Research Article
Identification of non-zein proteins in BR473
maize protein bodies by LC-nanoESI-MS/MS
The nutritional value of maize seed is limited due to its high content of storage proteins (zeins), which are deficient in essential amino acids such as lysine and tryptophan. In a previous paper, we showed that protein bodies obtained from BR473 maize variety, developed by Embrapa (Brazilian Agricultural Research Corporation), were mainly constituted by Z27 and a smaller quantity of Z50 g-zeins. Besides zein proteins, other not identified protein band in the SDS/PAGE was also observed, which could indicate the presence of non-zein proteins additionally to g-zeins. In the present paper, we have demonstrated the presence of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS and database searching. This fact could be related to the excellent energetic value and higher protein quality of BR473 maize grains, since high lysine concentration in some maize varieties has been related to the presence of cytoskeleton proteins that are non-zeins. We have identified the following proteins: Brittle-1 protein (chloroplast precursor), Legumin-1, glyceroldehyde-3-phosphate dehydrogenase, and elongation factor 1-a.Keywords: BR473 maize protein bodies / LC-nanoESI-MS/MS / Non-zein proteins / Protein identification
DOI 10.1002/jssc.200900339
1
Introduction
Despite the traditional consumption of maize in Brazil, this grain is known by its poor amino acid essential content. In order to improve the maize protein quality, Embrapa (Brazilian Agricultural Research Corporation, Brazil) devel-oped the BR473 maize variety, which has excellent energetic value and higher protein content than the normal maize. BR473 maize variety has 0.9 and 4.0 g/kg of grain of tryptophan and lysine (lys), respectively, against the 0.6 and 2.6 from normal maize varieties (http://www.cnpms.em-brapa.br/produtos/produtos/br473.html).
The high lys content in some maize varieties as opaque-2, for example, has been related to the presence of cyto-skeleton proteins attached to polysomes surrounding the protein bodies (PB) [1–3]. PB are the organelles where zeins, the maize storage proteins, are deposited. Zeins are synthesized by polysomes bound to the rough
endoplas-matic reticulum and transported to the lumen of these organelles where they aggregate in an organized regular way of development [1, 4].
Zeins are classified into four protein groups according to their sequence homology and solubility as a-, b-, g-, and d-zeins [5, 6]. Though zeins represent 50% of the maize proteins, they are deficient in essential aminoacids as lys and tryptophan [7–10] and, therefore, they may not be responsible for the high protein quality observed in BR473 maize variety.
In our previous researches [11, 12], we demonstrated that BR473 maize PB were mainly constituted of Z27 g-zein, as indicated by the most intense band in SDS/PAGE at about 30 kDa. Besides, it was possible to observe the presence of other less-intense bands around 40 and 50 kDa. The band around 50 kDa can be assigned to Z50 g-zein protein and the other band, at about 40 kDa, has already been observed in zeins SDS/PAGE [13], but it was not mentioned by the authors. This unidentified band could indicate the presence of non-zein proteins in addition to zeins.
In order to identify these non-zein proteins, which could explain the high protein content of BR473 maize variety, we analyzed the PB of these grains and those of a normal maize variety as control experiment. For this purpose, LC-nanoESI-MS/MS was used, since MS has become a powerful tool for protein sequencing and identi-fication [14–18].
