Characterization of Selenium Incorporation into Wheat Proteins by

Technical Note pubs.acs.org/ac

Characterization of Selenium Incorporation into Wheat Proteins by Two-Dimensional Gel Electrophoresis−Laser Ablation ICP MS followed by capillary HPLC−ICP MS and Electrospray Linear Trap Quadrupole Orbitrap MS Juliusz Bianga,† Espen Govasmark,‡ and Joanna Szpunar*,† †

CNRS-UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement, UMR5254, Hélioparc, 2, Av. Angot, 64053 Pau, France ‡ Department of Plant and Environmental Science, Norwegian University of Life Sciences, PO-5003, N-1432 Ås, Norway S Supporting Information *

ABSTRACT: A method has been developed for a rapid and precise location of selenium-containing proteins in large two-dimensional (2D) electrophoresis gels. A sample was divided into four aliquots which were analyzed in parallel by 1D isoelectric focusing electrophoresis (IEF)−laser ablation (LA) inductively coupled plasma mass spectrometry (ICP MS), 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE)−LA ICP MS, and, in duplicate, by 2D IEF-PAGE. On the basis of the 1 D electropherograms obtained, areas supposed to contain the largest concentrations of Se were subjected to LA ICP MS imaging to locate precisely the position of Secontaining proteins which were then identified in the parallel 2D gel by electrospray Orbitrap MS/MS. The method was applied to the identification and semiquantitative determination of selenium storage proteins in wheat. MS evidence is presented for the Se−S substitution in plants not only in methionine but also in cysteine. membranes of plant roots4 and incorporated mainly as selenomethionine into storage proteins,7,8 but a formal identification of the Se-wheat storage proteins is missing. Selenium in wheat, present at the low parts per million level, is distributed in ca. a 1:2 ratio between the water-soluble fraction containing small molecular weight metabolites and the water-insoluble fraction.9 The speciation of selenium in the latter, presumably containing storage proteins, is a daunting task and remains, to our knowledge, unknown. To date, the only studies on Se-proteins in cereals concerned rice. A fractionation scheme showed that the largest Se content (31.3%) was found in the glutelin fraction of rice grain.10 The other study was based on 1D SDS-PAGE separation of albumins, glutelins, globulins, and prolamins followed by offline Se determination in digested gel bands without identifying the proteins.11 Three ca. 15 kDa Se-containing proteins were tentatively identified in rice on the basis of the simultaneous Sespecific and MALDI analysis 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) gel bands with no evidence however of the presence of selenium in mass spectra.12

Heteroatom-targeted proteomics offers a way to detect, identify, and quantify metallo- and selenoproteins which elude canonical proteomics protocols either because the original metalloprotein is dissociated during the analytical procedure or because it is not abundant enough to be detected in the presence of large quantities of other proteins.1 The approach involves specific and sensitive detection of the protein(s) of interest by inductively coupled plasma mass spectrometry (ICP MS) owing to the attomole detection limits, large dynamic range, and, in contrast to soft ionization MS techniques, virtual independence of the signal of concomitant molecules.2 ICP MS is also a convenient tool to quantitatively track the target proteins during purification protocols, enabling, in fine, their detection and hence identification by electrospray MS/MS. Selenium, an essential element in human and animal nutrition, is deficient in many countries, requiring supplementation of the diet.3 This can be most conveniently achieved by the enrichment of staple food, e.g. wheat, by selenate fertilization (a practice termed agronomic biofortification4−6) and/or modification of the crop phenotype increasing its ability to accumulate more Se in the edible portion of the grain.4 The design of the optimal supplementation pathways requires the understanding of the mechanisms and pathways of Se uptake and accumulation. Selenate, as a sulfate analogue, is supposed to be taken up through sulfate transporters in the plasma © 2013 American Chemical Society

Received: November 21, 2012 Accepted: January 18, 2013 Published: January 18, 2013 2037

