A preliminary matrix‐assisted laser desorption/ionization time‐of‐flight approach for the characterization of Italian lentil varieties

Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) was used in this study to obtain protein fingerprints of seven different lentil varieties, to characterize their differences and similarities. Two different matrices have been tested in order to obtain reproducible and significant mass spectra. Extraction with water containing 0.1% of trifluoroacetic acid has been used as preparative step to obtain hydrophilic protein samples of lentil seeds. The obtained MALDI protein profiles identified clear differences between the seven studied lentil varieties. Moreover, considering the high complexity of the obtained MALDI spectra, multivariate techniques of data analysis were employed to find further classification details. These multivariate analyses confirmed the possibility of a clear classification of the seven lentil varieties, indicating that the proposed procedure can be a valid taxonomic tool, and a method to certify the origin of lentils, useful for high added value lentils (Italian lentils). Copyright © 2010 John Wiley & Sons, Ltd.     

New Phenolic Compounds from the Roots of Lentil (Lens culinaris)

While lentil (Lens culinaris) seeds are phytochemically well characterized, very little is known about secondary metabolites from lentil roots. Our research on lentil roots led to isolation of five phenolic compounds and five group B soyasaponins. Their structures were established using NMR spectroscopy and mass spectrometry. Four phenolics have not been hitherto described in the literature: 4‐O‐β‐d ‐glucopyranosyl‐2‐methoxybenzoic acid, (αS)‐4,4′‐di‐O‐β‐d ‐glucopyranosyl‐α,2′‐dihydroxydihydrochalcone, (αS)‐4′‐O‐β‐d ‐glucopyranosyl‐α,2′,4‐trihydroxydihydrochalcone, and keto‐2‐hydroxyglycitein. The DPPH^? radical‐scavenging activity of the purified phenolic compounds was additionally evaluated.     

Global mapping of rat plasma proteins with a native proteomic approach using nondenaturing micro 2DE and quantitative LC‐MS/MS

Plasma samples from adult male rats were separated by nondenaturing micro 2DE and a reference gel was selected, on which 136 CBB‐stained spots were numbered and subjected to in‐gel digestion and quantitative LC‐MS/MS. The analysis provided the assignment of 1–25 (average eight) non‐redundant proteins in each spot and totally 199 proteins were assigned in the 136 spots. About 40% of the proteins were detected in more than one spot and 15% in more than ten spots. We speculate this complexity arose from multiple causes, including protein heterogeneity, overlapping of protein locations and formation of protein complexes. Consequently, such results could not be appropriately presented as a conventional 2DE map, i.e. a list or a gel pattern with one or a few proteins annotated to each spot. Therefore, the LC‐MS/MS quantity data was used to reconstruct the gel distribution of each protein and a library containing 199 native protein maps was established for rat plasma. Since proteins that formed a complex would migrate together during the nondenaturing 2DE and thus show similar gel distributions, correlation analysis was attempted for similarity comparison between the maps. The protein pairs showing high correlation coefficients included some well‐known complexes, suggesting the promising application of native protein mapping for interaction analysis. With the importance of rat as the most commonly used laboratory animal in biomedical research, we expect this work would facilitate relevant studies by providing not only a reference library of rat plasma protein maps but a means for functional and interaction analysis.     

Concentration and pattern changes of porcine serum apolipoprotein A‐I in four different infectious diseases

Apolipoprotein A‐I (Apo A‐I) is a major protein in lipid/lipoprotein metabolism and decreased serum levels have been observed in many species in response to inflammatory and infectious challenges. Little is known about the porcine homologue, therefore in this work we have characterized it through biochemical and proteomic techniques. In 2DE, porcine serum Apo A‐I is found as three spots, the two more acidic ones corresponding to the mature protein, the more basic spot to the protein precursor. Despite high sequence coverage in LC‐MS/MS, we did not find a sequence or PTM difference between the two mature protein species. Besides this biochemical characterization, we measured overall levels and relative species abundance of serum Apo A‐I in four different viral and bacterial porcine infectious diseases. Lower overall amounts of Apo A‐I were observed in Salmonella typhimurium and Escherichia coli infections. In the 2DE protein pattern, an increase of the protein precursor together with a lower level of mature protein species were detected in the porcine circovirus type 2‐systemic disease and S. typhimurium infection. These results reveal that both the porcine serum Apo A‐I concentration and the species pattern are influenced by the nature of the infectious disease.     