The LC-nanoESI-MS/MS technique requests the protein enzymatic digestion and the chromatographic Roge´rio Campos Bicudo1
Tatiana Campos Bicudo2 Lucimara A. Forato3 Guilherme M. Titato1 Luiz A. Colnago3
Fernando M. Lanc-as1
1
Instituto de Quı´mica de Sa˜o Carlos, Laborato´rio de
Cromatografia-, Universidade de Sa˜o Paulo, Sa˜o Carlos/SP, Brazil
2Escola de Cieˆncias e Tecnologia,
Universidade Federal do Rio Grande do Norte, Natal/RN, Brazil
3Embrapa Instrumentac-a˜o
Agropecua´ria, Sa˜o Carlos/SP, Brazil
Received May 11, 2009 Revised July 23, 2009 Accepted July 28, 2009
Abbreviations: eEF1A, elongation factor 1-a; GAPC, cytosolic
glyceraldehydes-3-phosphate dehydrogenase; lys, lysine;
MoWSe, molecular weight search; NCBI, National Center
for Biotechnology Information; PB, protein bodies
Correspondence: Dr. Fernando Mauro Lanc-as, Instituto de
Quı´mica de Sa˜o Carlos, Laborato´rio de Cromatografia, Universi-dade de Sa˜o Paulo, Av. Trabalhador Sa˜o-carlense 400, Caixa Postal 780, CEP 13560-970, Sa˜o Carlos – SP, Brazil
E-mail: flancas@iqsc.usp.br Fax: 155-16-33739983
separation of generated peptides, followed by MS and MS/ MS analyses [19, 20].
At this moment, there are two ways for protein identi-fication using MS data: PMF [21, 22] and protein identifi-cation from MS/MS information. The last one is less ambiguous than PMF because the MS/MS spectra are highly specific. The amino acid sequence represented in each spectrum form exclusive signatures for each protein in the sample. For this reason, we used the MS/MS ion search approach supported by MASCOT [23] program to identify the target proteins.
Therefore, in this article we present the non-zein proteins identification results from BR473 maize variety PB obtained by means of LC-nanoESI-MS/MS analysis and database searching.
2
Materials and methods
2.1 Chemicals and reagentsAll solutions were prepared using Milli-Q water from Millipore (Bedford, MA, USA). Acetonitrile (HPLC-grade), formic acid, acetic acid, hydrochloric acid, urea, sucrose, tris (hydroxymethyl)-aminomethane (tris base), potassium chloride, magnesium chloride, and DTT were obtained from Mallinckrodt Baker (Paris, KY, France); iodoacetic acid was obtained from Acros Organics (NJ, USA).
Sequencing-grade modified trypsin was purchased from Promega (Madison, WI, USA) and BR473 maize variety was obtained from Embrapa.
2.2 PB purification
PB from BR473 and normal maize grains were ground in a blender, separately, with buffer A (200 mM Tris-HCl pH 8.5), 200 mM sucrose, 60 mM KCl, 50 mM MgCl2, and centrifuged at 500 g for 20 min. The
supernatant was centrifuged at 40 000 g for 1.5 h. The residue from this last centrifugation was frozen and lyophilized [11].
2.3 Protein in-solution digestion
BR473 and normal maize PB were in-solution digested, individually, following a standard protocol [24]. Briefly, the PB was resuspended in 6 M urea, 100 mM Tris-HCl buffer (pH 7.8) at 10 mg/mL. A 100 mL aliquot of the sample, containing 1 mg of PB, was reduced with 5 mL of DTT (200 mM) for 1 h at room temperature. Then, the sample was alkylated with 20 mL of iodoacetic acid (200 mM) for 1 h at room temperature. To consume any unreacted iodoacetic acid 20 mL of DTT was added to the proteins solution and the reaction was carried out at room temperature for 1 h.
Finally, the urea concentration was reduced to 0.6 M (a concentration at which the trypsin retains its activity) and the protein sample was digested with 100 mL of trypsin solution (200 ng/mL), containing 20 mg of sequencing-grade modified trypsin, at an enzyme/substrate ratio of 1:50 w/w. The digestion was carried out overnight at 371C. The enzymatic reaction was stopped by lowering solution pH with concentrated acetic acid too6.
The digest was analyzed directly by LC-nanoESI MS/MS.
2.4 LC-nanoESI-MS/MS
For LC-nanoESI-MS/MS analysis, a capillary LC system (CapLC, Micromass/Waters, UK) was directly interfaced to ESI-quadrupole/ToF Ultima API tandem mass spectro-meter (Micromass/Waters). The CapLC system was config-ured with two columns including a C18desalting column
(Waters Symmetry 300, 350 mm id 5 mm length, 5 mm particle size) and a C18analytical column (Waters Symmetry
300, 75 mm id 150 mm length, 3 mm particle size) both at 201C. The mobile phase used was (A) water/0.1% formic acid, (B) acetonitrile/0.1% formic acid, and (C) water. The injection volume was 6.4 mL. Peptide separation was carried out at 200 nL/min with a linear gradient of mobile phase (B) initiated from 10–45% v/v in 60 min followed by a linear gradient to 60% v/v in 10 min.