dx.doi.org/10.1021/ac3033799 | Anal. Chem. 2013, 85, 2037−2043

Analytical Chemistry

Technical Note

For the peptide identification, the HPLC system was connected with an ESI-LTQ-Orbitrap Velos (Thermo Scientific, Bremen, Germany) operated in the positive ionization mode. Material. A growth chamber study was conducted using a mixture of 88.4% sand, 4.4% peat, and 7.2% clay with the pH adjusted by CaCO3 to 6.9. Three pots of 19 L were filled with 9 kg soil, fertilized with a basal application of 5.5 g of NPK 21-410 (Yara), and 9 mg of selenium as selenate diluted in water. 56 seeds per pot in three replicates were grown to maturity in a 14 h day period at 20 °C and an 8 h dark period at 16 °C. All plants were harvested manually at maturity and dried at 45 °C for 48 h and milled on a mixer mill (Retsch Mixer Mill MM 200, Retsch, Germany) provided with zirconium jars prior to storage in plastic vials at 4 °C. Reagents. Analytical reagent grade chemicals purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and water (18 MΩ cm) obtained with a Milli-Q system (Millipore, Bedford, MA) were used throughout unless stated otherwise. HNO3 (69−70%) and H2O2 (30%) were purchased from Baker (Deventer, Holland). Calibration solutions used for total Se measurements were obtained from standard certified solutions with a content of 1 mg mL−1 (SPEX CertiPrep (Matuchen, NJ) by dilution with 0.1% HNO3 as necessary. Procedures. Total Selenium Determination. A 0.1 g sample (flour or protein pellet) was predigested overnight with 2.5 mL HNO3. A volume of 1.25 mL H2O2 was added and the digestion was completed by raising the temperature to 65 °C during 30 min and maintaining it for 4 h. After cooling down, the digest was made up to 50 mL with water and analyzed by ICP-MS (78Se) using a 4-point standard addition calibration. The quality was assured by analyzing a NIST 8436 Durum Wheat Flour SRM. Protein Extraction. A 0.1 g sample was extracted three times with 5 mL of water in an ice bath (4 °C) using an ultrasonic probe (model S450-A, Branson Ultrasons, Rungis, France) (35% of energy, 3 s pulse, 5 s pause) for 1 min. The sample was centrifuged after each extraction for 10 min at 2860 g. The pellet was resuspended in water after the first and second extractions, and the supernatant was discarded. The final pellet was extracted twice with 5 mL of a solution containing 8 M urea, 2.5 M thiourea, 4% CHAPS, and 0.04 M Tris-HCl at pH 6.8 using the same sonication program. After centrifugation (10 min, 2860g), the supernatant was transferred to 1.5 mL Eppendorff tubes and centrifuged again at 10 000g for 10 min. Isoelectric Focusing Separation. An aliquot of 200 μL of the extract was mixed with 140 μL desteak rehydration buffer (GE Healthcare) prior to isoelectric focusing separation; the IEF strip was rehydrated overnight under mineral oil. The voltage program was 500 V 1 h, 500−1000 V 1 h, 1000−5000 V 3 h, 5000 V 1 h, 5000−6000 V 3 h, and 6000 V 7 h. 1D SDS-PAGE Separation. A pellet was extracted with a solution containing 5% SDS, 40 mM dithiotreitol (DTT), and 0.1 M Tris-HCl at pH 6.8. The extract was mixed with a 5-fold excess Laemmli dye stock solution. A 20 μL extract aliquot was then loaded onto the gel. The separation was done under 160 V for 7 h. IEF/SDS-PAGE 2D Gel Electrophoresis Separation. Proteins separated in the first dimension were reduced by soaking the strip for 15 min in 65 mM DTT and on-strip derivatized by soaking in 74 mM iodoacetamide (IAM) solution. Then, the gel was fixed with 0.5% agarose on top of 12% tris-glycine SDS-

Because of the need for chaotropic media for protein solubilization, gel electrophoresis seems to be privileged to chromatography for the separation of water-insoluble selenium proteins. Selenium in 1D gels (isoelectric focusing (IEF) and SDS) can be conveniently detected by laser ablation (LA) ICP MS.13−21 The analysis is rapid (1 cm of gel/min) and sensitive (attomolar detection limits can be achieved).14 In the case of 2D gels, single-point analysis of the most intense spots in the gel21 or ablating lines passing them13,22 was reported, but a comprehensive specific detection of selenium-containing proteins in large (400 cm2) 2D gels remains problematic without the use of radioactive tracers. A true alternative for autoradiography23−25 can be the imaging mode in which a gel is scanned in a raster mode and then a 2D concentration “heat map” is constructed.26,27 The analysis in the imaging mode is, however, very time-consuming. Scanning a gel of 5 cm2 with a 100-μm resolution takes about 15 h. The gels of 25 cm × 25 cm are typical for high resolution proteomics; a dedicated large volume ablation cell was developed for heteroatom imaging protein spots blotted onto flexible membranes.28 It solved the problem of gel size, and the analysis was automated but remained time-consuming.28 Also the efficiency of the blotting is irreproducible and protein dependent therefore rendering the quantification impossible.14 The imaging of a whole gel is hardly useful as the proteins of interest are localized in particular areas representing a small percentage of the gel surface. The concept developed in this work uses the combination of two rapid IEF and SDS PAGE LA ICP MS scans to locate such areas which are then subject to fine 2D LA ICP MS imaging to locate precisely the Secontaining proteins. A capillary HPLC method with dual ICP MS and Orbitrap MS/MS detection was then developed to speciate the incorporated selenium and to identify the storage proteins in wheat with a focus on measuring the Se−S substitution in cysteine and methionine.

