DOI: 10.1515/achi-2017-0008 ACTA CHEMICA IASI, 25_1, 73-86 (2017) MALDI-TOF MASS SPECTROMETRIC ANALYSIS OF ZEINS EXTRACTED FROM MAIZE SEEDS Raluca Ştefănescu* and Sabina Băncilă Faculty of Chemistry, “Al. I. Cuza” University of Iasi, 11 Carol I Bd, Iasi 700506, Romania Abstract: We report the mass spectrometric analysis of zeins extracted from degreased maize flour with either an aqueous 70% ethanol solution or 60% acetonitrile containing 10 mM dithiothreitol. The analysis was performed on a MALDI-TOF mass spectrometer using α-cyano-4-hydroxycinnamic acid as matrix. The method allowed the detection of α-, β- and γ-zeins, but not δ-zeins and was used for the analysis of zein content in the maize inbred KWS 3381 and in commercial flour at different flour particles sizes: between 710 μm and 1.0 mm, between 250 μm and 355 μm and smaller than 100 μm. Keywords: MALDI-TOF; prolamins; Zea mays; zeins Introduction Zeins belong to the storage proteins named prolamins, which contain a high number of proline and glutamine units. Prolamins are located in the endosperm of many cereal seeds and are soluble in 70% alcohol.1-3 There are four groups of zeins: α-zeins, β-zeins, γ-zeins and δ-zeins. According to the electrophoretic migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the first group belongs to α-zeins, which are * Raluca Ștefănescu, e-mail: rallstef@gmail.com 74 R. Ștefănescu and S. Băncilă comprised of 19 kDa α-zeins and 22 kDa α-zeins, the second one, β-zeins, is represented by 14 kDa β-zeins, the third one contains γ-zeins that are comprised of 16 kDa and 27 kDa γ-zeins and, finally, δ-zeins are represented by 10 kDa δ-zein.1 Uniprot accession no. Total no. of amino acids* (no. of cysteine residues) AF371266 Q41881 129 (5) 15-kDa β- zein AF371264 Q946W0 158 (7) 17148 17125 12 25 16-kDa γ- zein AF371262 Q548E8 164 (12) 17751 17714 25 31 18-kDa δ- zein AF371265 Q946V9 190 (3) 27-kDa γ- zein AF371261 Q548E9 204 (15) 21823 21793 51 30 19-kDa α- zein B1 AF371269 Q946V6 213 (2) 23360 23318 23 41 19-kDa α- zein B3 AF371271 Q548E6 219 (2) 24088 24069 22 43 19-kDa α- zein D2 AF371268 Q946V7 220 (1) 24707 24515 18 47 19-kDa α- zein D1 AF371267 Q946V8 222 (1) 24819 24644 19 45 22-kDa α-zein Z1 AF371274 Q9SBC4 242 (1) 26360 26308 22 50 22-kDa α-zein Z5 AF371277 Q9SYT3 245 (1) 26711 - 22-kDa α-zein Z3 AF371275 O48966 245 (1) 26752 26741 22 51 22-kDa α-zein Z4 AF371276 Q946V4 246 (1) 26924 - 21 53 19-kDa α-zein B2 AF371270 Q548E7 246 (2) 27129 - 22 47 *without the signal sequence **considering all cysteine residues reduced ***reported by Adams W.R. and collaborators [M+H]+ [M+H]+exp.*** GenBank accession no. 10-kDa δ-zein P Q 14432 14432 20 15 calc.** Zein type No. of prolamine specific amino acids Table 1. List of the GenBank and Uniprot accession numbers for the zein proteins identified in the B73 maize inbred by Woo, Y.-M. and collaborators and comparison between the calculated and observed masses of the zeins from the spectra as reported by Adams W.R. and collaborators. 21222 - 35 14 19 50 MALDI-TOF mass spectrometric analysis of zeins extracted from maize seeds 75 Adams W.R. and the collaborators reported the identification of the zeins from the maize inbred B73 based on the molecular weights calculated for the amino acid sequences that have the signal peptide removed and which are found at the GenBank accession numbers reported by Woo, Y.M. et al. and the molecular weights obtained by MALDI-TOF mass spectrometric analysis (see Table 1).4-6 Moreover, the authors treated the zein extracts with iodoacetamide in order to alkylate the cysteine residues after the disulfide