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Quantitative analysis of bovine β-casein hydrolysates obtained using glutamyl endopeptidase
LWT - Food Science and Technology xxx (2015) 1e5
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Quantitative analysis of bovine b-casein hydrolysates obtained using glutamyl endopeptidase Yi-shen Zhu a, b, *, Phanindra Kalyankar b, Richard J. FitzGerald b a b
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China Department of Life Sciences, University of Limerick, Limerick, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2014 Received in revised form 31 March 2015 Accepted 9 April 2015 Available online xxx
A bovine b-casein preparation hydrolysed with glutamyl endopeptidase (GE) at 37 and 50 C was quantitatively analysed with the isobaric tag for relative and absolute quantification (iTRAQ) technique using nano-LC-ESI-QTOF-MS/MS. Protein hydrolysis was affected by incubation temperature. MS analysis of the enzymatic hydrolysates indicated that phosphorylated peptides were less detectable than nonphosphorylated peptides according to the MS intensities. However, there is no difference in hydrolysing rates between phosphorylated peptides and non-phosphorylated peptides. Both the high temperature during hydrolysation, i.e., at 50 C, and the iTRAQ-labeling procedure of samples introduced in Met oxidation. The slow rate of Asp cleavage with GE was further demonstrated with iTRAQ analysis. The results herein setup a quantification methodology to confirm the precise process of b-casein hydrolysis with GE, which is significant for quantifying the process of bioactive peptides from industry of food protein hydrolysate. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Glutamyl endopeptidase Substrate specificity Bovin b-casein Isobaric tag for relative/absolute quantification LC-MS
1. Introduction As a serine proteinase and a sub-family of the chymotrypsin-like proteinase, Glutamyl endopeptidase (GE), which is mainly found in Bacillus species, specifically cleaves negatively charged amino acid residues (Glu/Asp) (Yokoi et al., 2001). Madsen et al. (Madsen & Qvist, 1997) reported that GE had better ability of hydrolysis on caseins than on whey proteins. GE was purified from a commercial food-grade proteinase preparation from Bacillus licheniformis, Alcalase™ (Spellman, Kenny, O'Cuinn, & FitzGerald, 2005). The substrate specificity of GE with bovine b-casein, one of the major proteins in bovine milk accounting for approximately 33% of caseins, has been qualitatively studied (Kalyankar, Zhu, O'Cuinn, & FitzGerald, 2013). However, the hydrolysis process needs to be further characterised in quantitation analysis. A quantitative
Abbreviations: ACN, acetonitrile; FDR, false discovery rates; GE, glutamyl endopeptidase; HIC, hydrophobic interaction chromatography; ICAT, Isotope Coded Affinity Tag; iTRAQ, Isobaric tag for relative and absolute quantification; LCMS, Liquid chromatography-mass spectrometry. * Corresponding author. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, China. Tel.: þ86 18900660563; fax: þ86 25 58139910. E-mail address: [email protected] (Y.-s. Zhu).
