Research article Received: 25 February 2012
Revised: 8 May 2012
Accepted: 15 May 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3031
MS-based characterization of as2-casein isoforms in donkey’s milk† Rosaria Saletti,a* Vera Muccilli,a Vincenzo Cunsolo,a Debora Fontanini,b Antonella Capocchib and Salvatore Fotia The primary structure of four as2-casein (CN) isoforms, present as minor components in the dephosphorylated CN fraction of a milk sample collected in Eastern Sicily from an individual donkey belonging to the Ragusano breed at middle lactation stage, was determined, using the known donkey’s as2-CN (GenBank Acc. No. CAV00691; Mr 26 028 Da) as reference. Proteins, with experimentally measured Mr of 25 429, 21 939, 25 203 and 21 713 Da, were isolated by the combined use of reversed-phase high-performance liquid chromatography (RP-HPLC) and two-dimensional polyacrylamide gel electrophoresis. The major spot of each gel, corresponding to a single protein, was digested by trypsin, a-chymotrypsin and endoproteinase Glu-C. The resulting peptide mixtures were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry, and the data obtained were used for the sequence determination. The isoforms are produced from differential splicing events involving exons 4, 5 and 6 and parts of the exon 17. Copyright © 2012 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this article. Keywords: donkey milk; as2-casein; milk allergy; RP-HPLC/nESI-MSMS; 2D-PAGE
Introduction
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The efficacy of donkey’s milk, which has been traditionally used to feed children affected by milk allergy, has been recently confirmed by clinical investigations on children with severe cow’s milk protein allergy (CMPA).[1–4] These studies have shown that for a large number (83%–88%) of the investigated subjects, milk from donkey represents the unique safe and resolving “natural” treatment in both immunoglobulin E (IgE)-mediated and non-IgE-mediated CMPA, providing nutritional adequacy and good palatability. Although the mechanism of this tolerance has not yet been clarified, it is reasonable to assume that the reduced allergenic properties of donkey’s milk are related to structural differences of its protein components with respect to bovine milk. The high lactose content, the characteristic protein profile with a lower casein (CN) content (50%) with respect to cow’s milk (80%)[5] and the relevant percentage of essential amino acids make this milk a potential new dietetic food and a promising breast milk substitute. However, although composition, physicochemical and nutritional properties of donkey’s milk are well known, few data are available for its genetic polymorphism. The major information concerns whey proteins,[6–9] whereas the investigation of CN components is still at a relatively early stage of progress. In particular, the cDNA deduced amino acid sequences of a k- (GenBank Acc. No. ACA42445)[10] and two as2-CNs (GenBank Acc. No. CAV00691, Mr 26 028 Da and CAX65660, Mr 16 721 Da) are reported.[7,11] The correctness of the cDNA-derived sequence of as2-CN (GenBank Acc. No. CAV00691) was confirmed in a recent MS-based investigation of donkey caseome.[12] Furthermore, the primary structure of two b-CNs (UniProtKB/Swiss-Prot Acc. No. P86273)[13] and four donkey’s as1-CN isoforms (UniProtKB/Swiss-Prot Acc. No. P86272)[14] were directly determined by MS data and using the known counterparts from mare as reference.
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In the frame of our research line oriented to the characterization of the donkey’s milk proteins,[8,9,13–16] we report here the identification and direct sequence determination of the primary structure of four as2-CN isoforms by coupling reversed-phase high-performance liquid chromatography (RP-HPLC), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), enzymatic digestions and mass spectrometry (MS) methods.
Experimental Materials and methods Acetic acid (AA), formic acid (FA), trifluoracetic acid (TFA), sinapinic acid (SA), a-cyano-4-hydroxycinnamic acid (CHCA), cytochrome c from bovine heart, myoglobin from horse skeletal muscle, trypsinogen from bovine pancreas, endoproteinase Glu-C (Staphylococcus aureus strain V8), a-chymotrypsin from bovine pancreas, alkaline phosphatase from bovine intestinal mucosa and b-lactoglobulin from bovine milk were obtained from Sigma-Aldrich (Milan, Italy). Modified porcine trypsin was purchased from Promega (Madison, WI, USA). Ammonium bicarbonate, sodium acetate, HPLC-grade H2O and CH3CN were provided by Carlo Erba (Milan, Italy). * Correspondence to: Rosaria Saletti, Department of Chemical Sciences, University of Catania, Viale A. Doria, 6, I-95125 Catania, Italy. E-mail:
[email protected] †
This article is part of the Journal of Mass Spectrometry special issue entitled “2nd MS Food Day” edited by Gianluca Giorgi.
a Department of Chemical Sciences, University of Catania, Viale A. Doria 6, I-95125, Catania, Italy b Department of Biology, University of Pisa, Via L. Ghini 5, I-56126, Pisa, Italy
Copyright © 2012 John Wiley & Sons, Ltd.