Combination of virtual and experimental 2DE together with ESI LC‐MS/MS gives a clearer view about proteomes of human cells and plasma

Virtual and experimental 2DE coupled with ESI LC‐MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by 2DE. After staining and protein spot identification by MALDI‐TOF MS, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC‐MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw ( http://web.expasy.org/compute_pi/pi_tool‐doc.html ). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome.     

Affinity chromatographic purification of lentil lectin using immobilized yeast cells

A model system for evaluating macroporous supports containing immobilized whole cells in affinity Chromatographic applications is described. Whole cells were immobilized in a polyacrylamide network in a two-step polymerization process. The affinity system discussed consists of immobilized cells ofSaccharomyces cervisiae in the purification of lentil lectin fromLens culinaris.     

Proteins from Erwinia asparaginase Erwinase® and E. coli asparaginase 2 MEDAC® for treatment of human leukemia,show a multitude of modifications for which the consequences are completely unclear

L ‐Asparaginase from Erwinia chrysanthemi (ASPG_ERWCH; UniProtKB accession number P06608 (Erwinase^®)) and L ‐asparaginase 2 from Escherichia coli (ASPG2_ECOLI; UniProtKB accession number P00805 (Medac^®)), both L ‐asparagine amidohydrolases, are widely used for the treatment of acute lymphoblastic leukemia. A series of serious side effects have been reported and this warrants studies into the protein chemistry of the medical products sold. Mass spectrometry (MS) data on ASPG_ERWCH and ASPG2_ECOLI have not been published so far and herein a gel‐based proteomics study was performed to provide information about sequence and modifications of the commercially available medical products. ASPG_ERWCH and ASPG2_ECOLI were applied onto two‐dimensional gel electrophoresis, spots were in‐gel digested with several proteases and resulting peptides and protein modifications were analysed by nano‐ESI‐LC‐MS/MS. Four spots were observed for ASPG_ERWCH, six spots were observed for ASPG2_ECOLI and the identified proteins showed high sequence coverage without sequence conflicts. Several protein modifications including technical and posttranslational modifications were demonstrated. Protein modifications are known to change physicochemical, immunochemical, biological and pharmacological properties and results from this work may challenge re‐designing of the product including possible removal of the modifications by the manufacturer because it is not known whether they are contributing to the serious adverse effects of the protein drug.     

Chemical profiling and separation of bioactive secondary metabolites in Maca (Lepidium peruvianum) by normal and reverse phase thin layer chromatography coupled to desorption electrospray ionization‐mass spectrometry

Maca is a Peruvian tuberous root of the Brassicaceae family grown in the central Andes between altitudes of 4000 and 4500 m. The medicinal plant is a nutraceutical with important biological activities and health effects. In this study, we report a rapid high‐performance thin layer chromatography (HPTLC)‐(?)desorption electrospray ionization (DESI)‐mass spectrometry (MS) method to profile and separate intact glucosinolates without prior biochemical modifications from the hydromethanolic extracts of two phenotypes, red and black Maca (Lepidium peruvianum) seeds. In the first stage of the plant's life cycle, aromatic glucosinolates were the main chemical constituents whereby six aromatic, three indole, and one aliphatic glucosinolate were tentatively identified. At the seedling stage, glucolepigramin/Glucosinalbin was the most predominant precursor, rather than Glucotropaeolin, which is mainly found in hypocotyls and roots. These findings lead us to suggest that glucolepigramin/glucosinalbin play a major role as active precursors in the biosynthetic pathways of other secondary metabolites in the early stages of plant development. Between red and black Maca seeds, only minor differences in the relative abundances of glucosinolates were observed rather than different plant metabolites. For the first time, we report six potential plant antibiotics, phytoanticipins: glycosylated ascorbigens and dihydroascorbigens from Maca seeds. We also investigated a targeted reverse phase C₁₈ functionalized TLC‐DESI‐MS method with high sensitivity and specificity for Brassicaceae fatty acids in Maca seeds and health supplements such as black Maca root lyophilized powder and tinctures. The investigation of secondary metabolites by normal and reverse phase TLC‐DESI‐MS methods, described in this study, can aid in their identification as they begin to emerge in later stages of development in plant tissues such as leaves, hypocotyls, and roots.     