analysis on the GE b-casein hydrolysis process can provide more detailed information about how the b-casein is enzymatically hydrolysed by GE. Two isotopic labeling techniques are applied widely in quantitative analysis. The first technology is ICAT, which is using isotopiclabeled cysteine amino acid residues by a 8 Da different tag containing biotinylated reagents (Gygi et al., 1999). However, 15% of total proteins, which didn't contain any cysteine amino acid residues, can't be labeled with ICAT technique. Another technique called iTRAQ, is labeled on primary amines on the arginine and lysine side chains and on the N-terminal of the peptides with 2e8 isotope encoded reporter ions, which permits relative quantitation of 2e8 samples simultaneously. Quantitation with iTRAQ methodology is based on the abundance of low mass reporter ions, e.g., m/z 114e117, as iTRAQ4plex reagents, observed in tandem mass spectrometry (MS/MS) fragmentation of iTRAQ-labeled peptides (Treumann & Thiede, 2010). In a single iTARQ analysis, both identification of the peptides and their quantification can be achieved. Proteomics applications of the iTRAQ technique have been reported by several authors (Kristjansdottir & Kron, 2010; Sickmann, Burkhart, Vaudel, Zahedi, & Martens, 2011; Wright, Gan, Chong, & Pham, 2007). However, quantitative analysis was not widely applied in the field of food chemistry. Quantitative analysis of enzymatic hydrolysis of milk proteins using iTRAQ methodologies
http://dx.doi.org/10.1016/j.lwt.2015.04.021 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhu, Y., et al., Quantitative analysis of bovine b-casein hydrolysates obtained using glutamyl endopeptidase, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.04.021
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Y.-s. Zhu et al. / LWT - Food Science and Technology xxx (2015) 1e5
is reported on bacterial enzymes released in ripening cheese (Liu, Han, Sun, & Geng, 2014). The work herein was aimed to investigate the substrate specificity of GE with one of principal proteins in bovine milk, i.e., bcasein. The substrate specificity of GE is investigated on b-casein hydrolysation. The relative quantitation using the iTRAQ technology is applied on the hydrolysates of b-casein with GE. 2. Materials and methods 2.1. Materials and reagents
b-Casein was purified from bovine acid caseinate (Arrabawn Coop Society Ltd., Tipperary, Ireland) following calcium chloride treatment, IEX chromatography using DEAE column, dialysis, and centrifugation (Kalyankar, 2011). GE was purified from Alcalase™ 2.4 L (Novozyme, Bagsvaerd, Denmark) with HIC of phenyl Sepharose® (100 16 mm I.D, column volume 20 mL, Pharmacia Biotech, Cambridge, England) and IEX of HiTrap™-CM FF (column volume 5 mL, GE Healthcare, Bucks, UK) (Kalyankar et al., 2013). The activity of the purified GE was 256 nmol/min/mL of protein. The iTRAQ® Reagents-4plex Applications Kit and ICAT® Cation Exchange Buffer Pack were supplied by Applied Biosystems (Toronto, Canada). Other analytical reagents were obtained from Sigma Aldrich (Dublin, Ireland). 2.2. Digestion of b-casein with GE The enzymatic hydrolysis of b-casein (15 mg mL1) with GE (5 mg) was carried out in an aqueous solution (2 mL, 1.5% (w/v)) at 37 and 50 C, respectively. Samples (250 mL) were withdrawn at 0, 15, 60, 120 min and were diluted with 450 mL of 0.1% (v/v) formic acid in HPLC grade H2O (Kalyankar et al., 2013). 2.3. The iTRAQ labeling process Each of diluted samples was labeled with iTRAQ reporter ions following the manufacturer's protocol (Applied Biosystems, Toronto, Canada). The procedure was: 30 mL dissolution buffer and 15 mL of ethanol diluted iTRAQ reagent were transferred to each of the 5 mL diluted samples, which was sampled at 0, 15, 60 and 120 min from the GE hydrolysates; the mixture was incubated at room temperature for 1 h; ethanol was removed by vacuum vortex (RC10-22 and RCT-90, Analytica Ltd, Dublin, Ireland) for 1 h. The four labeled samples were mixed in a 1:1:1:1 (v/v) ratio. The samples were loaded and washed on an ICAT cation exchange cartridge according to the manufacturer's protocol (Applied Biosystems) to remove the excess iTRAQ reagent. Desalting procedure included three steps: 1. loading diluted sample mixture onto the cation-exchange cartridge, 2. washing salt and excess iTRAQ reagents from the cartridge, and 3. eluting the peptides with eluting buffer. The iTRAQ-labeled samples were ready to be injected into LC-MS after desalting. 