Characterization of as2-caseins in donkey’s milk Preparation of milk and enzymatic dephosphorylation of CN fraction The individual milk sample was collected in Eastern Sicily from a donkey belonging to the Ragusano breed, at middle lactation stage. After milking, the sample was immediately frozen and stored at 20 C until used. Milk skimming, CNs precipitation and dephosphorylation of the CN fraction by alkaline phosphatase from bovine intestinal mucosa were performed as previously reported.[14] Isolation of dephosphorylated as2-CNs by RP-HPLC Separation of the dephosphorylated CN mixture was carried out on a Varian 9010 HPLC (Sunnyvale, CA, USA), equipped with a Varian 9050 UV detector, and the data were acquired with a PC using the software system Peak Simple II. The dephosphorylated CN fraction solution was dissolved in H2O + 0.1% TFA : CH3CN + 0.1% TFA (65 : 35 v/v), filtered on micro-spin filters (Alltech, Milan, Italy) and loaded onto a reversed-phase Vydac C4 column (4.6 250 mm, 300 Å, 5 mm). The column was eluted at 50 C with a linear gradient of solvent B (CH3CN + 0.1% TFA) in A (H2O + 0.1% TFA) from 35% to 40% in 45 min at a flow rate of 1 ml/min. Peaks were detected by their absorption at 224 nm, manually collected and freeze-dried. Four enriched fractions of as2-CNs were obtained. Two-dimensional electrophoresis
J. Mass Spectrom. 2012, 47, 1150–1159
In-gel digestion of the protein spots was carried out on selected gel pieces manually excised from the Coomassie blue-stained two-dimensional electrophoresis (2-DE) gels. The excised gel pieces were transferred to 1.5 ml microcentrifuge tubes, washed three times with H2O : CH3CN (1 : 1 v/v) at 37 C and subjected to in-gel digestion using modified porcine trypsin or a-chymotrypsin from bovine pancreas or endoproteinase Glu-C, according to the method of Shevchenko et al.[17] Trypsin and a-chymotrypsin digestions were allowed to proceed overnight at 37 C, whereas endoproteinase Glu-C reaction was carried out for 48 h at 37 C. All the enzymatic digestions were stopped by cooling the gel pieces and supernatant solution at 24 C. After in-gel digestion, the digested solution was transferred into a clean 0.5-ml tube. The peptides were extracted from gel pieces with 0.1% TFA solution and subsequently with CH3CN. This extraction procedure was repeated three times. The total extracts were pooled, joined with the first supernatant, lyophilized and redissolved in 20 ml of 0.1% TFA. Matrix-assisted laser desorption/ionization time-of-flight MS analysis Matrix-assisted laser desorption/ionization (MALDI) mass spectra were acquired using a Voyager DE-PRO time-of-flight (TOF) mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with a UV nitrogen laser (337 nm). For MALDI-TOF analysis of the intact proteins, samples of the chromatographic fractions isolated from the RP-HPLC run were prepared according to the dried droplet method[18] using SA (dissolved in CH3CN : 0.1% TFA, 40 : 60 v/v at a concentration of 10 mg/ml) as matrix. Spectra were obtained in linear positive ion mode over an m/z range 15 000–30 000 and were averaged from about 150 laser shots to improve the signal-to-noise (S/N) ratio. Mass calibration was made using a mixture (1 : 1 : 5 v/v/v) of cytochrome c (m/z 12 232), myoglobin (m/z 16 952) and trypsinogen (m/z 23 982) as external standards. The accuracy obtained by MALDI-TOF measurement of the molecular masses of the proteins was 0.02%. The mixtures of enzymatic peptides were subjected to a desalting/concentration step[19] prior to analysis by MALDI-TOF MS, using a homemade 5-mm nanocolumn packed with C18 resin (POROS R2; PerSeptive Biosystems, Foster City, CA, USA) in a constricted GELoader tip (Eppendorf Scientific, Westbury, NY, USA). The bound peptides were eluted directly onto the MALDI target with 0.6 ml of matrix solution [10 mg/ml CHCA in 70% (v/v) CH3CN and 0.1% (v/v) TFA]. Spectra were obtained in reflectron positive ion mode over an m/z range 500–3000 and were averaged from about 150 laser shots to improve the S/N ratio. The MoverZ software (Proteometrics Ltd, New York, NY, USA) was used to analyze the MALDI-TOF spectra, which were internally calibrated using porcine trypsin autolysis products (m/z 842.51 and 2211.10). When trypsin autolysis peaks could not be detected, bovine b-lactoglobulin tryptic peptides (m/z 837.48, 2313.26 and 2707.38) were used for external mass calibration. Capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry analysis Capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analysis of the in-gel enzymatic digests
Copyright © 2012 John Wiley & Sons, Ltd.
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The four enriched as2-CNs fractions, obtained from four HPLC runs as described earlier, were solubilized in 25 mM Tris–HCl, pH 9.0, containing 2% CHAPS, 6 M urea, 2 M thiourea, 0.5% IPG buffer (pH 4–7; GE Healthcare, Milan, Italy) and traces of bromophenol blue (BPB), to a protein concentration of approximately 0.6 mg/ml. The solubilized proteins were reduced with 43 mM DTT for 2 h and then alkylated for 1 h with 60 mM acrylamide. The pH 4–7 linear IPG strips (13 cm; GE Healthcare, Milan, Italy) to be used for the first dimension separation were rehydrated overnight in 250 ml of the solubilized protein samples (approximately 150 mg), reduced and alkylated as described earlier. Focusing was performed with a Multiphor apparatus (GE Healthcare, Milan, Italy) at 20 C with the following program: 1 min at 200 V followed by a linear voltage gradient that went to 3500 V in 90 min and by 3 h of constant voltage (3500 V). The strips were equilibrated for the second dimension separation under continuous agitation in 50 mM Tris–HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS and traces of BPB (equilibration buffer) added with 25 mM DTT. After 20 min, the strips were alkylated in equilibration buffer containing 360 mM acrylamide from 20 min under continuous agitation and applied on 12% SDS–PAGE gels (14 16 cm). The second dimension separation was performed at 25 mA/gel to allow for the tracking dye to exit the IPG strip and penetrate the resolving gel and then at 40 mA/gel until the tracking dye reached the gel bottom. The gels were stained with CBB R-350 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) in methanol : H2O : acetic acid (3 : 6 : 1). The gel images were acquired using an Epson 1680 Pro scanner equipped with a transparency adapter. After image acquisition, the slab gels were stored in 7% acetic acid at 4 C until used for spot excision.