bridges reduction with dithiothreitol. The number of modified cysteine residues was estimated by comparing the mass spectra of the alkylated zeins with the mass spectra of the underivatized zeins. The peak assignment to α-, β-, γ- or δ-zeins was possible due to the different content in cysteine residues of the sequences of zeins (Table 1). Most of the proteins contain a number of lysine and arginine amino acid residues that allows the enzymatic cleavage of the protein using trypsin. In the case of the zeins, due to the low content in the amino acids lysine and arginine, a proteolytic cleavage using trypsin followed by the mass spectrometric analysis of resulted fragments will lead to difficulties in sequence assignment caused by the high length of these fragments (Table 2). The endoprotease Glu-C (from Staphylococcus aureus strain V8) is cleaving the peptide bonds C-terminally at glutamic or aspartic acid if the reaction is carried out in phosphate buffer saline (PBS). However, the number of these two specific amino acids is very low in all zeins. Alphachymotrypsin is a proteolytic enzyme which cleaves at the carboxyl end of tyrosine, phenylalanine, tryptophan, and leucine. Hydrolysis may also occur on the C-terminal side of methionine, isoleucine, serine, threonine, valine, histidine, glycine, and alanine. Due to the fact of huge number of cleavage 76 R. Ștefănescu and S. Băncilă sites, a plenty of very short sequences would be generated after cleavage with alpha-chymotrypsin. Table 2. The number of cleavage sites of zeins in the presence of trypsin, Glu-C or alpha-chymotrypsin identified by Woo Y.-M and collaborators.6 Zein type 10-kDa δ-zein Trypsin Glu-C cleavage cleavage sites sites K R D E 0 0 1 0 Alpha-chymotrypsin cleavage sites Y 1 F 5 W 0 L 15 M 29 I 3 15-kDa β- zein 0 5 1 3 12 1 0 16 18 1 16-kDa γ- zein 0 3 0 3 8 7 1 14 3 1 18-kDa δ- zein 1 0 1 0 2 5 2 13 48 10 27-kDa γ- zein 0 5 0 2 4 2 0 19 1 4 19-kDa α- zein B1 0 2 0 1 8 13 0 42 0 9 19-kDa α- zein B3 0 3 1 1 8 12 0 43 0 12 19-kDa α- zein D2 0 5 1 2 9 13 0 39 1 12 19-kDa α- zein D1 0 3 0 1 10 14 0 38 1 12 22-kDa α-zein Z1 0 2 0 1 8 9 0 44 4 8 22-kDa α-zein Z5 0 3 1 2 8 9 0 44 3 12 22-kDa α-zein Z3 0 3 0 1 7 9 0 42 4 12 22-kDa α-zein Z4 0 2 0 2 8 8 1 44 4 12 19-kDa α-zein B2 0 4 1 1 9 13 0 52 1 10 In the present study we identified the signals observed in the mass spectra acquired for the zein extracts from maize inbred KWS 3381 with the corresponding molecular weights for the singly charged ions of the zein calculated based on primary sequences available at GenBank and Uniprot, which are determined for the maize inbred B73 by Woo, Y.-M. and collaborators. MALDI-TOF mass spectrometric analysis of zeins extracted from maize seeds 77 Results and Discussion Using MALDI-TOF mass spectrometry, we first analyzed the zeins extracted with 70% ethanol in water from the maize inbred KWS 3381 flour with the particles size between 710 μm and 1.0 mm. The mass spectrum shown in Figure 1 displays peaks at m/z 23340, 24007 and 17731 that are assigned to the singly charged ions of the 19 kDa α-zein B1, 19 kDa α-zein B3 and 16 kDa γ-zein, respectively, and peaks at m/z 11670 and 8868 that correspond to the doubly charged ions of the 19 kDa α-zein B1 and 16 kDa γ-zein. Figure 1. MALDI-TOF mass spectrum of the zeins from the maize inbred KWS 3381 flour having the particle size between 710 μm and 1.0 mm with 70% ethanol in water. The zeins were extracted by ultrasonication for 30 minutes in an ultrasonic water bath. Figure 2 displays the mass spectrum acquired for the zeins extracted with 70% ethanol from the commercial maize flour that has the size of the particles between 710 μm and 1.0 mm. Based on the m/z values of the peaks present in the mass spectrum, three characteristic signals 19 kDa α-zein B1 (at m/z 23356), the 19 kDa α-zein B3 (at m/z 24035) and a 22 kDa α-zein (at m/z 26757) were identified. 