The analytical column outlet was coupled to the elec-trospray interface (nano Z-spray ion source) of the mass spectrometer on which peptides were analyzed, in the positive-ion mode, using a cone voltage of 35 V, a capillary voltage of 3500 V, and a source temperature of 1001C. The MS survey scan was m/z 200–2000 with a scan time of 1.0 s and an interscan time of 0.1 s. The MS/MS survey scan was m/z 50–2000 with a scan time of 1.0 s and an interscan time of 0.1 s. Mass spectrometer was operated in the data direct analysis mode, in which the instrument automatically selected peptides for MS/MS based on their charge state (12, 13, and 14) and intensity (three most abundant ions detected in the MS scan with a dynamic exclusion time of 30 s). The system switched back from MS/MS to MS mode after a maximum of 3 s of acquisition or when the precursor intensity fell below a predefined threshold (8 counts/s). All data were recorded by MassLynx software version 4.0 (Micromass/Waters).
2.5 Database searching
The data obtained from LC-nanoESI-MS/MS experiment was converted from raw instrument output to the .pkl format using ProteinLynx Browser version 2.0 from Micromass/Waters (smooth 2/3 Savitzky Golay and center 3 channels/80% centroid).
Subsequently, the .pkl file was used to search against the National Center for Biotechnology Information (NCBI)
J. Sep. Sci. 2009, 32, 3579–3584
non-redundant protein database using the ‘‘MS/MS ion search’’ approach supported by MASCOT program version 2.1 (Matrix Science, London, UK). The search included the use of monoisotopic atomic masses, 0.1 Da for peptide mass tolerance, and 0.05 Da for fragment mass tolerance. The taxonomy chosen was all entries and trypsin was specified as protease. One missed cleavage site per peptide was allowed. The search was performed with cysteine carbami-domethylation as fixed modification and methionine oxidation as variable modification.
3
Results and discussion
3.1 Protein identification by LC-nanoESI-MS/MS and database searching
The BR473 maize PB were in-solution digested with trypsin and the digest was subjected to LC-nanoESI-MS/MS (Fig. 1). Digestion process, which produces similar-sized peptides, collaborates to overcome the solubility and handling problems related to proteins. Besides, peptides are easily ionized and fragmented in the tandem mass spectrometer [25].
Chromatogram 1 (top panel) shows sample components detected by MS. The three most intense peaks that refer to the most abundant peptide ions detected in each MS survey scan were selected to fragment in the collision cell of the mass spectrometer. The resulting chromatograms, from 2 to 4, report only these selected peptides.
The data obtained from LC-nanoESI-MS/MS run were processed and submitted to the MASCOT search engine. MASCOT program, which is a development of the MoWSe (Molecular Weight Search) computer program, was used to
compare the experimental MS/MS spectra with theoretical spectra. From this comparison, a probability-based MoWSe score that reflects the statistical significance of the match between the experimental and predicted spectra was calcu-lated [23, 26]. The best match, or matches, was then repor-ted.
Theoretical spectra were generated by virtual dissocia-tion of peptides derived from proteins present in the non-redundant protein sequence database at the NCBI [27].
Thereby, sequences of thirteen peptides were success-fully obtained by MS/MS and database searching. Based on these peptide sequences, four unique proteins were quickly identified when compared with the normal maize PB analyses (Table 1). In Table 1, only the results with protein score above 43 are shown.
The quality and reliability of the match between the experimental and predicted spectra can be evaluated on the basis of the score reported or by visual inspection of the actual MS/MS spectrum. In terms of visual inspection, an MS/MS spectrum in which over half of the predicted b- and/ or y-ions in a peptide match the major signals in the spec-trum is a reliable match [28]. All the matches obtained in our analyses were evaluated according to the criteria cited above.