In-gel digestion of protein spots
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% Intensity % Intensity
80
a
b
% Intensity
100
c
% Intensity
was performed using an Ultimate 3000 LC system combined with an autosampler and a flow splitter 1 : 100 (Dionex Corporation, Sunnyvale, CA, USA), coupled online with a linear ion trap nano-electrospray mass spectrometer (LTQ, Thermo Fischer Scientific, San Jose, CA). Ionization was performed with liquid junction using an uncoated capillary probe (30 mm i.d., New Objective, Woburn, MA, USA). Ten microliters of enzymatic digest solution for each spot was loaded onto a m precolumn C18 (0.3 5 mm, 100 , 5 mm, PepMap, Dionex) equilibrated with 0.5% FA aqueous solution at a flow rate of 20 ml/min for 4 min. Then, the solution was switched onto a reversed-phase C18 column (0.18 150 mm, 300 Å, 5 mm, BioBasic, ThermoFisher Scientific), and peptides were separated by elution at room temperature with a linear gradient of solvent B (CH3CN + 0.5% FA) in A (H2O + 0.5% FA) from 20% to 55% in 40 min at a flow rate of 1.5 ml/min. The nESI source operated under the following conditions: source temperature 220 C, source voltage 1.9 kV and capillary voltage 42 V. Repetitive mass spectra were acquired in positive ion mode in the m/z range 350–2000. Characterization of peptide ions was performed by the datadependent method as follows: (i) full-scan MS in the m/z range 350–2000; (ii) zoom scan of the five most intense ions (isolation width: 2); and (iii) MS/MS analysis of the five most intense ions (normalized collision energy: 30 a.u., activation Q: 0.250). Mass calibration was made using a standard mixture of caffeine (Mr 194.1 Da), MRFA peptide (Mr 423.6 Da) and Ultramark (Mr 1621 Da). Data acquisition and data analysis were performed using the Excalibur v. 1.4 and Bioworks Browser v. 3.2 software (ThermoFisher Scientific), respectively. The General Protein/Mass Analysis for Windows (GPMAW) software (http:// welcome.to/gpmaw) was used for all sequence handling and storage. De novo amino acid sequencing of the peptides investigated was also performed by manual interpretation of the MS/MS spectra assisted by the use of the AminoCalc v.1.13 software (Protana, Denmark).
d
Results and discussion Characterization of the primary structure of the as2-CNs Figure 1 shows the RP-HPLC chromatogram of the dephosphorylated CN mixture in which the peaks named F1, F4 and F5 correspond, respectively, to as1-CN isoforms (A, A1, B and B1),[14] as2-CN[12] and b-CN isoforms (A and AΔ5),[13] previously characterized. The MALDI-TOF MS analysis of the collected F2, F20 , F3 and F30 fractions allowed to detect four unknown components, having an experimentally measured Mr of 25 429, 21 939, 25 203 and 21 713 (0.02%) Da, respectively (Fig. 1, inset a–d). Comparing the experimental masses, a difference of 226 Da between the components with Mr of 25 429 and 25 203 Da and those with Mr of 21 939 and 21 713 Da was observed, whereas the two proteins having an Mr of 25 429 and 21 939 Da and those with Mr of 25 203 and 21 713 Da differ with each other for 3490 Da. To identify these unknown proteins, the corresponding chromatographic fractions were manually collected, digested with trypsin and the resulting peptide mixtures analyzed by MALDI-TOF MS (see supplementary material). Most of the signals were identical in the four mass spectra and could be interpreted as arising from the theoretical cleavages of the as2-CN of donkey (GenBank Acc. No. CAV00691; 221 amino acids and Mr 26 028 Da) (Table 1). This finding clearly indicated that the proteins present in all these fractions are four different donkey’s as2-CN isoforms. The mass spectra of the fractions F2, F20 , F3 and F30 (Fig. 1, inset a–d) also showed that the fractions are cross-contaminated. To improve the separation of the four proteins, each chromatographic fraction was further resolved by 2-DE. The 2-DE map of each fraction revealed the presence of a main protein spot in each gel, labeled as spot F2A, F20 A, F3A and F30 A, respectively, showing an apparent molecular mass of 25 kDa (F2A and F3A) and 21 kDa (F20 A and F30 A) and pI 5.6 (data not reported). As an example, the 2D-PAGE separation of the chromatographic fraction F30 A is
25430
F5
21940
25430
ABS λ=224 nm (%)
F3
60
25204
F1
F3’
21714
40
0 15000
F2
25204
F2’ 18000
21000
24000
27000
30000
(m/z)
20 F4
0 0
5
10
15
20
25
30
35
40
45
time (min)
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Figure 1. UV profile (l = 224 nm) of the RP-HPLC run of the dephosphorylated casein fraction obtained from milk of an individual donkey belonging to the Ragusano breed. Insets a–d show the MALDI-TOF mass spectra of the chromatographic fraction F2 (a), F20 (b), F3 (c) and F30 (d). Inset e shows the 2D-PAGE of the chromatographic fraction F30 . The spot labeled as F30 A corresponds to the isoform having Mr 21 713 Da.
