 | 90.1 | 3.4 | NDYGWSVEVDGDDYEPLK |
| Jacalin-related lectin 34-like (XP_022000240.1) | 55.76 | 790.3864 | 2369.1229 | 3 | 2.82E+06 | 13.10 | 71.4 | 9.2 | GTGTGTFGTGGHEGLGTNIGHVEGR |
| 897.7587 | 2691.2427 | 3 | 5.97E+05 | 15.87 | 63.1 | 6.9 | SIGQTGSQGLGTEPGSHGGIMGDYGATR | ||
| 792.8624 | 1584.7071 | 2 | 1.54E+06 | 11.05 | 81.4 | 6.6 | TGGIMGEYGSTGQGNR | ||
| Oleosin, partial (CAA44224.1) | 48.65 | 669.3257 | 1337.6332 | 2 | 4.42E+06 | 8.73 | 98.2 | 8.1 | GTLQDAGEYAGQK |
| 747.3840 | 1493.7482 | 2 | 2.11E+07 | 15.60 | 71.9 | 8.3 | QTAGSVPESLDYVK | ||
| Oleosin (CAA55348.1) | 31.67 | 684.3487 | 1367.6815 | 2 | 1.83E+06 | 2.62 | 73.7 | 6.3 | HHVTTTQPQYR |
| 619.3114 | 1237.6059 | 2 | 2.07E+06 | 8.98 | 89.5 | 7.8 | LQDVGEYTGQK | ||
Unique peptides derived mainly from major seed storage proteins (i.e. cocosin, conlinin, edestin, other specific legumin-like or vicilin-like globulin subunits, and 2S albumins), and oleosins which are structural proteins found in plant parts with high oil content. Mass spectra of two peptide markers specific to coconut and hempseed obtained from 11S globulins are shown in Fig. 4. Product spectra of another three peptides unique to pumpkin oleosin 18.2 kDa-like, flax oleosin high molecular weight isoform, and black cumin nigellin-1.1 chain A, are shown in Fig. 5. In the present study, most of the identified and selected peptides were specific to the species investigated, but in some cases the peptides were assigned to another species of a given genus, i.e. N. damascena or C. moschata, and thus, the specificity of the protein for the genus can be confirmed.
Figure 4.
Mass spectra of peptide markers specific to coconut and hempseed obtained from 11S globulins extracted from: (A) coconut oil (11S globulin isoform 2, AKS26849.1); (B) hemp oil (edestin 3, SNQ45160.1).
Figure 5.
Mass spectra of species-specific peptide markers obtained from proteins extracted from cold-pressed oils. (A) Pumpkin oleosin 18.2 kDa-like (XP_022928435.1); (B) flax oleosin high molecular weight isoform (ABB01624.1); (C) Nigella nigellin-1.1 chain A (pdb|2NB2|A).
To date, only a few comprehensive proteomic studies of oilseeds having application to food science have been published. Many protein sequences submitted to the NCBI database were obtained by methods of DNA sequencing. Several peptides released from hemp edestin 1 and edestin 2 identified in our study (Supplementary material 2) have been reported previously in hempseed defatted flour24.
Regarding allergens with food route exposure, proteins derived from four of the ten species investigated, namely pumpkin, rapeseed, sesame, and sunflower, have been included in the database of officially recognized allergens maintained by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee25. In the present study, some food allergens derived from pumpkin, sesame, and sunflower cold-pressed oils were detected. These allergens include pumpkin Cuc ma 5, sunflower Hel a 3, and six sesame allergens (Ses i 1, Ses i 2, Ses i 3, Ses i 4, Ses i 6, and Ses i 7). Details of the allergens and detected peptides are shown in Table 2. Recently, seven sesame allergens have been detected in raw sesame seeds and quantified in food products such as sauces, cookies, cake, and candy26 based on selected signature peptides. Eight out of twelve of those previously selected signature peptides for sesame allergens were identified in the present study.
Table 2.
Allergens identified in cold-pressed oils.