The use of ESR technique for assessment of heating temperatures of archaeological lentil samples

Heat-induced paramagnetic centers in modern and archaeological lentils (Lens culinaris, Medik.) were studied by X-band (9.3 GHz) electron spin resonance (ESR) technique. The modern red lentil samples were heated in an electrical furnace at increasing temperatures in the range 70–500 °C. The ESR spectral parameters (the intensity, g-value and peak-to-peak line width) of the heat-induced organic radicals were investigated for modern red lentil (Lens culinaris, Medik.) samples. The obtained ESR spectra indicate that the relative number of heat-induced paramagnetic species and peak-to-peak line widths depends on the temperature and heating time of the modern lentil. The g-values also depend on the heating temperature but not heating time. Heated modern red lentils produced a range of organic radicals with g-values from g = 2.0062 to 2.0035. ESR signals of carbonised archaeological lentil samples from two archaeological deposits of the Van province in Turkey were studied and g-values, peak-to-peak line widths, intensities and elemental compositions were compared with those obtained for modern samples in order to assess at which temperature these archaeological lentils were heated in prehistoric sites. The maximum temperatures of the previous heating of carbonised UA5 and Y11 lentil seeds are as follows about 500 °C and above 500 °C, respectively.     

Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells

Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Indeed, such understanding would enable dedifferentiation and reprogrammation of any cell type from a patient needing a cell therapy treatment. Proteome analysis has emerged as an important technology for deciphering these biological processes and thereby ESC and EGC proteomes are increasingly studied. Nevertheless, their nuclear proteomes have only been poorly investigated up to now. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC nuclear proteins. Nuclei from ESCs and EGCs at undifferentiated stage were purified by subcellular fractionation. After 2‐D separation, a subtractive strategy (subtracting culture environment contaminating spots) was applied and a comparison of ESC, (8.5 day post coïtum (dpc))‐EGC and (11.5 dpc)‐EGC specific nuclear proteomes was performed. A total of 33 ESC, 53 (8.5 dpc)‐EGC, and 36 (11.5 dpc)‐EGC spots were identified by MALDI‐TOF‐MS and/or nano‐LC‐MS/MS. This approach led to the identification of two isoforms (with and without N‐terminal acetylation) of a known pluripotency marker, namely developmental pluripotency associated 5 (DPPA5), which has never been identified before in 2‐D gel‐MS studies of ESCs and EGCs. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (protein arginine N‐methyltransferase 7; PRMT7) behaving as proteins involved in pluripotency.     

Acute‐phase proteins investigation based on lectins affinity capture prior to 2‐DE separation: Application to serum from multiple sclerosis patients

Plasma acute‐phase proteins (APPs) glyco‐isoforms are important biomarkers of inflammatory processes such as those occurring in multiple sclerosis (MS). Specific analysis of these proteins is often hampered by sample biochemical complexity. The aim of our study was to set up a method to accurately visualize, identify and quantify APPs glyco‐isoforms in human serum. An enrichment strategy based on affinity chromatography using the carbohydrate‐binding proteins concanavalin A (ConA) and erythrina cristagalli lectin (ECL) was applied to pooled serum samples from 15 patients and 9 healthy individuals. Image analysis of 2‐DE detected 30 spots with a fold change higher than 1.5. A total of 14 were statistically significant (p value<0.05): 7 up‐regulated and 7 down‐regulated in MS samples. ESI LC‐Nanospray IT mass spectrometry analysis confirmed that all of them were APPs isoforms supporting the idea that the accurate analysis of differential glycosylation profiles in these biomarkers is instrumental to distinguish between MS patients and healthy subjects. Additionally, overlaps in ConA/ECL maps protein patterns suggest how the used lectins are able to bind sugars harbored by the same oligosaccharide structure. Among identified proteins, the presence of complex and/or hybrid type N‐linked sugar structures is well known. Performing galectin‐3 binding and Western blotting, we were able to demonstrate a correlation between hybrid type glyco‐isoforms of β‐haptoglobin and MS. In conclusion, although the patho‐physiological role of the identified species still remains unclear and further validations are needed, these findings may have a relevant impact on disease‐specific marker identification approaches.     