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2012, 47, 1150–1159
J. Mass Spectrom. 2012, 47, 1150–1159
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
1–1 2–7 8–20 21–22 23–25 26–33 34–46 47–55 56–72 47–72 47–72 73–77 47–77 47–77 56–77 78–80 56–80 81–85 73–85 86–89 73–89 90–93 94–100 101–111 94–111 112–114 112–124 115–124 125–147 148–148 125–148 149–161 149–161 148–161 148–161 162–169 148–169 149–169 149–169 170–173
T1 T2 T3 T4 T5 T6 T7 T8 T9 T(8+9) T(8+9)*Met Ox T10 T(8+9+10) T(8+9+10)*Met Ox T(9+10) T11 T(9+10+11) T12 T(10+11+12) T13 T(10+11+12+13) T14 T15 T16 T(15+16) T17 T(16+17) T18 T19 T20 T(19+20) T21 T21*Met Ox T(20+21) T(20+21)*Met Ox T22 T(20+21+22)*Met Ox T(21+22) T(21+22)*Met Ox T23
147.1 823.4 1409.6 294.2 404.2 906.5 1557.6 1035.5 1821.8 2838.3 2854.3 649.3 3468.6 3484.6 2452.1 405.2 2838.3 633.3 1649.8 534.3 2165.1 502.3 941.5 1378.8 2301.2 400.2 1760.0 1200.7 2518.3 147.1 2646.4 1467.7 1483.7 1595.8 1611.8 978.5 2571.2 2427.1 2443.1 537.3
Position Calculated MH+
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Fragment — — — — — — — — 1821.0 (2); 1821.7a 2838.5 (2); 2838.2a 2854.3 (2) — 3468.4 (3) 3485.0 (3) 2452.1 (3); 2452.0a — 2839.0 (3); 2838.2a — 1649.2 (2) — 2164.9 (3) — — 1378.5 (2); 1378.7a 2300.6 (2) — 1759.4 (2) 1200.4 (2); 1200.7a 2518.6 (2); 2518.1a — 2646.7 (2) 1467.0 (2) 1483.0 (2) 1595.0 (2) 1611.0 (2) — 2572.0 (3) — — —
— — 1409.5 (2) — — 906.6 (1) 1557.1 (2) 1035.2 (1) 1821.2 (2) 2837.6 (3) 2854.7 (3) — — — 2451.6 (3) — 2838.0 (3) — 1650.4 (2) — — — — 1378.6 (2); 1378.7a — 1759.9a 1200.2 (2) 2518.3 (2) — 2646.5 (2) ; 2646.4a 1467.1 (2) 1483. 2 (2) 1595.2 (2) 1611.2 (2) — — 2426.7 (3) 2442.7 (3) —
F20 A Measured MH+ by ESI-MS/MS (z); MALDI-MSa
F2A Measured MH+ by ESI-MS/MS (z); MALDI-MSa — — 1409.5 (2) — — — 1557.5 (2) — 1821.5 (2) 2838.6 (2) 2854.5 (2) — — — 2452.4 (3) — 2838.3 — 1649.2 (2) — — — 941.4 (1) 1378.4 (2) ; 1378.7a — — 1759.9a 1200.5 (2) 2518.7 (2) — 2647.0 (2) 1467.4 (2) 1483.3 (2) 1595.2 (2) 1611.3 (2) — — — — —
F3A Measured MH+ by ESI-MS/MS (z); MALDI-MSa — — — — — — — — 1821.4 (3) 2838.0 (3) 2854.3 (3) — — 3485.7 (3) 2451.6 (2) — 2838.6 (3) — — — — — 941.6 (1) 1378.6 (2) ; 1378.6a — — 1759.9a 1200.3 (2) 2518.7 (2) — 2646.6 (3) 1467.2 (2) 1483.8 (2) 1595.2 (2) 1611.3 (3) — — — — —
F30 A Measured MH+ by ESI-MS/MS (z); MALDI-MSa
QPR LNFLQYLQALRQPR IVLTPWDQTK TGASPFIPIVNTEQLFTSEEIPK K TGASPFIPIVNTEQLFTSEEIPKK TVDMESTEVVTEK TVDMESTEVVTEK KTVDMESTEVVTEK KTVDMESTEVVTEK TELTEEEK KTVDMESTEVVTEKKTELTEEEK TVDMESTEVVTEKTELTEEEK TVDMESTEVVTEKTELTEEEK NYLK (Continues)
K HNMEHR SSSEDSVNISQEK FK QEK YVVIPTSK ESICSTSCEEATR NINEMESAK FPTEVYSSSSSSEESAK NINEMESAKFPTEVYSSSSSSEESAK NINEMESAKFPTEVYSSSSSSEESAK FPTER NINEMESAKFPTEVYSSSSSSEESAKFPTER NINEMESAKFPTEVYSSSSSSEESAKFPTER FPTEVYSSSSSSEESAKFPTER EEK FPTEVYSSSSSSEESAKFPTEREEK EVEEK FPTEREEKEVEEK HHLK FPTEREEKEVEEKHHLK QLNK INQFYEK LNFLQYLQALR
Donkey’s as2-CN reference sequence (GenBank Acc. No. CAV00691)
Table 1. Fragments, position, calculated monoisotopic and experimentally measured MH+ of tryptic fragments of donkey’s as2-CNs present in spots F2A, F20 A, F3 and F30 A
Characterization of as2-caseins in donkey’s milk
KTVDMESTEVVTEKTELTEEEKNYLK TELTEEEKNYLK TVDMESTEVVTEKTELTEEEKNYLK TVDMESTEVVTEKTELTEEEKNYLK LLNK INQYYEK FTLPQYFK IVHQHQTTMDPQSHSK TNSYQIIPVLR YF 3073.3 (3) 1469.3 (2) 2946.4 (3) 2961.8 (3) — — 1043.3 (2); 1043.5a — — — — 1496.4 (2) — — — — 1043.4 (2); 1043.5a — — — 3074.2 (3) 1496.4 (2) 2945.4 (3) 2961.2 (3) — — 1043.4 (2); 1043.5a 1873.7a 1303.4 (2); 1303.7a — Ox
3073.5 1496.7 2945.4 2961.4 487.3 957.5 1043.5 1873.9 1303.7 329.2 148–173 162–173 149–173 149–173 174–177 178–184 185–192 193–208 209–219 220–221 T(20+21+22+23) T(22+23) T(21+22+23) T(21+22+23)*Met T24 T25 T26 T27 T28 T29
3073.4 (4) 1496.1 (2) 2944.8 (3) — — — 1043.1 (2) — 1303.4 (2) —
F30 A Measured MH+ by ESI-MS/MS (z); MALDI-MSa F3A Measured MH+ by ESI-MS/MS (z); MALDI-MSa Position Calculated MH+ Fragment
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Table 1. (Continued)
F2A Measured MH+ by ESI-MS/MS (z); MALDI-MSa
F20 A Measured MH+ by ESI-MS/MS (z); MALDI-MSa
Donkey’s as2-CN reference sequence (GenBank Acc. No. CAV00691)