2.4. LCMS analysis of iTRAQ-labeled digests of b-casein The iTRAQ-labeled samples were detected on the micrOTOFQ-II tandem mass spectrometry system (Bruker Daltonics, Bremen, Germany) coupled to an Ultimate 3000 nano-flow HPLC (Dionex, Sunnyvale, USA). First of all, the iTRAQ samples were desalted online using a C18 PepMap 100 precolumn cartridge (Dionex) with 0.1% TFA at 25 mL/min for 30 min. After desalting, samples were eluted to a 15 cm, 75 mm ID C18 PepMap analytical column (Dionex) in 0.1% formic acid. Elution was then performed on a predefined 60 min LC-MS/MS gradient program (2e40% ACN
containing 0.1% formic acid) and further eluted for 5 min at 95% ACN. Duplicate samples of the different b-casein digests were analysed on separate LC-MS/MS runs. MS measurements were all performed on a predefined 50e2400 m/z acquisition window. Tandem MS data were processed via Compass Data Analysis v 4.0 SP4 (Bruker Daltonics). Parameters of peak finder were set to S/ N at least 3 and minimum 10 counts intensity. Deconvolution of MS and MS/MS charges between 200 and 2500 m/z, maximum of 5 þ for MS and maximum of 3 þ for MS/MS spectra. All iTRAQ data were searched against the NCBInr database (downloaded at Nov 11th, 2010) with MASCOT (v2.30, Matrix Science, London, UK). The iTRAQ modifications were set to include 4-plex iTRAQ mass shifts (iTRAQ ions were labeled at Lys (K), Tyr (Y) and N-term), phosphorylation of serine and threonine (þ80 Da, Ser(S)/Thr(T)) and oxidation of methionine (þ16 Da, Met(M)) as variable modifications. Mass tolerances for all identifications were set to 0.06 Da for MS and 0.1 Da for MS/MS. Peptide level filters were set to a MASCOT score of 20 and at a p 0.05 (Zhang, Ficarro, Li, & Marto, 2009). The identified iTRAQ peptides (MS/MS) were automatically calculated using WARP-LC 1.2 (Bruker Daltonics).
3. Results and discussion Based on the fragments released from CID, i.e., 4-plex iTRAQ reporter ions at 114.1, 115.1, 116.1 and 117.1, iTRAQ-labeled peptides were counted in WARP-LC® 1.2. The sequence coverage is 53.1% and 27.8% of the identified b-casein at 37 and 50 C, respectively (Tables 1 and 2). The FDR were less than 4%. To improve sequence coverage, sequences of iTRAQ-labeled samples from two parallel experiments were combined (Wright, Chong, Gan, & Pham, 2006). The ratios of 115/114, 116/114 and 117/114 in all 19 peptide fragments, which represented the ratios of concentrations at different sampling intervals, indicated the maximum concentrations were reached at 60 min in the samples hydrolysed with GE at 50 C (Table 2, Fig. 1). In the samples hydrolysed with GE at 37 C (Table 1), two peptides (f1-14, f48-91) reached their maximal concentrations at the beginning of sampling, four peptides (f32-42, f92-100, f92-121, f122-131) reached maximal concentrations at 60 min and the rest 10 peptides were still increasing at 120 min, according to the ratios of 115/114, 116/114 and 117/114 in 16 peptide fragments. None of the peptides identified in the GE hydrolysed bcaseins’ samples matched the reported bioactive sequences in BIOPEP database (Dziuba & Dziuba, 2009). b-Casein f1-14 and f48-91 were only identified in the samples GE hydrolysed at 37 C. These iTRAQ reporter ions 114.1, which represents sampling at 0 min, were maximum among the four iTRAQ reporter ions. The identification indicated that f1-14 might already be cleaved by GE at the beginning of the hydrolysis at 50 C, and f1-14 was also rapidly hydrolysed at 37 C. b-Casein f1-5, RELEE, was identified in both samples hydrolysed with GE at 37 and 50 C. The relative quantitation analysis showed that f1-5 was rising at 37 C from 0 min to 120 min, and the sequence reached maximum concentration in the sample hydrolysed with GE at 50 C for 60 min. A hydrophobic peptide with molecular weight of 5315 Da (Ward, 1998), b-casein f48-91, was observed with low intensity (data not shown) in the sample hydrolysed with GE at 37 C. The cleavage on C-terminal of Asp with GE was already reported as 1000-fold slower than the cleavage on C-terminal of Glu (Breddam & Meldal, 1992). This is a critical factor of the low intensity identification on f48-91 in the sample hydrolysed with GE at 37 C. Interestingly, even with low intensity, the sequence was identified with MASCOT score of 30. Therefore, GE cleavage on both Glu and Asp was further demonstrated.