■

EXPERIMENTAL SECTION Instrumentation. Horizontal (IEF-SYS) and vertical TV400 (maxi gels 16.5 cm × 17.5 cm) and TV100 (mini gels 7.5 cm × 8 cm) electrophoresis systems were used (Biostep, Jahnsdorf, Germany). IEF separation was done on 18 cm Immobiline DryStrip pH 3−10 NL gel (GE Healthcare Life Sciences). Tris-glycine SDS-PAGE gels were cast on site. For the LA ICP MS scanning, gels were dried using a Hoefer Slab gel dryer GD2000 (Amersham Biosciences). A NewWave Research (Fremont, CA) UP-213 laser was used. The ablated aerosol was mixed in the spray chamber with a 1 ppb Y solution in 2% HNO3 prior to ICP MS. An Agilent 7500cs ICP MS (Agilent, Hachi-oji, Japan) was fitted with a 1.5-mm i.d. injector and Pt sampler and skimmer cones. For capHPLC−ICP MS experiments, an HPLC Agilent 1260 Infinity system (Agilent) equipped with a bioinert autosampler and a switching valve was used. The system consisted of two pumps: a binary pump with the flow range between 1 and 20 μL/min (capillary) and an isocratic pump which was used just for loading the sample on a preconcentration cartridge. The exit of the column was connected to ICP MS (Agilent 7500cs) by means of a total consumption nebulizer as described in detail elsewhere.29 A 1 mm i.d. injector and platinum cones were used. A flow of 32 mL/min oxygen was introduced into plasma. The ICP MS was tuned using a 20 ppb standard solution in 2% HNO3 at 4 μL/min supplied by a syringe pump. 2038

dx.doi.org/10.1021/ac3033799 | Anal. Chem. 2013, 85, 2037−2043

Analytical Chemistry

Technical Note

Figure 1. Gel electrophoresis with laser ablation ICP MS detection: (a) IEF-ICP MS electropherogram. (b) SDS PAGE LA ICP MS electropherogram. (c) Coumassie blue-stained 2D gel. The red square determines the area of interest for LA ICP MS. (d) Concentration heat map of selenium in the imaged parts of the gel. (e and f) Closeups of the discussed parts of the gel.

Se-Containing Peptide Mapping and Protein Identification. Spots in which selenium was detected were excised with a scalpel from a gel run in parallel and placed in 1.5 mL Eppendorf tubes. Pieces of gel were washed with 200 μL of 200 mM ammonium bicarbonate 40% acetonitrile (ACN) and dried. The proteins were digested with 20 μL of trypsin solution (containing 0.4 μg of proteomic grade trypsin in 40 mM ammonium acetate; 9% ACN) for 6 h at 37 °C. Then, the solution was removed from the tube with a pipet. It was then 10 kDa cutoff filtered (Vivacone 500, Sartorius, Goettingen, Germany) by centrifugation for 20 min at 10 000g. A volume of 2 μL of 1 M DTT solution was added. Am 8 μL aliquot of the filtrate was loaded onto a C18 peptide cartridge (35 mm × 0.5 mm × 5 μm, Zorbax 300SB, Agilent, Germany) at 20 μL/min by injection into a flow of 0.1% formic acid in 2% acetonitrile for 4 min (at least 2 min