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reported in Fig. 1(inset e). These main spots were manually excised and subjected to in-gel digestion with modified porcine trypsin, a-chymotrypsin from bovine pancreas and endoproteinase Glu-C. The enzymatic mixtures were then analyzed by capillary RPHPLC/nESI-MS/MS and the results interpreted using the known donkey’s as2-CN as reference. Characterization of the primary structure of the protein with Mr 25 429 (spot F2A) The capillary RP-HPLC/nESI-MS/MS analysis of the tryptic digest of the spot F2A enabled the identification of the sequence of most of the fragments of the as2-CN of donkey already detected by MALDI-TOF MS and also the characterization of peptides previously undetected (see Table 1). To obtain complementary information about the primary structure, the spot F2A was also subjected to a-chymotrypsin and endoproteinase Glu-C digestion. Selected fragments identified in the chymotrypsin and Glu-C digestion mixtures used to complete the sequence coverage are reported in Table 2(I). In particular, the MS/MS spectrum of the doubly charged molecular ion at m/z 927.2 (MH+ 1853.4) in the chymotryptic digest (Fig. 2a) allowed to deduce the sequence KLLYYEKFTLPQYF, which corresponds to the theoretical fragment 173 KLLNKINQYYEKFTLPQYF191 of the reference donkey’s as2-CN without the pentapeptide N176KINQ180. The deletion of this peptide was confirmed by the characterization of the three fragments KNYLKLLYYE (MH+ 1346.8), EKNYLKLLYYE (MH+ 1475.0) and KTELTEEEKNYLKLLYYE (MH+ 2306.2), present in the Glu-C digested mixture, as shown in Table 2(I). In summary, the results obtained revealed that the donkey’s as2-CN corresponding to the spot F2A presents 216 amino acids, and it is the isoform with Mr 25 429 Da. This isoform differs from the known donkey’s as2-CN (Mr 26 028 Da) for the absence of the pentapeptide N176KINQ180 that accounts, within the experimental errors, for the decrease of 599 Da in its Mr (Table 3 and Fig. 3). Characterization of the primary structure of the protein with Mr 21 939 (spot F20 A) The sequence of most of the fragments already detected by MALDI-TOF MS was confirmed by capillary RP-HPLC/nESI-MS/MS analysis of the tryptic digest of the spot F20 A, and, in addition, tryptic peptides previously undetected were identified and sequenced (Table 1). It was also possible to identify uncharacterized traits of the chain by complementary chymotryptic cleavage, as summarized in Table 2(II). Particularly, interpretation of the MS/MS spectrum of the triply charged molecular ion at m/z 1185.2 (MH+ 3553.6) allowed the characterization of the sequence KHNMEHRSSSEEATRNINEMESAKFPTEVY, which corresponds to the N-terminal region Lys1–Tyr61 of the reference donkey’s as2-CN without the segment 12Asp–Glu42 (Fig. 2b). In addition, the characterization of the sequence KLLYYEKFTLPQYF allowed to deduce that also in the CN isoform present in the spot F20 A, the N176KINQ180 pentapeptide is absent. Selected fragments identified in the chymotrypsin digestion mixture used to complete the sequence coverage are reported in Table 2(II). These findings showed that this protein consists of 185 amino acids. The absence of the segment Asp12–Glu42 together with the absence of the pentapeptide Asn176–Gln180 is in agreement with the experimentally measured Mr of 21 939 Da and explains the difference of 3490 Da between the molecular mass of this isoform and that corresponding to the spot F2A (Table 3 and Fig. 3).
Copyright © 2012 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2012, 47, 1150–1159
Characterization of as2-caseins in donkey’s milk Table 2. Position, calculated monoisotopic MH+ and ESI-MS measured MH+ of selected fragments identified in the chymotryptic (A) and Glu-C (B) digestion mixtures of donkey’s as2-CN present in the spot F2A (I), F20 A (II), F3A (III) and F30 A (IV) Position I A 1–26 92–110 173–186 192–221 B 85–99 161–178 168–178 169–178 II A 1–30 62–92 92 –107 173–186 207–221 III A 22–61 29–61 30–61 62–88 173–185 173–191 176–191 192–214 B 1–19 184–202 IV A 1–30 62–88 62–91 92–106 176–191 192–214
Donkey’s as2-CN reference sequence (GenBank Acc. No. CAV00691)
Calculated MH+
Measured MH+
3181.5 2387.3 1853.0 3612.8
3181.3 (3) 2386.4 (2) 1853.4 (2) 3613.4 (4)
KHNM*EHRSSSEDSVNISQEKFKQEKY NKINQFYEKLNFLQYLQAL KLLYYEKFTLPQYFa KIVHQHQTTM*DPQSHSKTNSYQIIPVLRYF
1940.0 2306.2 1475.8 1346.7
1939.2 (2) 2306.2 (3) 1475.0 (2) 1346.8 (2)
KHHLKQLNKINQFYE KTELTEEEKNYLKLLYYEa EKNYLKLLYYEa KNYLKLLYYEa
3551.6 3586.7 2075.1 1853.0 1829.0
3553.6 (3) 3587.0 (3) 2075.5 (2) 1853.4 (2) 1829.5 (2)
KHNMEHRSSSEEATRNINEMESAKFPTEVYb SSSSSSEESAKFPTEREEKEVEEKHHLKQLN NKINQFYEKLNFLQYL KLLYYEKFTLPQYFa SKTNSYQIIPVLRYF
4711.3 3852.8 3723.7 3103.4 1701.0 2450.3 2096.0 2786.3
4712.3 (4) 3853.4 (3) 3723.0 (3) 3103.3 (3) 1700.5 (2) 2450.7 (2) 2095.5 (2) 2786.5 (3)