Please cite this article in press as: Zhu, Y., et al., Quantitative analysis of bovine b-casein hydrolysates obtained using glutamyl endopeptidase, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.04.021
Y.-s. Zhu et al. / LWT - Food Science and Technology xxx (2015) 1e5
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Table 1 iTRAQ-labeled peptide sequences identified upon incubation of b-casein with glutamyl endopeptidase for 0, 15, 60 and 120 min at 37 C. Fragment f1-5 f1-14
Sequence
Ion selected for MSMS (charge)
MASCOT scores.
115/114
116/114
RELEE-L
410.2201 (2)
23.3
1.0200
1.2651
117/114 1.4454
RELEELNVPGEIVE-S
885.4737 (2)
44.25
0.6524
0.7983
0.3047 14.0303
f6-11
E-LNVPGE-I
772.4321 (1)
21.84
2.5152
9.0000
f22-31
E-SITRINKKIE-K
745.4688 (2)
23.69
2.6184
4.8289
5.3947
f22-31
E-SITRINKKIE-K
817.5199 (2)
29.07
1.8739
5.0000
11.2941
f32-37
E-KFQS*EE-Q
568.2674 (2)
21.33
2.3045
6.9701
11.1537
f32-42
E-KFQS*EEQQQTE-D
875.4004 (2)
66.5
0.7544
4.6140
0.01
f32-44
E-KFQS*EEQQQTEDE-L
665.2925 (3)
30.22
1.7123
2.8082
3.4110
f48-91
D-KIHPFAQTQSLVYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPE-V
1063.9928 (5)
30.62
0.5550
0.7450
0.4500
f92-100
E-VMGVSKVKE-A
632.8805 (2)
30.62
2.4455
4.0091
3.6182
f92-100
E-VMGVSKVKE-A
704.9315 (2)
49.37
1.6727
5.3673
9.5908
f92-121
E-VMoGVSKVKEAMAPKHKEMoPFPKYPVEPFTE-S
1086.8438 (4)
39.27
1.2870
3.7685
2.2500
f101-121
E-AMAPKHKEMPFPKYPVEPFTE-S
1017.5492 (3)
52.39
0.9545
2.1894
3.7576
f101-121
E-AMAPKHKEMoPFPKYPVEPFTE-S
767.4124 (4)
39.25
1.3333
4.3977
6.1520
f101-121
E-AMAPKHKEMPFPKYPVEPFTE-S
799.4392 (4)
36.09
1.5630
4.3810
4.5294
f122-131
E-SQSLTLTDVE-N
618.8256 (2)
40.78
1.6095
3.0714
2.3939
Note: S* represents phosphorylated serine; the amino acid residue with dash line means it is an iTRAQ labeled amino acid residues; Mo represents oxidised methionine. 114, 115, 116 and 117 represent the iTRAQ ions' intensity labeled on samples digested at 0, 15, 60 and 120 min respectively. The 115/114, 116/114 and 117/114 represent the ratios of iTRAQ ions intensity labeled on samples digested at 15, 60 and 120 min (in relation to 0 min) respectively.
Table 2 iTRAQ-labeled peptide sequences identified upon incubation of b-casein with glutamyl endopeptidase for 0, 15, 60 and 120 min at 50 C. Sequence
Ion selected for MSMS (charge)
MASCOT scores.