PAGE. Proteins were detected by Coomassie blue staining. For further LA-ICPMS analysis, the gel was dried on 3 MM Whatmann Chromatography under vacuum at 80 °C. Selenium Detection and Imaging by Laser Ablation ICP MS. Laser ablation conditions were optimized for the maximum sensitivity using a lab-made glutathione peroxidase (GPx) loaded standard gel. The optimum conditions were the following: spot size 250 μm, repetition rate 20 Hz, output energy 100%, fluence 7 J/cm2. Scan speed was 100 and 50 μm/ s for SDS and IEF gels. Two gels were prepared at the same time in identical conditions. One of them was dried on 3 MM Whatmann Chromatography under vacuum at 80 °C and served for LA ICP MS analysis, the other one was used for tryptic digestion followed by capillary HPLC−ICP MS/ESI MS. 2039

dx.doi.org/10.1021/ac3033799 | Anal. Chem. 2013, 85, 2037−2043

Analytical Chemistry

Technical Note

washing time). Then, the flow was reversed and the retained peptides were eluted at 4 μL/min onto a separation column (100 mm × 0.3 mm × 3.5 μm, Zorbax 300SB, Agilent, Germany) using the following gradient: 0−2 min 2% B, 2−5 min 2−5% B, 5−35 min 5−2 5% B; 35−40 min 25−40% B; 40−45 min 40−97% B; 45−50 min 97% B; 50−55 min 97−2% B, where A was 0.1% formic acid in H2O and B was 0.1% formic acid in acetonitrile. For electrospray ionization (ESI) MS analysis, a 300−1100 m/z scan at 60 000 resolution was performed. The most intense double- or triple-charged ions were automatically fragmented. The fragmentation spectra for these ions were acquired in the high energy collision induced dissociation (HCD) mode within 80−2000 m/z at a resolution 30 000. The results were examined versus SwissProt and the NCBI database with the use of the Mascot software. When the proteins were identified, Se-containing analogues of all the detected sulfur-containing peptides were searched for by mass difference (+47.94446/z). In a second run, fragmentation of detected Se-containing peptides was performed.

concentrated in a limited number of spots corresponding to a group of proteins with the molecular weight of 32 kDa, separated into two groups according to pI 7−8 and 9−10. Note the absence of the largest peak at pI 7.8 which was likely to correspond to noncovalent Se−protein complexes which dissociated during the SDS analysis resulting in the release of inorganic selenium eluting in the front. Several other areas of the gel where spots were visible by Coomassie staining were imaged for the purpose of validation of the approach, but no selenium was detected. Identification of the Selenium-Rich Proteins. The spots most abundant in selenium (cf spots in the closeups in Figure 1e and f) were excised, digested with trypsin, and analyzed by HPLC with parallel detection with ICP MS and electrospray Orbitrap MS/MS. The presence of Se-containing peptides among all the species present in the tryptic digest was demonstrated by the use of selenium-selective ICP MS detection. The chromatograms of the spots are given in Figure 2 (spot 1), Supporting Information Figure 1SI (spots 2−6),

■

RESULTS AND DISCUSSION The total Se content in the investigated sample was 43.4 ± 0.67 μg/g (dry weight). A 53% portion of Se was found in the waterinsoluble fraction. The water-soluble fraction contained 28% of selenium which is slightly higher than the values reported by Moreno et al.,30 who found an average yield of 23% using water as extractant for the wheat flour SEAS-5 test material (total Se 0.69 ± 0.09 μg Se·g−1), and Govasmark et al.,8 who extracted 19% of Se-containing metabolites from Se-enriched wheat flour (total Se 1.1 ± 0.02 μg Se·g−1) using 0.9% NaCl. After precipitation, 97% of Se present in the extract was recovered in the pellet. The solid residue after water and protein extraction contained 6% of the total selenium. Selenium Imaging in 2D Gels by Laser Ablation ICP MS. The principle of the approach developed is illustrated in Figure 1. Four aliquots of the sample were subjected, in parallel, to 1D IEF, 1D SDS PAGE and, in duplicate, to 2D SDS PAGE. The 1D IEF and 1D SDS gels were analyzed by LA ICP MS to produce electropherograms shown in Figures 1a and b, respectively. Figure 1a indicates that most of the selenium can be found at pI 7−8 with three smaller peaks at pI 8.4, 8.7, and 9.0, respectively. These peaks can correspond to simple proteins containing selenomethionine (SeMet) and selenocysteine (SeCys) residues, to Se−S or S−Se−S bridged dimers, or simply to proteins binding selenium by coordination. The parallel SDS PAGE ICP MS analysis indicates the presence of abundant Se proteins in the 22−26 and 30−32 kDa ranges. Note that in contrast to the IEF, only proteins with covalently bound selenium are visible, the bridges and Se bound by coordination are cleaved due to the presence of DTT (reducing agent). Consequently, the part of selenium bound as SeO4− by coordination to cysteine sulfur or selenocystein selenium elutes in the low molecular mass region of the 1D SDS gel. The analysis of the characteristics of the Se-containing proteins (pI, Mr) allowed the determination of two rectangle areas in which the Se-rich proteins were expected. These areas (Figure 1c) were imaged in order to locate the protein spots precisely. Note that they represented ca. 10% of the whole gel which resulted in a considerable economy of time and running costs. A concentration heat image of selenium is represented in Figure 1e and f. It shows that most of the selenium, ca. 80%, is