To illustrate all the analyses we considered the MS/MS spectrum (Fig. 2) of the peptide DVYDNVAHAFVK from Brittle-1 protein (Table 1). Over half of the predicted y-ions matched the major signals in the spectrum. The results indicated that this peptide sequence was unambiguously determined and represent the real sequence of this peptide.
3.2 Non-zein proteins identification
According to the results reported in Table 1, the proteins identified are non-zein proteins. As previously mentioned, we have demonstrated that BR473 maize PB were mainly constituted by Z27 g-zein. However, this zein was not identified by MS, which can be explained by the intentional use of trypsin as protease. The main objective of protein digestion in a protein identification approach is to generate peptides of optimal length (6–20 amino acids) for MS analysis and database searching. It is hard to get sequence information from peptides longer than about 20 amino acids while in a peptide too short (less than about six amino acids) it is difficult to produce unique sequence matches in database searches [28]. Trypsin cleaves proteins at lys and arginine residues, unless either of these is followed by a proline residue in the C-terminal direction. As zeins, mainly Z27 g-zein, are rich in proline, poor in lys, and contain a few number of arginine residues, the digestion process can have generated peptides longer than 20 residues. Moreover, the presence of many other peptides, from non-zein proteins, can have suppressed the few peptides of a length well suited to MS analysis obtained from zeins. Thus, the intentional use of trypsin to digest BR473 maize PB allowed the identification of only non-zein proteins.
Figure 1. Base peak intensity (BPI) chromatograms from
LC-nanoESI-MS/MS run. BPI chromatograms were obtained from BR473 PB tryptic peptides. Numbers from 1 to 4 refer to chromatograms related to data direct analysis mode of acquisi-tion. Chromatogram 1: MS acquisition; chromatograms 2–4: MS/ MS acquisition.
3.3 Identified proteins
The non-zein proteins identified in BR473 PB were not observed in the normal PB because these proteins may not be present in normal maize or they are found in very low concentrations, undetectable under the experimental condi-tions applied.
The non-zein proteins identified in BR473 PB were already observed by other authors as described below.
According to Clore et al. [1] in maize endosperm cells between 10 and 16 days after pollination there is a reorga-nization of the cytoskeleton, which is consistent with a modification of cytoskeleton structure and function coin-cident with the onset of starch and storage protein
synth-Table 1. Database searching results obtained from MASCOT MS/MS ion search analysis from BR473 maize PB
Protein name/NCBI accession number Mr/pIa) Sequence
coverage (%)/scoreb) Matched peptide sequencesc) Exp./Calc. Mr(Da)d) Rt(min)e)
Brittle-1 protein, chloroplast precursor [Zea mays]/(gi|231654)
47054/8.51 15/186 1. R.LVSGAIAGAVSR.T 1099.6496/1099.6349 19.91(4) 2. R.TFVAPLETIR.T 1145.6644/1145.6444 24.20(2) 3. K.DVYDNVAHAFVK.I 1376.6816/1376.6724 25.11(2) 4. R.RPGADVGPVATLLIGSA AGAIASSATFPLEVAR.K 3134.6200/3134.7087 44.39(2)
Legumin 1 [Zea mays]/(gi|16305144) 53346/6.31 9/127 1. R.VVVDAMGLLLPR.Y 1281.7368/1281.7478 33.38(2)
2. K.FLLAGGFSK.G 938.5554/938.5225 24.18(2)
3. K.GQPHFAENIFK.G 1286.6582/1286.6407 21.44(2)
4. R.AGQLLIVPQGYLVATK.A 1669.9374/1669.9766 30.66(2)
Cytosolic GAPC [Zea mays]/(giI1184774) 36597/7.01 16/87 1. K.FGIVEGLMTTVHAITATQK.T 2016.0604/2016.0713 39.14(2)
2. R.AASFNIIPSSTGAAK.A 1433.7754/1433.7514 22.41(2)
3. K.GILGYVEEDLVSTDFQGDSR.S 2199.0078/2199.0331 49.34(2)
a Subunit of translation elongation factor 1 [Zea mays]/(giI1321656)
49544/9.19 4/61 1. R.LPLQDVYK.I 974.5542/974.5800 20.92(3)
2. K.IGGIGTVPVGR.V 1024.6018/1024.6029 20.17(2)
a) Mr(Dalton): protein nominal mass.