115/114
RELEE-L
410.2201 (2)
20.51
0.6693
116/114 2.7511
117/114 0.6347
E-SITRINKKIE-K
745.4688 (2)
29.34
1.1280
12.1611
2.1943
f1-5 f22-31 f22-31
E-SITRINKKIE-K
817.5199 (2)
57.68
1.0683
7.6211
4.3851
f32-42
E-KFQS*EEQQQTE-D
875.4004 (2)
40.78
0.0100
4.2586
0.6897
f32-44
E-KFQS*EEQQQTEDE-L
997.4351 (2)
71.09
1.9796
4.4694
2.4490
f92-100
E-VMGVSKVKE-A
560.8295 (2)
21.32
0.5862
3.6207
1.6034
f92-100
E-VMoGVSKVKE-A
568.8269 (2)
29.11
0.0100
16.8077
2.3462
f92-100
E-VMGVSKVKE-A
632.8805 (2)
35.87
0.5976
8.2948
1.3068
f92-100
E-VMoGVSKVKE-A
640.8779 (2)
33.07
4.0678
29.9322
7.4576
f92-100
E-VMGVSKVKE-A
704.9315 (2)
38.22
0.7550
7.0860
2.2890
f92-100
E-VMoGVSKVKE-A
712.9290 (2)
41.97
1.5040
18.0887
4.9718
f92-121
E-VMoGVSKVKEAMoAPKHKEMoPFPKYPVEPFTE-S
1050.8183 (4)
25.91
1.7250
4.9375
0.0100
f92-121
E-VMoGVSKVKEAMoAPKHKEMoPFPKYPVEPFTE-S
1086.8438 (4)
29.35
0.6637
2.4159
0.2389
f101-121
E-AMAPKHKEMPFPKYPVEPFTE-S
1017.5492 (3)
62.43
1.1653
6.4959
1.2562
f101-121
E-AMAPKHKEMoPFPKYPVEPFTE-S
767.4124 (4)
31.08
1.4203
11.7953
2.5647
f101-121
E-AMoAPKHKEMoPFPKYPVEPFTE-S
771.4111 (4)
20.47
1.5339
14.7119
5.5847
f101-121
E-AMAPKHKEMPFPKYPVEPFTE-S
799.4392 (4)
34.26
0.7783
5.2544
1.1436
f122-131
E-SQSLTLTDVE-N
618.8256 (2)
62.61
0.7001
2.7142
0.9740
*
o
Note: S represents phosphorylated serine; the amino acid residue with dash line means it is an iTRAQ labeled amino acid residues; M represents oxidised methionine. 114, 115, 116 and 117 represent the iTRAQ ions' intensity labeled on samples digested at 0, 15, 60 and 120 min respectively. The 115/114, 116/114 and 117/114 represent the ratios of iTRAQ ions intensity labeled on samples digested at 15, 60 and 120 min (in relation to 0 min) respectively.
There are five phosphorylated serines in b-casein. However, only phosphorylated Ser35 was identified in the samples hydrolysed with GE at 37 and 50 C, i.e., f32-42 and f32-44 of b-casein. The identification indicates that phosphorylated peptides were less detectable from the non-phosphorylated peptides with iTRAQlabels. Interestingly, although the phosphorylated peptides with iTRAQ-labels were less detectable in the identification, there is no difference in digesting rates from the ratios of 115/114, 116/114 and 117/114 (representing the ratios of iTRAQ ions intensity labeled on samples enzymatically hydrolysed at 15, 60 and 120 min (in relation to 0 min) respectively) between phosphorylated peptides and non-phosphorylated peptides (Tables 1 and 2), which indicated that phosphorylation would not be affected by the iTRAQ-labeling procedure.