Figure 2. HPLC−ICP MS chromatogram of a tryptic digest of high molecular weight glutenin subunit (cf Figure 1, spot 1).

and Figure 3 (spot 7). The identity of the selenopeptides was confirmed by ESI MS/MS analysis; the morphology of the ESI MS extracted ion chromatogram (XIC) including all the Secontaining peptide ions matched that of the chromatogram with the ICP MS detection (Figure 2). The elution times of corresponding sulfur-containing analogues were slightly different, and their intensity was significantly higher. Table 1 lists the Se-containing peptides detected. The identification of proteins was completed by the completion of the sequence coverage by noncontaining selenium trypticpeptides, the sequence coverage was higher than 40%; the modifications taken into account included carbamidomethylation of cysteine and oxidation of methionine. The identified Se-rich proteins belong to two groups: glutenins (6 proteins including 5 low molecular weight subunits and a high molecular weight one) and a gamma-gliadin. Both classes altogether represent ca. 80% of storage proteins in wheat.31 Glutenins are multichained proteins rich in glutamine and proline with low levels of charged amino acids with subunits being linked through disulfide bridges.32 The four α-, β-, γ-, and ω-gliadins, also rich in proline and glutamine, have a low level of charged amino acids but are single chained.31 Both classes contain a significant number of cystein residues. Indeed, cystein bridges were found responsible for linking the subunits of glutenins and are present as intrachain bridges in gliadins.32 Selenium was found not only in the form of selenomethionine but also of selenocysteine. Whereas the substitution of 2040

dx.doi.org/10.1021/ac3033799 | Anal. Chem. 2013, 85, 2037−2043

Analytical Chemistry

Technical Note

Figure 3. Capillary HPLC with dual ESI MS and ICP MS detection of the VFLQQQCSPVAMPQR peptide: (a) HPLC−ICP MS chromatogram (1) peptide with Se−S substitution in cysteine; (2) peptide with Se−S substitution in methionine. (b) ESI MS (blue line) XIC of Se-substituted peptide, (gray line) XIC of unsubstituted peptide, (inset) comparison of the theoretical and experimental isotopic patterns. (c) MS/MS of the SeCys-containing peptide ion. (d) MS/MS of the SeMet-containing peptide ion.

Se−S in methionine is largely described for yeast18,22and crops,31 this is to our knowledge the first mass spectrometry evidence for an unspecific selenocysteine/cysteine substitution in a plant. Also, the MS data reported so far to identify SeMetcontaining proteins concerned samples with Se concentrations higher by 1−2 orders of magnitude, such as Se-rich yeast22 or Brazil nuts.33 Selenium−Sulfur Substitution in Cysteine. In order to investigate quantitatively the Se−S substitution, a peptide produced by tryptic digestion of a gi121102 protein containing both methionine and cysteine residues in its structure (VFLQQQCSPVAMPQR) was examined in detail. HPLC with parallel ICP MS (Figure 3a) and electrospray MS (Figure 3b) detection of different species corresponded to this peptide: the S-containing peptide (black line) and two selenized peptides (blue line). The MS/MS analysis allowed the identification of the peptide with substituted cysteine (mass difference of characteristic of carbamidoylmethylated selenocysteine 207.97452 cf Figure 3c) and the peptide with substituted methionine (mass difference 178.98459 characteristic of SeMet, cf Figure 3d). The inset in Figure 3 shows the correspondence of the theoretical and experimental isotopic Se

pattern (double charged). No peptide with S substituted by Se in both amino acids was detected. A chromatogram obtained with ICP MS detection (Figure 3a) allows the quantification of selenium and estimation of the degree of substitution of cysteine as one-third of that of methionine. Interestingly, the same ratio was found when comparing the intensity of the most abundant peaks in electrospray mass spectra. The latter suggests that an exchange of an amino acid in this peptide for selenium does not result in a change of response which offers the possibility of relative quantification of the nonsubstituted peptide. The corresponding peak height is equal to that of the peptide with substitute methionine. These observations allow the conclusion that about 4% of sulfur was substituted by selenium (1% as selenocysteine). The conclusion can be generalized to the other proteins by comparison of the intensity of the substituted and unsubstituted peptides (SeCys 0.64 ± 0.5% and SeMet 2.15 ± 1.1%) (cf Table 1) The observed Se−S substitution in glutenins may be of importance as glutenins are technologically active proteins with a function of retaining gas in the formation of dough producing spongy baked products.34 As the quality of dough depends on the number and distribution of inter- and intrachain disulfide 2041