78 R. Ștefănescu and S. Băncilă Figure 2. MALDI-TOF mass spectrum of the zeins extracted from commercial maize flour having the particle size between 710 μm and 1.0 mm, with 70% ethanol in water for 30 minutes using an ultrasound bath. Figure 3. MALDI-TOF mass spectrum of the zeins extracted from commercial maize flour having the particle size between 250 μm and 355 μm with 70% ethanol in water for 30 minutes using an ultrasound bath. Similarly, the mass spectrum of extracted proteins in 70% ethanol from commercial flour with particle size between 250 μm and 355 μm, MALDI-TOF mass spectrometric analysis of zeins extracted from maize seeds 79 displays two signals at 19 kDa (α-zein B1 at m/z 23363 and α-zein B3 at m/z 24054) and one signal at 22 kDa α-zein (at m/z 26783) (see Figure 3). Figure 4 shows the MALDI-TOF mass spectrum of the zeins extracted with 60% acetonitrile in water and 10 mM DTT from the maize inbred KWS 3381 that has the particle size between 710 μm and 1.0 mm. In addition to previous observed signals from above investigated extracts, 19 kDa α-zein B1 (at m/z 23345), the 19kDa α-zein B3 (at m/z 24014) and a 22 kDa α-zein (at m/z 26722), two supplementary signals 15 kDa β-zein (at m/z 17453) and the 16 kDa γ-zein (at m/z 17740) were noticed in the mass spectrum. Figure 4. MALDI-TOF mass spectrum of zeins extracted from the maize inbred KWS 3381 flour, having the particle size between 710 μm and 1.0 mm, with 60% acetonitrile in water and 10 mM DTT for 60 minutes at 60°C. The mass spectrum depicted in Figure 5 contains peaks that correspond to the zeins extracted with 60% acetonitrile and 10 mM DTT from the commercial flour with particle size between 710 μm and 1.0 mm. The peaks at m/z 23351, 24045 and 26751 were assigned to the singly charged ions of the 19 kDa α-zein B1, 19 kDa α-zein B3 and a 22 kDa 80 R. Ștefănescu and S. Băncilă α-zein, respectively. The peaks at m/z 21847 and at m/z 17737 correspond to the 27 kDa γ-zein and to the 16 kDa γ-zein while the peaks at m/z 17443 and m/z 17143 correspond to the 15 kDa β-zein. Figure 5. MALDI-TOF mass spectrum of the zeins extracted from commercial maize flour having the particle size between 710 μm and 1.0 mm, with 60% acetonitrile in water and 10 mM DTT for 60 minutes at 60°C. The difference between the sample preparation for the mass spectrum presented in the Figure 5 and the mass spectrum presented in the Figure 6 consists only in that in the later the size of the commercial flour is between 250 μm and 355 m. The results show that the peak corresponding to the 15 kDa β-zein and the 16 kDa γ-zein is less abundant and the peak assigned to the 27 kDa γ-zein is missing. MALDI-TOF mass spectrometric analysis of zeins extracted from maize seeds 81 Figure 6. MALDI-TOF mass spectrum of the zeins extracted from commercial maize flour having the particle size between 250 μm and 355 μm with 60% acetonitrile in water and 10 mM DTT for 60 minutes at 60°C. Figure 7. MALDI-TOF mass spectrum of the zeins extracted from the maize inbred KWS 3381, having the particle size 100 μm, with 70% ethanol in water for 30 minutes using an ultrasound bath. In the Figures 7 and 8, the mass spectra of the zein extracted with ethanol 70% and acetonitrile 60% containing 10 mM DTT respectively from the maize inbred KWS 3381 that has the flour particle size smaller than 100 μm are presented. 