Oxidation of Met was a common reaction during heat treatment of proteins and peptides (Li, Schoneich, & Borchardt, 1995). Zhu et al. (Zhu & FitzGerald, 2010) reported that pH-dependent heat effect on methionine oxidation was observed. From Tables 1 and 2, temperature effect was also observed on the oxidation of Met. Three Met containing sequences, i.e., triply oxidised f92-121, singly oxidised f92-100 and singly/doubly oxidised f101-121, were identified in the samples hydrolysed with GE at 50 C. Singly oxidised f101-121 and doubly oxidised f92-121 were identified in the samples hydrolysed with GE at 37 C. In the same procedure of the sample treatment, more Met were oxidised in the samples GE hydrolysed at higher temperature, i.e., 50 C. From the previous report, the Met of f92-100 was identified as non-oxidised. However, three different iTRAQ-labeled sequences of singly oxidised f92-100
Please cite this article in press as: Zhu, Y., et al., Quantitative analysis of bovine b-casein hydrolysates obtained using glutamyl endopeptidase, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.04.021
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Y.-s. Zhu et al. / LWT - Food Science and Technology xxx (2015) 1e5
Fig. 1. The fragment ion spectra and the sequence with identified b and y ions of iTRAQ-labeled f77-85 in the b-casein hydrolysate sample digested by glutimyl endopeptidase at 50 C with the quantification of the iTRAQ reporter ions.
Fig. 2. The iTRAQ reporter ions of iTRAQ-labeled b-casein f185-209 in the samples digested with GE at 37 (A) and 50 (B) C identified in BioTools.
were identified in the samples hydrolysed with GE at 50 C, which suggests that the procedure of the iTRAQ-labeling introduced oxidation to Met. The C-terminal peptides from f132 to f209 contain only single glutamyl residue (E195) and single aspartic acid residue (D184). None peptides in the range was identified with iTRAQ-labels in all samples hydrolysed with GE at 37 and 50 C in WARP-LC® (Bruker Daltonics). However, iTRAQ-labeled f185-209 was identified in BioTools (Bruker Daltonics). The MASCOT scores were 32 and 18 for the sequence in the sample hydrolysed at 37 and 50 C, respectively. Interestingly, the intensity results of iTRAQ reporter ions reached maximum at 115 and 116 in the MSMS data of sequences in the sample hydrolysed at 37 and 50 C, respectively (Fig. 2). The results indicate that the f185-209 was hydrolysed at 37 C quicker than hydrolysed at 50 C. It might be a reason that the sequence f185-209 was hydrolysed faster with GE in lower temperature i.e., 37 C. Partially and fully iTRAQ-labeled peptides were identified in the samples of GE hydrolysed both at 37 and 50 C, i.e., f22-31, f92-100 and f101-121 of b-casein. The result demonstrated that incubation for 1 h at room temperature may not a suitable duration for the iTRAQ-labeling process, which is suggested in the iTRAQ application protocol (Applied Biosystems). The study on incubation time should be further investigated to provide an optimised incubation time to fully labeling peptides.
4. Conclusion Relative quantification analysis indicates detail hydrolysing process of b-casein with GE at 37 and 50 C. Incubation temperature was important in enhancing the hydrolysis rate, e.g., iTRAQ-labeled f1-14 and f48-91 were not identified in the sample hydrolysed with GE at 50 C. The facts that GE cleaves on Asp/Glu and the rate of Asp cleavage was very slow, were further demonstrated with iTRAQ analysis. High temperature during hydrolysation, i.e., at 50 C, could generate more oxidised Met. In the iTRAQ-labeling procedure of samples, oxidised Met was generated as well. The quantification methodology herein provides an opportunity to specifically study the release procedure of bioactive peptides from industry of food protein hydrolysate. Acknowledgements This study was part of Irish Food and Health Research Alliance project which is financially supported by the Higher Education Authority under the Programme for Research in Third Level Institutions (cycle 4) as part of the National Development Plan 2007e2013, Ireland. This work was funded under the National Development Plan (2006e2010), through the Food Institutional Research Measure, administered by the Irish Department of Agriculture and Food. And it was part of A Project Funded by the Priority