dx.doi.org/10.1021/ac3033799 | Anal. Chem. 2013, 85, 2037−2043

Analytical Chemistry

Technical Note

Table 1. Selenized Peptides Detected in Protein Spots Marked in Figure 1a protein (spot nr) HMW glutenin, subunit DY10, GLT0_WHEAT (spot 1)

LMW glutenin subunit, gi|169666949 (spot 3, 4)

LMW glutenin subunit, gi|121455 (spot 3, 4)

LMW glutenin subunit, gi|44885910 (spot 6)

LMW glutenin subunit, gi|886965 (spot 5)

LMW glutenin subunit, gi|17425192 (spot 2) Gamma-gliadin, gi|121102 (spot 7)

m/z detected (charge)

selenized peptide sequence CUQQLR or UCQQLRc QLQUER SeMEGGDALSASQ ELQESSLEAUR AQQPATQLPTVUR LPWSTGLQSeMR TLPTMUR TLPTSeMCR VFLQQQUSPVAMPQSLAR VFLQQQCSPVASeMPQSLAR VFLQQQUIPVAMQR VFLQQQCIPVASeMQR SQMLQQSIUHVMQQQCCQQLRc SQSeMLQQSICHVMQQQCCQQLRc TLPMMUR TLPSeMMCRc VFLQQQUSPVAMPQSLAR VFLQQQCSPVASeMPQSLAR SQMLWQSSUHVMQQQCCRc SQMLWQSSCHVSeMQQQCCRc SQMLWQSSCHVSeMoxQQQCCRc VFLQQQUSPVAMPQSLAR VFLQQQCSPVASeMPQSLAR VFLQQQCSPVASeMPQSLAR VFLQQQCSPVASeMoxPQSLAR VFLQQQUSHVAMSQR VFLQQQCSHVASeMSQR VFLQQQUSPVAMPQR VFLQQQCSPVASeMPQR VFLQQQCSPVASeMoxPQR ILPTSeMoxCSVNVPLYR ILPTSeMoxCSVNVPLYR

456.6666 441.1717 554.2011 685.2768 759.3503 618.7852 463.6867 463.6867 1054.4937 1054.4937 883.4187 883.4187 913.7209 913.7209 478.6839 478.6839 1054.4994 1054.4994 801.3114 801.3114 605.2339 1054.4986 703.3338 1054.4986 708.6649 933.9123 933.9123 918.9206 918.9206 618.2823 863.9091 576.2759

(2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (3+) (2+) (2+) (2+) (2+) (3+) (3+) (4+) (2+) (3+) (2+) (3+) (2+) (2+) (2+) (2+) (3+) (2+) (3+)

experimental mass, Da

theoretical mass, Da

Δ, ppm

RT, min

Se−Sb ratio

911.3176 880.3278 1106.387 1368.538 1516.685 1235.555 925.3578 925.3578 2106.972 2106.972 1764.822 1764.822 2738.139 2738.139 955.3522 955.3522 2106.983 2106.983 2400.911 2400.911 2416.904 2106.982 2106.978 2106.982 2122.971 1865.809 1865.809 1835.826 1835.826 1851.824 1725.803 1725.804

911.3176 880.3296 1106.388 1368.541 1516.689 1235.556 925.3584 925.3584 2106.978 2106.978 1764.824 1764.824 2738.14 2738.14 955.3512 955.3512 2106.978 2106.978 2400.907 2400.907 2416.902 2106.978 2106.977 2106.978 2122.972 1865.81 1865.81 1835.824 1835.824 1851.819 1725.802 1725.801

0.03 2.1 1.2 2.5 2.7 0.7 0.7 0.7 2.8 2.8 1.1 1.1 0.2 0.2 1.0 1.0 2.6 2.6 1.5 1.5 1.1 1.8 0.4 1.8 0.4 0.5 0.5 0.7 0.7 2.5 0.6 1.9

9.89 9.89 13.59 15.52 17.17 34.02 14.33 15.70 31.06 32.04 30.01 30.80 31.74 32.54 19.14 20.40 31.14 32.02 25.43 26.25 26.25 31.08 31.95 32.00 32.00