KQEKYVVIPTSKESICSTSCEEATRNINEM ESAKFPTEVY IPTSKESICSTSCEEATRNINEM*ESAKFPTEVY PTSKESICSTSCEEATRNINEMESAKFPTEVY SSSSSSEESAKFPTEREEKEVEEKHHL KLLNKINQYYEKF KLLNKINQYYEKFTLPQYF NKINQYYEKFTLPQYF KIVHQHQTTM*DPQSHSKTNSRYFc
2214.0 2362.2
2213.3 (2) 2362.3 (2)
KHNMEHRSSSEDSVNISQE KFTLPQYFKIVHQHQTTMD
3567.6 3103.4 3472.7 1962.0 2096.0 2786.3
3568.6 (3) 3103.4 (3) 3473.7 (3) 1961.4 (2) 2096.4 (2) 2787.1 (3)
KHNM*EHRSSSEEATRNINEMESAKFPTEVYb SSSSSSEESAKFPTEREEKEVEEKHHL SSSSSSEESAKFPTEREEKEVEEKHHLKQL NKINQFYEKLNFLQY NKINQYYEKFTLPQYF KIVHQHQTTM*DPQSHSKTNSRYFc
Sequences not previously covered by tryptic cleavage are reported in bold. Numbers in brackets indicate the charge state of the parent ions selected for peptide sequence determination by MS/MS experiments. Asterisk indicates that the methionine residue is present as methionine sulfoxide. a Fragment in which the pentapeptide N176KINQ180 of the donkey’s reference as2-CN is absent. b Fragment in which the sequence D12SVNISQEKFKQEKYVVIPTSKESICSTSCE42 of the donkey’s reference as2-CN is absent. c Fragment in which the heptapeptide Y212QIIPVL218 of the donkey’s reference as2-CN is absent.
Characterization of the primary structure of the protein with Mr 25 203 (spot F3A)
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Capillary RP-HPLC/nESI-MS/MS analysis of the tryptic digestion mixture of the protein present in the spot confirmed the sequence of most of the fragments of the as2-CN of donkey already detected by MALDI-TOF MS and also resulted in the identification of others fragments previously undetected (see Table 1), although the sequence coverage of the entire protein still remained incomplete. To obtain additional data, spot F3A was digested with chymotrypsin and endoproteinase Glu-C and the resulting peptide mixture was analyzed by capillary RP-HPLC/nESI-MS/MS. Selected
fragments identified in the peptide hydrolysates used to complete the sequence coverage are reported in Table 2(III). In particular, the MS/MS spectrum of the triply charged molecular ion at m/z 929.5 (MH+ 2786.5) (Fig. 2c) allowed to deduce the sequence KIVHQHQTTMoxDPQSHSKTNSRYF, corresponding to the Lys192-Phe221 trait of the reference donkey’s as2-CN without the heptapeptide Y212QIIPVL218. In conclusion, these data reveal that this isoform differs from the known donkey’s as2-CN (Mr 26 028 Da) for the absence of the heptapeptide Y212QIIPVL218 that accounts, within the experimental errors, for the decrease of 825 Da in its Mr (Table 3 and Fig. 3).
R. Saletti et al. b ions 100
Relative Abundance
5
y ions
13 12 11 10 9
10 11
9
12
b10
8
5
4
1299.4
3
70 60
b6
50 40
20 10
762.7
844.2 909.3
b11 y9 1396.4 y8 1172. b9 1043.3 y12 1186.3 y10 y11 b8 1611.5 y13 1335.2 1085.3 1499.4 1724.7
681.2
600
1000 m/z
800 4
b ions
y ions
b132+
667.1b 5
0 400
b122+
y5
y3 457. b4 1 518.1
5
a
b12 1524.5
810.2
y4 554.0
30
Relative Abundance
8
6
KLLYYEKFTLPQYF
90 80
4
1400
1200
1600
1800
21
13
7
23 24 25 26
28 29
KHNMEHRSSSEEATRNI NEMESAKFPTEVY 26
29 28 27
7
100
b293+
90
1124.9
6
5
4
80 70
y293+ 1142.5
60 50
30
y6 b4/ y4 608.06 b 5 755.1 b233+ 511.9 640.1 890.7
b13 1644.0 1523.3 y272+ 1586.7
b232+ b21 1256.1
b253+ 982.9
y26/b262+ 1521.4
0
400
600
800 4
b ions
1000
1200 m/z
1400 11 12
6
5
b y282+
2+1335.3
y7 883.3
y5
20 10
b242+ 1399.3
y7/b243+ b283+ 933.5 1091.5
40
b252+ 1472.7
y292+ 1712.2
1800
1600
14
K I V H Q H Q T T Mox D P Q S HS K T N S R Y F 100
y ions
22 21
20
18
13 12
15
10
908.3 y152+
90
Relative Abundance
80 y122+
70
726.1
60
1335.3 2+
40
y222+
30
886.6 y202+ b5 606.3 b6 816.0 742. 9 3+ y212+ b4/b123+ b14 b112+ 848.9 549.1 668.1 478.5
20 10
c
b11
50
b20 1223.7 y10 1226.6 b222+ 1329.6 y182+ 1091.0
0 400
600
800
1000
m/z
1200
y12 y13 1451.6 1566..5 1400
1600
1800
2000
Figure 2. ESI-MS/MS spectrum of the (a) doubly charged molecular ion at m/z 927.2 (MH+ 1853.4) present in the chymotryptic digest of the spot F2A, demonstrating that the peptide N176KINQ180, present in the sequence of the parent as2-CN, is missed; (b) triply charged molecular ion at m/z 1185.2 (MH+ 3553.6) present in the chymotryptic digest of the spot F20 A, demonstrating that the peptide D12SVNISQEKFKQEKYVVIPTSKESICSTSCE42, present in the sequence of the parent as2-CN, is missed; (c) triply charged molecular ion at m/z 929.5 (MH+ 2786.5) present in the chymotryptic digest of the spot F3A, demonstrating that the peptide Y212QIIPVL218, present in the sequence of the parent as2-CN, is missed. The sequences of the peptides are displayed with the fragment ions observed in the spectrum.