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Quantitative analysis of bovine b-casein hydrolysates obtained using glutamyl endopeptidase Yi-shen Zhu a, b, *, Phanindra Kalyankar b, Richard J. FitzGerald b a b
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China Department of Life Sciences, University of Limerick, Limerick, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2014 Received in revised form 31 March 2015 Accepted 9 April 2015 Available online xxx
A bovine b-casein preparation hydrolysed with glutamyl endopeptidase (GE) at 37 and 50 C was quantitatively analysed with the isobaric tag for relative and absolute quantification (iTRAQ) technique using nano-LC-ESI-QTOF-MS/MS. Protein hydrolysis was affected by incubation temperature. MS analysis of the enzymatic hydrolysates indicated that phosphorylated peptides were less detectable than nonphosphorylated peptides according to the MS intensities. However, there is no difference in hydrolysing rates between phosphorylated peptides and non-phosphorylated peptides. Both the high temperature during hydrolysation, i.e., at 50 C, and the iTRAQ-labeling procedure of samples introduced in Met oxidation. The slow rate of Asp cleavage with GE was further demonstrated with iTRAQ analysis. The results herein setup a quantification methodology to confirm the precise process of b-casein hydrolysis with GE, which is significant for quantifying the process of bioactive peptides from industry of food protein hydrolysate. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Glutamyl endopeptidase Substrate specificity Bovin b-casein Isobaric tag for relative/absolute quantification LC-MS
1. Introduction As a serine proteinase and a sub-family of the chymotrypsin-like proteinase, Glutamyl endopeptidase (GE), which is mainly found in Bacillus species, specifically cleaves negatively charged amino acid residues (Glu/Asp) (Yokoi et al., 2001). Madsen et al. (Madsen & Qvist, 1997) reported that GE had better ability of hydrolysis on caseins than on whey proteins. GE was purified from a commercial food-grade proteinase preparation from Bacillus licheniformis, Alcalase™ (Spellman, Kenny, O'Cuinn, & FitzGerald, 2005). The substrate specificity of GE with bovine b-casein, one of the major proteins in bovine milk accounting for approximately 33% of caseins, has been qualitatively studied (Kalyankar, Zhu, O'Cuinn, & FitzGerald, 2013). However, the hydrolysis process needs to be further characterised in quantitation analysis. A quantitative
Abbreviations: ACN, acetonitrile; FDR, false discovery rates; GE, glutamyl endopeptidase; HIC, hydrophobic interaction chromatography; ICAT, Isotope Coded Affinity Tag; iTRAQ, Isobaric tag for relative and absolute quantification; LCMS, Liquid chromatography-mass spectrometry. * Corresponding author. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, China. Tel.: þ86 18900660563; fax: þ86 25 58139910. E-mail address: [email protected] (Y.-s. Zhu).
analysis on the GE b-casein hydrolysis process can provide more detailed information about how the b-casein is enzymatically hydrolysed by GE. Two isotopic labeling techniques are applied widely in quantitative analysis. The first technology is ICAT, which is using isotopiclabeled cysteine amino acid residues by a 8 Da different tag containing biotinylated reagents (Gygi et al., 1999). However, 15% of total proteins, which didn't contain any cysteine amino acid residues, can't be labeled with ICAT technique. Another technique called iTRAQ, is labeled on primary amines on the arginine and lysine side chains and on the N-terminal of the peptides with 2e8 isotope encoded reporter ions, which permits relative quantitation of 2e8 samples simultaneously. Quantitation with iTRAQ methodology is based on the abundance of low mass reporter ions, e.g., m/z 114e117, as iTRAQ4plex reagents, observed in tandem mass spectrometry (MS/MS) fragmentation of iTRAQ-labeled peptides (Treumann & Thiede, 2010). In a single iTARQ analysis, both identification of the peptides and their quantification can be achieved. Proteomics applications of the iTRAQ technique have been reported by several authors (Kristjansdottir & Kron, 2010; Sickmann, Burkhart, Vaudel, Zahedi, & Martens, 2011; Wright, Gan, Chong, & Pham, 2007). However, quantitative analysis was not widely applied in the field of food chemistry. Quantitative analysis of enzymatic hydrolysis of milk proteins using iTRAQ methodologies