0.005 0.010 0.018 0.006 0.004 0.026 0.003 0.007 0.002 0.009 0.005 0.020 0.021 0.034 0.003 0.008 0.004 0.014 0.008 0.020d 0.200d 0.003 0.016d 0.016d 0.016d

19.35 25.34 26.21 26.21 36.16 36.21

0.027 0.008 0.036d 0.036d 0.045d 0.045d

a

U (selenocystein), SeM (selenomethionine), SeMox (oxidized selenomethione). bApproximate Se−S substitution ratio evaluated according to Se/S = [(∑ISe/ASe)/(∑IS/AS)] × 1/n where ∑ISe is a sum of intensities of all detected forms of a selenized peptide corresponding to 80Se, ∑IS is a sum intensities of all detected forms of unsubstituted peptide corresponding to 32S, n is number of selenoamino acids, and ASe and AS correspond to abundances of 80Se and 32S, respectively cSe−S substitution can occur for any of the methionine or cystein residues. None of the Met or Cys positions were found to be favored. dValue corresponding to all the forms of the peptide

■

bonds, the manipulation of expression of genes specific for these subunits is associated with good bread-making quality and of the cross-linking by altering cystine residues.35

■

ASSOCIATED CONTENT

S Supporting Information *

Figure 1S: HPLC−ICP MS chromatograms of tryptic digests corresponding to spots 2−6 (cf Figure 1). Figure 2S: ESI MS/ MS fragmentation spectra showing Se/S substitution of the CCQQLR peptide confirming the presence of UCQQLR and CUQQLR monosubstituted forms (closeups of y1/y′1 and b1/b′1 ions are shown in the inset). This material is available free of charge via the Internet at http://pubs.acs.org.

CONCLUSIONS

The time-consuming analytical heteratom-tagged proteomics using large 2D gels can be largely accelerated by using information from two short 1D IEF and SDS PAGE laser ablation ICP MS analyses to delimit areas in the 2D gel worth analyzing in the laser ablation ICP MS imaging mode. The developed approach was demonstrated to describe a family of selenium-accumulating proteins (Se-rich glutenins) in wheat. For the first time in plants, mass spectrometric evidence is produced for the substitution of sulfur by selenium not only in methionine but also in cysteine. The method can be readily extended to the rapid detection of any heteratom-containing protein, either naturally occurring (metalloproteins) or artificially tagged with metals.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge the support of the National Research Agency (ANR) Project SELENOPROTEOME, that of the Aquitaine Region, and FEDER (20071303002PFP). J.B. 2042

dx.doi.org/10.1021/ac3033799 | Anal. Chem. 2013, 85, 2037−2043

Analytical Chemistry

Technical Note

(33) Dernovics, M.; Giusti, P.; Lobinski, R. J. Anal. Atom. Spectrom. 2007, 22, 41−50. (34) Belderok, B.; Mesdag, H.; Donner, D. A. Bread-Making Quality of Wheat; Springer: New York, 2000. (35) Anjum, F. M.; Khan, M. R.; Din, A.; Saeed, M.; Pasha, I.; Arshad, M. U. J. Food Sci. 2007, 72, R56−R63.

acknowledges the fellowship of the French Ministry of Education.