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J. Mass Spectrom. 2012, 47, 1150–1159
Characterization of as2-caseins in donkey’s milk Primary structure comparison of the donkey’s and cow’s as2-CNs
Table 3. Proposed nomenclature for the full-length and the internally deleted donkey’s as2-CNs Nomenclature of the isoforma A A1 AΔ4,5,6 1 A2 AΔ4,5,6 2
Calculated Mr
Amino acid differences
26 028 25 429 21 939 25 203 21 713
— –Asn176–Gln180 –Asp12–Glu42; –Asn176–Gln180 –Tyr212–Leu218 –Asp12–Glu42; –Tyr212–Leu218
As reported in the Introduction, cow’s milk is one of the most common causes of food allergy in children. It was demonstrated that whole CN fraction acts as a potent allergen and that each of the different CN types (as1-, as2-, b- and k-) can induce a marked IgE response. CNs show a reduced rigidity of the tertiary structure,[20] suggesting the presence of preferentially linear epitopes that have been characterized.[21] With respect to the bovine as2-CN, four major (AA 83–100, AA 143–158, AA 157–172 and AA 165–188) and six minor (AA 31–44, AA 43–56, AA 93–106, AA 105–114, AA 117–128 and AA 191–200) sequential IgE-binding regions were identified.[22] In addition, the trait 171–180 was exclusively recognized by patients with persistent cow’s milk allergy.[23] The knowledge of the primary structure of as2-CN from donkey and the determination of its internally deleted isoforms here reported provide the basis for some evaluation of their sequences. Comparison of the bovine and the full-length donkey’s as2-CNs reveals that these two proteins share a low sequence homology (57% of identity and 70% of similarity). In particular, the IgE-binding linear epitopes of cow’s as2-CN and the corresponding domains present in donkey’s as2-CN show remarkable differences in their amino acid sequences. Moreover, it is interesting to note that the amino acid sequences absent in the internally deleted as2-CN isoforms from donkey here characterized are traits of some IgE-binding epitopes of bovine as2-CN. Particularly, the NKINQ sequence absent in the as2-CN isoforms A1 and AΔ4,5,6 from donkey 1 is a trait of two major IgE-binding epitopes of bovine as2-CN. Consequently, the considerable differences between the primary structure of donkey and bovine as2-CNs could be related to the already demonstrated low allergenic properties of donkey’s milk. Although these considerations seem reasonable, of course, further biochemical studies and clinical experiences are needed to establish precise relationship between donkey’s milk protein structures and its low allergenic properties.
Amino acid differences with respect to the full-length donkey’s as2-CN (GenBank Acc. No. CAV00691) are reported in the third column. a ΔX = deletion of the region encoded by exon X.
Characterization of the primary structure of the protein with Mr 21 713 (spot F30 A) The sequence coverage of the protein corresponding to the spot F30 A, achieved by coupling tryptic digestion, MALDI-TOF MS and capillary RP-HPLC/nESI-MS/MS analysis, is reported in Table 1. To complete the sequence characterization of this isoform, spot F30 A was digested with chymotrypsin, and the resulting peptide mixture was analyzed by capillary RP-HPLC/nESI-MS/MS. The analysis of the chymotryptic digest confirmed most of the results already obtained by the analysis of the tryptic mixture. In addition, it was possible to complete the characterization of the sequence by the identification of the N- and C-terminal regions of the protein as reported in Table 2 (IV). Specifically, the identification of the peptide KHNMoxEHRSSSEEATRNINEMESAKFPTEVY, common to the component with Mr 21 939 Da, and the peptide KIVHQHQTTMoxDPQSHSKTNSRYF, common to the component with Mr 25 203, allowed to conclude that the sequences Tyr212–Leu218 and Asp12–Glu42 of the reference as2-CN are absent in the CN isoform with a molecular mass of 21 713 Da (Table 3 and Fig. 3).
A A A A A
A A A A A
Ex2
Ex3
Ex4
Ex5
Ex6
KH -----
NMEHRSSSE ---------------------------------
DSVNISQE --------
KFKQEKYVVIPTSK --------------
ESICSTSCE ---------
--------
--------------
---------
Ex7
Ex8
Ex9
Ex10
EATRNINEM ---------------------------------
ESAKFPTE -----------------------------
VYSSSSSSE ---------------------------------
ESAKFPTE -----------------------------
Ex11
REEKEVEEKHHLKQ -----------------------------------------------------
Ex12 A A A A A
A A A A A
A A A A A
Ex13
LNKINQFYEKLNFLQYLQALRQPRIVLTPWDQTKTGASPFIPIV ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ex14
Ex15
Ex16
EIPKKTVDM ---------------------------------
ESTEVVTE -----------------------------
KTELTEEEKNYLKLL ---------------------------------------------------------
NTEQLFTSE ---------------------------------
Ex17
Ex18
NKINQYYEKFTLPQYFKIVHQHQTTMDPQSHSKTNSYQIIPVL -------------------------------------------------------------------------------------------------------------------------------------------------
RYF ---------
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Figure 3. Exon modular structure of the primary structure of the as2-CN variant A, sequences and proposed nomenclatures for the four internally deleted donkey’s as2-CN isoforms. The exon nomenclature is that used by Cosenza et al.[24]