82 R. Ștefănescu and S. Băncilă Figure 8. MALDI-TOF mass spectrum of the zeins extracted from the maize inbred KWS 3381flour, having the particle size smaller than 100 μm, with 60% acetonitrile in water and 10 mM DTT for 60 minutes at 60 °C. In contrast to the other studies reporting the analysis by MALDITOF MS of zeins extracted from degreased maize flour using the matrices 2,5-dihydroxyl benzoic acid (DHB) or 2-(4-hydroxyphenylazo)benzoic acid (HABA), in the present study we employed the matrix α-cyano-4hydroxycinnamic acid.4,5 This matrix allowed the detection in the mass spectra of the 19 kDa α-zeins B1 and B3, a 22 kDa α-zein, the 27 kDa and the 16 kDa γ-zeins and the 15 kDa β-zein. The extraction of the zeins with 70% ethanol led to the identification in the mass spectra of the 19 kDa α-zeins B1 and B3 and of a 22 kDa α-zein. The extraction with 60% acetonitrile and 10 mM dithiothreitol allowed the extraction of the 27 kDa γ-zein and the 15 kDa β-zein in addition to the zeins extracted in 70% ethanol. MALDI-TOF mass spectrometric analysis of zeins extracted from maize seeds 83 Experimental Materials Maize seeds, inbred KWS 3381, were obtained from the company KWS (Germany). Commercial flour was obtained from a local grocery store. Petroleum ether and ethanol were purchased from Merck, acetonitrile, HPLC grade, and the matrix, α-cyano-4-hydroxycinnamic acid, were from Sigma-Aldrich. The trifluoroacetic acid, peptide synthesis grade, was from Scharlab. All solutions were prepared using deionized water from a MilliQ® Integral 3 system (Merck, Bucharest, Romania). Preparation of the flour for zein extraction Maize seeds from the inbred KWS 3381 were ground with a tripod portable mill (MB03, 1500 rpm, produced by IPEE, Romania). An amount of 20 g of the resulting flour was defatted with petroleum ether for 5 hours using a Soxhlet extractor. The flour was allowed to dry for 24 hours in a laboratory oven at 100°C. A sieve shaker (Retsch, Germany) was employed for selecting the flour with particles smaller than 710 μm. This flour was further ground using a laboratory electric mill (SAMAP F100, Andolsheim, France) until particles with the size lower than 100 μm were obtained. 20 g of commercial flour was defatted and dried as described above. For separating the flour particles with different sizes, sieves of 1.4 mm, 1 mm, 710 μm, 500 μm, 355 μm, 250 μm, 200 μm, 180 μm and 100 μm were employed using a sieve shaker. Zein extraction The extraction of the zeins from the maize flours was performed using two methods. In the first method, 150 mg of defatted flour that has selected particle size was mixed with 1.5 mL 70% ethanol in water for 30 84 R. Ștefănescu and S. Băncilă minutes in an ultrasound bath. The time necessary for the extraction was established in the work reported by Bancila S. et al. and by Drochioiu G. et al..7,8 The samples were centrifuged for 10 minutes at 16,000 rpm using a Hettich Mikro 22R centrifuge (from Andreas Hettich GmbH&Co. KG, Tuttlingen, Germany). In the second method, 50 mg of defatted flour that has a selected particle size was added to 2.5 mL 60% acetonitrile, 10 mM dithiothreitol in water and was kept at 60 °C while mixing at every 15 minutes. After 60 minutes, the sample was centrifuged for 10 minutes at 16,000 rpm. The extraction of the samples was performed prior to their application on the target plate for the analysis by MALDI-TOF mass spectrometry. Mass spectrometric analysis For MALDI-TOF mass spectrometric analysis, a saturated solution of α-cyano-4-hydroxycinnamic