■

REFERENCES

(1) Sanz-Medel, A. Anal. Bioanal. Chem. 2005, 381, 1−2. (2) Becker, J. S.; Jakubowski, N. Chem. Soc. Rev. 2009, 38, 1969− 1983. (3) Roman Vinas, B.; Ribas Barba, L.; Ngo, J.; Gurinovic, M.; Novakovic, R.; Cavelaars, A.; De Groot, L. C. P. G. M.; Van’T Veer, P.; Matthys, C.; Serra Majem, L. Annals Nutr. Metab. 2011, 59, 84−95. (4) Hawkesford, M. J.; Zhao, F. J. Cereal Sci. 2007, 46, 282−292. (5) Govasmark, E.; Salbu, B. J. Sci. Food Agric. 2011, 91, 1367−1372. (6) Govasmark, E.; Salbu, B. J. Plant Nutr. 2011, 34, 1919−1929. (7) Cubadda, F.; Aureli, F.; Ciardullo, S.; D’Amato, M.; Raggi, A.; Acharya, R.; Reddy, R. A. V.; Prakash, N. T. J. Agric. Food Chem. 2010, 58, 2295−2301. (8) Govasmark, E.; Brandt-Kjelsen, A.; Szpunar, J.; Bierla, K.; Vegarud, G.; Salbu, B. Pure Appl. Chem. 2010, 82, 461−471. (9) Aureli, F.; Ouerdane, L.; Bierla, K.; Prakask, T.; Cubadda, F.; Szpunar, J. Metallomics 2012, submitted. (10) Fang, Y.; Catron, B.; Zhang, Y.; Zhao, L.; Caruso, J. A.; Hu, Q. J. Agric. Food Chem. 2010, 58, 9731−9738. (11) Liu, K.; Chen, F.; Zhao, Y.; Gu, Z.; Yang, H. Food Chem. 2011, 127, 1526−1531. (12) Liu, K.; Zhao, Y.; Chen, F.; Gu, Z.; Bu, G. Eur. Food Res. Technol. 2012, 234, 61−68. (13) da Silva, M. A. O.; Arruda, M. A. Z. Microchim. Acta 2012, 176, 131−136. (14) Bianga, J.; Ballihaut, G.; Pecheyran, C.; Touat, Z.; Preud’Homme, H.; Mounicou, S.; Chavatte, L.; Lobinski, R.; Szpunar, J. J. Anal. Atom. Spectrom. 2012, 27, 25−32. (15) Ballihaut, G.; Kilpatrick, L. E.; Davis, W. C. Anal. Chem. 2011, 83, 8667−8674. (16) Ballihaut, G.; Kilpatrick, L. E.; Kilpatrick, E. L.; Davis, W. C. J. Anal. Atom. Spectrom. 2011, 26, 383−394. (17) Pedrero, Z.; Madrid, Y.; Camara, C.; Schram, E.; Luten, J. B.; Feldmann, I.; Waentig, L.; Hayen, H.; Jakubowski, N. J. Anal. Atom. Spectrom. 2009, 24, 775−784. (18) Tastet, L.; Schaumloeffel, D.; Bouyssiere, B.; Lobinski, R. Talanta 2008, 75, 1140−1145. (19) Ballihaut, G.; Mounicou, S.; Lobinski, R. Anal. Bioanal. Chem. 2007, 388, 585−591. (20) Ballihaut, G.; Pécheyran, C.; Mounicou, S.; Preud’homme, H.; Grimaud, R.; Lobinski, R.; Ballihaut, G.; Grimaud, R.; Lobinski, R. TrAC−Trends Anal. Chem. 2007, 26, 183−190. (21) Chassaigne, H.; Chéry, C. C.; Bordin, G.; Vanhaecke, F.; Rodriguez, A. R. J. Anal. Atom. Spectrom. 2004, 19, 85−95. (22) Tastet, L.; Schaumloeffel, D.; Lobinski, R. J. Anal. Atom. Spectrom. 2008, 23, 309−317. (23) Behne, D.; Alber, D.; Kyriakopoulos, A. J. Radioanal. Nuclear Chem. 2004, 259, 435−439. (24) Bertelsmann, H.; Behne, D.; Hammadeh, M.; Kyriakopoulos, A. Trace Elements Electrolytes 2010, 27, 232−234. (25) Bukalis, K.; Wolf, C.; Behne, D.; Kyriakopoulos, A. J. Chromatogr. A 2007, 1155, 180−186. (26) Becker, J. S.; Lobinski, R.; Becker, J. S. Metallomics 2009, 1, 312. (27) Tsang, C. N.; Bianga, J.; Sun, H.; Szpunar, J.; Lobinski, R. Metallomics 2012, 4, 277−283. (28) Feldmann, I.; Koehler, C. U.; Roos, P. H.; Jakubowski, N. J. Anal. Atom. Spectrom. 2006, 21, 1006−1015. (29) Schaumloeffel, D.; Encinar, J. R.; Lobinski, R. Anal. Chem. 2003, 75, 6837−6842. (30) Moreno, P.; Quijano, M. A.; Gutiérrez, A. M.; Pérez-Conde, M. C.; Cámara, C. Anal. Chim. Acta 2004, 315−327. (31) Dupont, F. M.; Vensel, W. H.; Tanaka, C. K.; Hurkman, W. J.; Altenbach, S. B. Prot. Sci. 2011, 9. (32) Veraverbeke, W. S.; Delcour, J. A. Crit. Rev. Food Sci. Nutr. 2002, 42, 179−208. 2043

dx.doi.org/10.1021/ac3033799 | Anal. Chem. 2013, 85, 2037−2043