R. Saletti et al.
Conclusions
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In this study, four proteins with experimentally measured Mr 25 429, 21 939, 25 203 and 21 713 (0.02%) Da were detected by RP-HPLC analysis and MALDI-TOF MS as minor components of the dephosphorylated CN fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed. By coupling RP-HPLC, 2D-PAGE, enzymatic digestions, MALDI-TOF MS and capillary RP-HPLC/nESI-MS/MS, the unknown components were identified as internally deleted donkey’s as2-CN isoforms and their primary structure totally characterized using the amino acid sequence of the known full-length donkey’s as2-CN as reference (GenBank Acc. No. CAV00691). In detail, the protein with Mr 25 429 Da differs from the full-length as2-CN for the deletion of the Asn176–Gln180 pentapeptide, whereas in the protein with Mr 21 939 Da, in addition to the same peptide, the sequence Asp12–Glu42 is also suppressed (Fig. 3 and Table 3). In the third isoform (Mr 25 203 Da), the Tyr212–Leu218 heptapeptide is absent in comparison with the full-length as2-CN, whereas in the primary structure of the component with Mr 21 713 Da, both the heptapeptide Tyr212–Leu218 and the sequence Asp12–Glu42 are missed. Taking into account the exon modular structure of the donkey’s as2-CN[24] (Fig. 3), it can be observed that (i) the sequence Asn176–Gln180, absent in the isoforms with Mr 25 429 and 21 939 Da, represents the first five amino acids encoded by the exon 17; (ii) the sequence Asp12–Glu42, absent in the primary structure of the isoforms with Mr 21 939 and 21 713 Da, is encoded by exons 4, 5 and 6; (iii) the heptapeptide Tyr212–Leu218, absent in the primary structure of the isoforms with Mr 25 203 and 21 713 Da, is the terminal part of the sequence encoded by the exon 17. As evidenced by our data, it can be hypothesized that exons 4, 5 and 6 are completely skipped in some events. On the other hand, cryptic 50 and 30 splicing sites inside exon 17 may determine the absence of the starting five amino acids or the final seven amino acids encoded by this exon in the final products. In light of these considerations, the full-length donkey’s as2-CN and the four internally deleted isoforms can be named as reported in Table 3. The existence in an individual milk sample of a full-length as2-CN (221 amino acids; Mr 26 028)[12] and its four different isoforms, resulting from differential splicing events, is analogous to what was already noted for the as1-type CNs.[14] This polymorphism seems to be correlated with the complex intron/exon modular structure of the genes encondig these proteins, consisting of a large number of short exons, that may undergone differential splicing events during primary transcript processing, thus originating isoforms with different amino acid lengths.[25] Comparative analysis of the primary structure of the as2-CN from donkey and other mammalians reveals that the differential splicing events involving the exons 4, 5, 6 and the last seven amino acids encoded by exon 17 seem unique of donkey species, whereas the first five amino acids (N176KINQ180) encoded by exon 17 are also constitutively spliced in the as2-CN from mare (NCBI Acc. No. NP_001164238).[26] Moreover, it is interesting to note that the amino acid sequences absent in the internally deleted as2-CN isoforms from donkey here characterized are traits of some IgE-binding epitopes of bovine as2-CN. In particular, the NKINQ sequence absent in the isoforms A1 and AΔ4,5,6 from 1 donkey and constitutively spliced in the as2-CN from mare is a trait of two major IgE-binding epitopes of bovine as2-CN and therefore could be related to the already demonstrated low allergenic properties of donkey’s milk. Although these considerations seem
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reasonable, of course, further biochemical studies and clinical experiences are needed to establish precise relationship between donkey’s milk protein structures and its low allergenic properties. The protein sequence data reported in this article will appear in the UniProt Knowledgebase under the accession number B7VGF9 (direct submission, 29 February 2012). Finally, the results here reported show the usefulness of the MS-based approached in the characterization of internally deleted protein isoforms, resulting from differential splicing events, which cannot be predicted at the genomic level. Acknowledgements This work was supported by grants from MURST (PRIN 2008, project number 20087ATS57) and from FIRB Italian Human ProteomeNet RBRN07BMCT. We are grateful to Dr D. Franchina and Dr K. Torrisi (ASILAT srl farm, Milo, Catania) for the gift of the individual Ragusano donkey’s milk coming from Eastern Sicily. Supporting information Supporting information may be found in the online version of this article.
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[20] P. F. Fox. Milk proteins as food ingredients. Int. J. Dairy Technol. 2001, 54, 41. [21] L. Monaci, V. Tregoat, A. J. van Hengel, E. Anklam. Milk allergens, their characteristics and their detection in food: a review. Eur. Food Res. Technol. 2006, 223, 149. [22] P. J. Busse, K. N. Jarvinen, L. Vila, K. Beyer, H. A. Sampson. Identification of sequential IgE-binding epitopes on bovine alpha(s2)-casein in cow’s milk allergic patients. Int. Arch. Allergy Immunol. 2002, 129, 93. [23] K. N. Jarvinen, K. Beyer, L. Vila, P. Chatchatee, P. J. Busse, H. A. Sampson. B-cell epitopes as a screening instrument for persistent cow’s milk allergy. J. Allergy Clin. Immunol. 2002, 110, 293. [24] G. Cosenza, A. Pauciullo, A. L. Annunziata, A. Rando, L. Chianese, D. Marletta, G. Iannolino, D. Nicodemo, D. Di Berardino, L. Ramunno. Identification and characterization of the donkey CSN1S2 I and II cDNAs. Ital. J. Anim. Sci. 2010, 9, 206. [25] T. Lenasi, I. Rogelj, P. Dovc. Characterization of equine cDNA sequences for as1-, b- and k-casein. J. Dairy Res. 2003, 70, 29. [26] P. D. Martin, L. Miclo, E. Rebours, A. Mateos, G. Miranda. Equus caballus mRNA encoding CSN1S2 (alphaS2-casein) precursor. Direct Submission in EMBL/GenBank/DDBJ Databases, December 2009.
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