b) Sequence coverage (%) – percentage of protein sequence covered by matched peptides; probability-based MoWSe score: 10
Log(p), where p is the probability that the observed match is a random event. Individual ions scores 443 indicate identity or ex-tensive homology (po0.05). Protein scores are derived from ion scores as a non-probabilistic basis for ranking protein hits. c) Matched peptide sequences: tryptic peptide sequences identified by MASCOT ‘‘MS/MS ion search’’.
d) Experimental and calculated Mr: monoisotopic mass of neutral peptide.
e) Retention time of the most intense peaks from chromatogram 1 (Fig. 1); MS/MS chromatogram for the selected peak (chromatograms 2–4 from Fig. 1).
Figure 2. MS/MS spectrum
of the [M12H]21ion at m/z
689.35 of the peptide
DVYDNVAHAFVK from Brit-tle-1 protein (Table 1). The
correspondence of the
experimental MS/MS spec-trum fragment ions with
predicted b- and y-ions
from selected peptide
sequence is shown. The number in parenthesis indi-cates the number of resi-dues in the fragment.
J. Sep. Sci. 2009, 32, 3579–3584
esis. Starch grains and PB begin to accumulate at about 10 days after pollination. These observations could explain the presence of the Brittle-1 protein (Table 1), since several studies support the possibility that Brittle-1 plays a signifi-cant role in starch accumulation in maize endosperm [29]. Yagamata et al. [30] analyzed PB fractions from sweet corn by SDS/PAGE and they found that beyond zeins bands, the gel showed the presence of larger molecular mass proteins. One of these was a 51 kDa protein that was identified as Legumin-1. This protein was previously iden-tified by Woo et al. [13] in B73 inbred in maize endosperm. Beyond the Brittle-1 and Legumin-1, the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) was also found. According to our results, the SDS/PAGE band at about 40 kDa, previously cited [11, 13], can be attributed to 36 kDa GAPC protein.
Elongation factor 1-a (eEF1A) is a multifunctional protein in eukaryotes, where it works as a protein synthesis factor as well as a cytoskeletal protein [31].
As previously mentioned and according to Wang et al. [32], ‘‘the nutritional value of maize seed is most limited by its protein quality because its storage proteins are devoid of the essential amino acid as lysine. The Lys content of the kernel can be significantly increased by the opaque-2 mutation, which reduces zein synthesis and increases accumulation of proteins that contain lysine. eEF1A is one of these proteins, and its concentration is highly correlated with the Lys content of the endosperm.’’
Another important remark is that eEF1A and GAPC are high in lys [3] so that they can contribute to the total lys in BR473 maize endosperm.
4
Concluding remarks
In order to identify the proteins described previously we employed the LC-MS/MS technique, which is a well-established technique for peptide and protein sequencing analysis. According to the reported results, the proteins identified in BR473 PB are non-zeins, which was already observed by other authors. From MS and database searching results we identified the following proteins: Brittle-1 protein (chlorosplast precursor), Legumin-1, GAPC, and eEF1A. The presence of these unique non-zein proteins, which are absent in the normal maize and probably upregulated in the BR473 maize, provide some support to explain the excellent energetic value and higher protein content of this strain of maize. Moreover, eEF1A and GAPC are high in lys content, which could contribute to the total lys in BR473 maize endosperm.
The authors thank FAPESP (Fundac-a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, process 02/08407-8) for the financial support. They also thank LNLS (Brazilian Synchro-tron Light Laboratory, research proposals: MAS3906 and MAS4540) for its support in MS experiments.
The authors have declared no conflict of interest.
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