| Species | Allergen name/isoallergen | Protein | Accession no | Peptide sequence |
|---|---|---|---|---|
| Pumpkin | Cuc ma 5/Cuc ma 5.0101 | 2S albumin | Q39649.1 | CDMLEEIAREEQR |
| EGGSFDECCR | ||||
| NLPSMCGIRPQR | ||||
| NVDEECRCDMLEEIAR | ||||
| Sunflower | Hel a 3/Hel a 3.0101 | Putative lipid transfer protein | AAP47226.1 | VRPDMASSLPGK |
| Sesame | Ses i 1/Ses i 1.0101 | 2S albumin | AAK15088.1 | QQQQEGGYQEGQSQQVYQR |
| Ses i 2/Ses i 2.0101 | 2S albumin | AAD42943.1 | MCGMSYPTECR | |
| Ses i 3/Ses i 3.0101 | 7S vicilin-like globulin | AAK15089.1 | IPYVFEDQHFITGFR | |
| Ses i 3/Ses i 3.0101 | 7S vicilin-like globulin | AAK15089.1 | SFSDEILEAAFNTR | |
| Ses i 4/Ses i 4.0101 | Oleosin | AAG23840.1 | GVQEGTLYVGEK | |
| Ses i 6/Ses i 6.0101 | 11S globulin | AAD42944.1 | IQSEGGTTELWDER | |
| Ses i 7/Ses i 7.0101 | 11S globulin | AAK15087.1 | FESEAGLTEFWDR | |
| Ses i 7/Ses i 7.0101 | 11S globulin | AAK15087.1 | EGQLIIVPQNYVVAK |
This study presents the discovery of unique peptide markers, but further, a large-scale study is needed to confirm their utility in the authentication of commercially manufactured cold-pressed oils. The specificity of the peptide marker should be also further verified using different cultivars, geographical locations, etc. for each species.
Conclusions
This study provides a set of specific proteins and species-specific peptide markers which can be helpful for food analysts to verify the declared composition of cold-pressed oil as well as the presence of food allergens. Ninety-two specific peptides unique to coconut, evening primrose, flax, hemp, milk thistle, nigella, pumpkin, rapeseed, sesame, and sunflower were detected and identified in cold-pressed oils. The unique peptides were released mainly from specific seed storage proteins and oleosins. Additionally, several food allergens, i.e. pumpkin, sunflower, and sesame allergenic proteins, were observed in the relevant cold-pressed oils. The results are undoubtedly beneficial in the era of widely used unlawful practices, mainly the modification of product composition with cheaper substitutes.
Methods
Reagents and samples
Acetonitrile (LC–MS grade) and formic acid (MS grade) were obtained from Sigma-Aldrich (Schnelldorf, Germany). Sequence-grade modified trypsin gold, lyophilized, was obtained from Promega GmbH (Mannheim, Germany). Reversed-phase Sep-Pak C18 Plus cartridges, sorbent weight 360 mg/0.7 mL, were obtained from Waters (Milford, MA, USA). All other chemicals were purchased from Sigma-Aldrich, at the best available purity grade.
The material for the study consisted of 10 selected oilseeds, namely, coconut (Cocos nucifera L.), evening primrose (Oenothera biennis L.), hemp (Cannabis sativa L.), flax (Linum usitatissimum L.), milk thistle (Silybum marianum L.), nigella/black cumin (Nigella sativa or N. indica), pumpkin (Cucurbita pepo L.), rapeseed (Brassica napus L.), sesame (Sesamum indicum L.), and sunflower (Helianthus annuus L.). The seeds were obtained from the Polish company SemCo Sp. z o.o. (Szamotuły near Poznań) which specializes in the production of oils. Coconut shreds were purchased at a supermarket. Seeds were stored at 4 °C until further proteomic analysis.
Preparation of samples
The oil was prepared in a cold-pressing process using a Yoda YD-ZY-02A oil press (Warsaw, Poland). The oil temperature during the production process was in the range of 38−50 °C and the efficiency of the pressing process in relation to the oil content was approximately 85%. Protein extraction with acetone was performed according to Martín-Hernández, Bénet, & Obert15 with some changes. To 50 g of oil, 125 mL of cold acetone was added. After shaking, the mixture was stirred on a magnetic stirrer at 500 rpm for 1 h at 4 °C. The mixture was then centrifuged at 11,000 RCF and 4 °C for 15 min, and the supernatant was discarded. The precipitate was washed twice with 5 mL of cold acetone. The pellet was dried overnight in drying oven at 40 °C, and then stored at − 20 °C until proteomic analysis. The samples were analysed in duplicate.
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)
SDS-PAGE was performed to compare the profiles of the proteins extracted from oil samples according to a previously described method27. Dried pellet (5 mg) was dissolved with lysis buffer (8 M urea, 2 M thiourea, 0.05 mM Tris, 75 mM dithiothreitol (DTT), 3% SDS, and 0.05% bromophenol blue, at pH = 6.8) and heated at 98 °C for 4 min. The protein concentration was determined using a 2-D Quant kit (GE Healthcare Bio-Sciences, Fairfield, CT, USA). Protein aliquots (12 μg) were loaded onto 15% polyacrylamide gels prepared in a Hoefer SE250 system (GE Healthcare Bio-Sciences). A reference broad-range molecular weight standard (Bio-Rad Laboratories, Inc., CA, USA) was applied. Gels were run at a constant current of 20 mA per gel, then stained with Coomassie brilliant blue and scanned (Gel Doc XR + System, Bio-Rad Laboratories, Inc., CA, USA).