Analysis of E. coli soluble proteins by non‐denaturing micro 2‐DE/3‐DE and MALDI‐MS‐PMF

Escherichia coli (strain K‐12)‐soluble proteins were analyzed by nondenaturing micro 2‐DE and MALDI‐MS‐PMF. The reported conditions of nondenaturing IEF in agarose column gels [Jin, Y., Manabe, T., Electrophoresis 2009, 30, 939–948] were modified to optimize the resolution of cellular soluble proteins. About 300 CBB‐stained spots, the apparent molecular masses of which ranged from ca. 6000 to 10 kDa, were detected. All the spots on two reference 2‐DE gels (one for wide mass range and one for low‐molecular‐mass range) were numbered and subjected to MALDI‐MS‐PMF for the assignment of constituting polypeptides. Most of the spots (310 spots out of 329) provided significant match (p<0.05) with polypeptides in Swiss‐Prot database and totally 228 polypeptide species were assigned. Activity staining of enzymes such as alkaline phosphatase and catalases was performed on the 2‐DE gels and the locations of the activity spots matched well with those of the MS‐assigned polypeptides of the enzymes. Most of the polypeptides with subunit information in Swiss‐Prot (119 polypeptides as homo‐multimers and 25 as hetero‐multimers out of the 228), such as pyruvate dehydrogenase complex which is composed of three enzymatic components, were detected at the apparent mass positions of their polymers, suggesting that the proteins were separated retaining their subunit structures. When a nondenaturing 2‐DE gel was vertically cut into 2 mm strips and one of the strips was subjected to a third‐dimension micro SDS‐PAGE (micro 3‐DE), about 190 CBB‐stained spots were detected. The assignment of the polypeptides separated on the 3‐DE gel would further provide information on protein/polypeptide interactions.     

Proteomic profiling of mature leaves from oil palm (Elaeis guineensis Jacq.)

Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI‐protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307.     

Applications of MALDI‐TOF MS to large‐scale human mtDNA population‐based studies

Analysis of the mitochondrial DNA variation in populations is commonly carried out in many fields of biomedical research. We propose the analysis of mitochondrial DNA coding region SNP (mtSNP) variation to a high level of phylogenetic resolution based on MALDI‐TOF MS. The African phylogeny has been chosen to test the applicability of the technique but any other part of the worldwide phylogeny (or any other mtSNP panel) could be equally suitable for MALDI‐TOF MS genotyping. SNP selection thus aimed to fully cover all the mtSNPs defining major and minor branches of the known African tree, including, macro‐haplogroup L, and haplogroups M1, and U6. A total of 230 mtSNPs were finally selected. We used tests samples collected from two different African locations, namely, Mozambique and Chad Basin. Different internal genotyping controls and other indirect approaches (e.g. phylogenetic checking coupled with automatic sequencing) were used in order to evaluate the reproducibility of the technique, which resulted to be 100% using samples previously subjected to whole genome amplification. The advantages of the MALDI‐TOF MS are also discussed in comparison with other popular methods such as minisequencing, highlighting its high‐throughput nature, which is particularly suitable for case–control medical studies, forensic databasing or population and anthropological studies.     

2D difference gel electrophoresis reference map of a Fusarium graminearum nivalenol producing strain

Fusarium graminearum is widely studied as a model for toxin production among plant pathogenic fungi. A 2D DIGE reference map for the nivalenol‐producing strain 453 was established. Based on a whole protein extract, all reproducible spots were systematically picked and analyzed by MALDI‐TOF/TOF, leading to the identification of 1102 protein species. The obtained map contributes to the annotation of the genome by identifying previously nondescribed hypothetical proteins and will serve as a reference for future studies aiming at deciphering F. graminearum biology and chemotype diversity.     

Searching for serum tumor markers for colorectal cancer using a 2‐D DIGE approach

Identification of specific protein markers for colorectal cancer (CRC) could provide a basis for its early diagnosis and detection, as well as clues to the molecular mechanisms governing cancer progression. In the present study, 2‐D DIGE coupled with MS was used to screen for biomarker candidates in the serum proteome of ten human CRC samples and ten healthy control samples. After pooling identical amounts of serum proteins (based on total protein concentration), albumin/IgG was depleted under partially denaturing conditions. Subsequently, the serum samples were labeled with three different CyDyes, and separated by 2‐D DIGE. After analysis with the biological variation analysis module of the DeCyder software, only three spots were found to be significantly elevated in all patient groups (with ratios from 1.52 to 9.08), whereas five spots were significantly down‐regulated in patients (with ratios from ?1.23 to ?10.21) (t‐test; p<0.05). Finally, two potential biomarkers, Transaldolase 1 and thyroid receptor interactor, were chosen for validation and analysis by ELISA with the serum of 30 CRC patients and 30 healthy controls. The serum levels of the two proteins correlated well with the 2‐D DIGE results. Thus, 2‐D DIGE approaches show great promise for biomarker discovery in CRC.     