acid in acetonitrile: 0.1% trifluoroacetic acid in water (2:1 v/v) was prepared. The matrix solution was kept in an ultrasound bath for 15 minutes and centrifuged for 1 minute at 6000 rpm with a SPROUT® centrifuge commercialized by Heathrow Scientific, Illinois, USA. The zein extract prepared by either method 1 or method 2 was diluted tenfold and 20 fold with the supernatant of the matrix solution. A volume of 1 μL of each sample-matrix solution was placed on a 384sample spots target plate and allowed to dry for 1 hour. The samples were analyzed in linear mode with the AB SCIEX TOF/TOF 5800 mass spectrometer equipped with a Nd:YAG laser that operates at 349 nm. External calibration was performed using the average mass of the singly charged ions of bovine insulin (5734.59), thioredoxin (11674.48) and apomyoglobin (16952.56). MALDI-TOF mass spectrometric analysis of zeins extracted from maize seeds 85 Conclusions In the present study, MALDI-TOF mass spectrometry was employed for rapid analysis of zeins extracted with solutions containing 70% ethanol in water or 60% acetonitrile in water containing 10 mM dithiothreitol. The matrix α-cyano-4-hydroxycinnamic acid provided a good detection of α-, βand γ-zeins. Amino acid sequence assignment was possible due to previous identification of zeins reported by Adams et al. which was based on the comparison between experimental and calculated molecular masses for the zein amino acid sequences determined by Woo et al. and on the calculation of the number of cysteine residues from the mass spectra of alkylated and underivatized zeins. Acknowledgements The authors thank for the permission to analyze the maize extracts using the AB Sciex TOF/TOF 5800 mass spectrometer at the Center for Advanced Research and Development in Experimental Medicine (CEMEX) from the University of Medicine and Pharmacy “Grigore T. Popa” from Iasi. Part of this work was supported by the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, Project PN-II-RU-TE-2014-4-0920. The maize seeds inbred KWS 3381 were a gift from ing. Nicolae Berea (SC Semconsult SRL). References 1. Shewry, P.R.; Halford, N. G. Cereal seed storage proteins: structures, properties and role in grain utilization. J. Exp. Bot. 2002, 53, 947-958. 2. Coleman, C.E., Larkins, B.A. The prolamins of maize. In Seed Proteins; P.R. Shewry, R. Casey, Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998, pp 109-139. 86 R. Ștefănescu and S. Băncilă 3. Wallace, J.C.; Lopes, M.A.; Paiva, E.; Larkins, B.A. New methods for extraction and quantitation of zeins reveal a high content of γ-zein in modified opaque-2 maize. Plant Physiol. 1990, 92, 191-196. 4. Wang, J.-F.; Geil, P.H.; Kolling, D.R.; Padua, G.W. Analysis of zein by matrix-assisted laser-desorption/ionization mass spectrometry. J. Agric. Food Chem. 2003, 51, 5849-5854. 5. Adams, W.R.; Huang, S.; Kriz, A.L.; Luethy, M.H. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of zeins in mature maize kernels. J. Agric. Food Chem. 2004, 52, 1842-1849. 6. Woo, Y.-M.; Hu, D. W.-N.; Larkins, B.A.; Jung, R. Genomics analysis of genes expressed in maize endosperm identifies novel seed proteins and clarifies patterns of zein gene expression. Plant Cell. 2001, 13, 2297-2317. 7. Drochioiu, G.; Ciobanu, C.I.; Bancila, S.; Ion, L.; Petre, B.A.; Andries, C.; Gradinaru, R.V., Murariu, M. Ultrasound-based protein determination in maize seeds. Ultrason. Sonochem. 2016, 29, 93-103. 8. Bancila, S.; Ciobanu, C. I.; Murariu, M.; Drochioiu, G. Ultrasoundassisted zein extraction and determination in some patented maize flours. Rev. Roum. Chim. 2016, 61, 725-731.