In-solution tryptic digestion
Protein digestion was carried out as previously described26 with slight modifications. Extracted proteins (3 mg) were rehydrated in 100 μL of 50 mM ammonium bicarbonate. The proteins were reduced by 200 mM DTT (56 °C for 1 h) and then alkylated using 200 mM iodoacetamide for 30 min in the dark at room temperature. The remaining iodoacetamide was quenched by the addition of 200 mM DTT and incubation at room temperature for 30 min. The samples were digested in an ammonium bicarbonate solution containing 0.083 μg/μL trypsin (Promega GmbH, Mannheim, Germany), at 37 °C for overnight. The digests were purified by reversed-phase extraction using Sep-Pak C18 Plus cartridges (Waters, Milford, MA, USA). The SPE column was equilibrated with solvent A consisting of 98% water, 2% acetonitrile, and 0.1% formic acid, then with solvent B consisting of 65% acetonitrile, 35% water, and 0.1% formic acid. The sample (0.6 mL) was then added to the cartridge and washed with solvent A. The peptides were eluted with solvent B and vacuum-dried in a centrifugal evaporator (miVacDuo Concentrator, Genevac Ltd., Suffolk, UK). Samples were resuspended in 2% acetonitrile in Milli-Q water containing 0.1% formic acid (solvent A) before UHPLC-Q-TOF–MS/MS analyses.
Proteins and peptides identification
Mass spectrometry analysis was according to a previously described procedure26 with slight modifications. UHPLC-Q-TOF–MS/MS analysis was performed on an Agilent Technologies 1290 Infinity series liquid chromatograph (Santa Clara, CA, USA) composed of a binary pump, a thermostat, and an autosampler, coupled to a 6550 UHD iFunnel Q-TOF LC/MS. Compounds were ionized by electrospray ionization (ESI) using a JestStream Technology ion source. Chromatographic separation was performed on a 2.1 × 150 mm, 1.8 μm particle-size Agilent RRHD Eclipse Plus C18 column. Instrument control and data acquisition were performed using Agilent MassHunter Workstation Software. The LC parameters were set as follows: 10 μL injection volume, 0.3 mL/min mobile phase flow. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Gradient steps were applied as follows: 0–2 min, 2% B; 2–40 min, to 32% B; 40–45 min, to 37% B; 45–50 min, to 90% B; 50–55 min, 90% B; and a 5-min post-run at 2% B. The ion source gas (nitrogen) temperature was 250 °C, the flow rate was 14 L/min, nebulizer pressure was 35 psi, sheath gas temperature was 250 °C, and sheath gas flow was 11 L/min. The capillary voltage was set at 3500 V, nozzle voltage to 1000 V, and the fragmentor to 400 V. Positive ions formed in an electrospray were acquired in the range of 100–3000 m/z in MS scan mode and in auto MS/MS mode, with a scan rate of 5 scan/s for MS and 3 scan/s for MS/MS. Internal mass calibration was enabled by using two reference masses at 121.0509 and 922.0098 m/z.
A National Center for Biotechnology Information (NCBI, U.S. National Library of Medicine) protein database search for protein and peptide identification was performed, using the Spectrum Mill MS Proteomics Workbench with > 50% score peak intensity and 10 ppm precursor mass tolerance, with the following parameters: trypsin enzyme, taxonomy green plants or a given taxonomy genus (i.e. Brassica, Cannabis, Cocos, Cucurbita, Helianthus, Nigella, Linum, Oenothera, Sesamum, Silybum), two missed cleavages, 50 ppm products mass tolerance, carbamidomethylation as fixed modification, and methionine oxidation as a variable modification. The matches and Spectrum Mill scores were evaluated at 1% of the false discovery rate (FDR), for identity and homology thresholds. Selected peptides, in FASTA format, were searched against the NCBInr database, using the protein Basic Local Alignment Search Tool (BLAST) and blastp algorithm (U.S. National Library of Medicine, Bethesda, MD), for species and protein specificity.
Supplementary information
Acknowledgements
The study was supported by the National Science Centre, Poland, under Project Number 2017/25/B/NZ9/02000.
Author contributions
M.M. and E.F. conceived and designed the study, K.K.M conducted the experiments, A.S. and E.F acquired the MS data, K.K.M and M.M. analysed the results and wrote the manuscript. All authors reviewed the manuscript.
Data availability
The datasets generated and analysed during this study are available from the corresponding author on reasonable request.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
is available for this paper at 10.1038/s41598-020-76944-z.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated and analysed during this study are available from the corresponding author on reasonable request.