Analysis of low‐density lipoprotein‐associated proteins using the method of digitized native protein mapping

The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS (in data‐independent acquisition mode, or MS^E), was improved by using a new MS/MS mode, ion mobility separation enhanced‐MS^E (HDMS^E), and applied to analyze the area of human plasma low‐density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid‐cut into 72 square gel pieces and subjected to quantitative LC‐MS/MS. Compared with MS^E, HDMS^E showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC‐HDMS^E and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B‐100 was the most abundant protein in the grid‐cut area, concentrated at pI ca. 5.4–6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39–42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B‐100. Twenty‐two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.     

Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray‐ tandem mass spectrometry

Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI‐QTOF‐MS and direct infusion ESI‐FTICR‐MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision‐induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl‐ and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed‐phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI‐MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class. Copyright © 2008 John Wiley & Sons, Ltd.     

A competent extraction method of plant proteins for 2-D gel electrophoresis

The efficient extraction of high‐quality proteins is a key factor for a successful proteomic analysis approach. In the method suggested here, absolute ethanol containing 10 mM DTT was used to precipitate the proteins in plant tissue homogenates followed by their resuspension in a urea‐/thiourea‐ and NP‐40‐containing solution. Protein profiles were examined on pH 3–11 non‐linear IEF strips and SDS‐PAGE and compared with extracts using the established method of acetone‐10% TCA/0.07% 2‐mercaptoethanol precipitation (V. Méchin et al., Methods Mol. Biol. 2006, 355, 1–8). In addition to protein profile similarity for the two extracts, the acidic part of the acetone containing 10% TCA/0.07% 2‐mercaptoethanol extraction showed protein spots with high molecular weight in the range of 250–150 kDa, while the ethanol containing 10 mM DTT extracts indicated extra proteins spots at the basic part of the gels with molecular weights in the range of 25–15 kDa. The MALDI‐TOF‐MS of differential spots from acetone containing 10% TCA/0.07% 2‐mercaptoethanol precipitation method and absolute ethanol containing 10mM DTT indicated no similarity, ruling out the possibility that the two clusters shown represent identical proteins. The described method is easy in implementation, chemicals used are less toxic and proteins are easier to resuspend therefore presents an additional choice to implement towards finding the optimum method for extraction.     

Effect of Elicitation with Iron Chelate and Sodium Metasilicate on Phenolic Compounds in Legume Sprouts

Seven-day-old sprouts of fenugreek (Trigonella foenum-graecum L.), lentil (Lens culinaris L.), and alfalfa (Medicago sativa L.) were studied. The legume seeds and then sprouts were soaked each day for 30 min during 6 days with water (control) or mixture of Fe-EDTA and sodium silicate (Optysil), or sodium silicate (Na-Sil) alone. Germination and sprout growing was carried out at temperature 20 ± 2 °C in 16/8 h (day/night) conditions. Phenolic compounds (free, ester, and glycosides) content were determined by HPLC-ESI-MS/MS using a multiple reaction monitoring of selected ions. Flavonoids and phenolic acids were released from their esters after acid hydrolysis and from glycosides by alkaline hydrolysis. The presence and high content of (−)-epicatechin (EC) in fenugreek sprouts was demonstrated for the first time. Applied elicitors decreased the level of free EC in fenugreek and alfalfa sprouts but enhanced the content of its esters. Besides, elicitors decreased the content of quercetin glycosides in lentil and fenugreek sprouts but increased the content of quercetin and apigenin glycosides in alfalfa sprouts. The applied elicitors decreased the glycoside levels of most phenolic acids in lentil and p-hydroxybenzoic acid in fenugreek, while they increased the content of this acid in alfalfa. The mixture of iron chelate and sodium silicate had less effect on changes in flavonoid and phenolic acid content in legume sprouts than silicate alone. In general, the used elicitors increased the content of total phenolic compounds in fenugreek and alfalfa sprouts and decreased the content in lentil sprouts. Among the evaluated elicitors, Optysil seems to be worth recommending due to the presence of iron chelate, which can be used to enrich sprouts with this element.