http://dx.doi.org/10.1016/j.lwt.2015.04.021 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhu, Y., et al., Quantitative analysis of bovine b-casein hydrolysates obtained using glutamyl endopeptidase, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.04.021
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Y.-s. Zhu et al. / LWT - Food Science and Technology xxx (2015) 1e5
is reported on bacterial enzymes released in ripening cheese (Liu, Han, Sun, & Geng, 2014). The work herein was aimed to investigate the substrate specificity of GE with one of principal proteins in bovine milk, i.e., bcasein. The substrate specificity of GE is investigated on b-casein hydrolysation. The relative quantitation using the iTRAQ technology is applied on the hydrolysates of b-casein with GE. 2. Materials and methods 2.1. Materials and reagents
b-Casein was purified from bovine acid caseinate (Arrabawn Coop Society Ltd., Tipperary, Ireland) following calcium chloride treatment, IEX chromatography using DEAE column, dialysis, and centrifugation (Kalyankar, 2011). GE was purified from Alcalase™ 2.4 L (Novozyme, Bagsvaerd, Denmark) with HIC of phenyl Sepharose® (100 16 mm I.D, column volume 20 mL, Pharmacia Biotech, Cambridge, England) and IEX of HiTrap™-CM FF (column volume 5 mL, GE Healthcare, Bucks, UK) (Kalyankar et al., 2013). The activity of the purified GE was 256 nmol/min/mL of protein. The iTRAQ® Reagents-4plex Applications Kit and ICAT® Cation Exchange Buffer Pack were supplied by Applied Biosystems (Toronto, Canada). Other analytical reagents were obtained from Sigma Aldrich (Dublin, Ireland). 2.2. Digestion of b-casein with GE The enzymatic hydrolysis of b-casein (15 mg mL1) with GE (5 mg) was carried out in an aqueous solution (2 mL, 1.5% (w/v)) at 37 and 50 C, respectively. Samples (250 mL) were withdrawn at 0, 15, 60, 120 min and were diluted with 450 mL of 0.1% (v/v) formic acid in HPLC grade H2O (Kalyankar et al., 2013). 2.3. The iTRAQ labeling process Each of diluted samples was labeled with iTRAQ reporter ions following the manufacturer's protocol (Applied Biosystems, Toronto, Canada). The procedure was: 30 mL dissolution buffer and 15 mL of ethanol diluted iTRAQ reagent were transferred to each of the 5 mL diluted samples, which was sampled at 0, 15, 60 and 120 min from the GE hydrolysates; the mixture was incubated at room temperature for 1 h; ethanol was removed by vacuum vortex (RC10-22 and RCT-90, Analytica Ltd, Dublin, Ireland) for 1 h. The four labeled samples were mixed in a 1:1:1:1 (v/v) ratio. The samples were loaded and washed on an ICAT cation exchange cartridge according to the manufacturer's protocol (Applied Biosystems) to remove the excess iTRAQ reagent. Desalting procedure included three steps: 1. loading diluted sample mixture onto the cation-exchange cartridge, 2. washing salt and excess iTRAQ reagents from the cartridge, and 3. eluting the peptides with eluting buffer. The iTRAQ-labeled samples were ready to be injected into LC-MS after desalting. 2.4. LCMS analysis of iTRAQ-labeled digests of b-casein The iTRAQ-labeled samples were detected on the micrOTOFQ-II tandem mass spectrometry system (Bruker Daltonics, Bremen, Germany) coupled to an Ultimate 3000 nano-flow HPLC (Dionex, Sunnyvale, USA). First of all, the iTRAQ samples were desalted online using a C18 PepMap 100 precolumn cartridge (Dionex) with 0.1% TFA at 25 mL/min for 30 min. After desalting, samples were eluted to a 15 cm, 75 mm ID C18 PepMap analytical column (Dionex) in 0.1% formic acid. Elution was then performed on a predefined 60 min LC-MS/MS gradient program (2e40% ACN
containing 0.1% formic acid) and further eluted for 5 min at 95% ACN. Duplicate samples of the different b-casein digests were analysed on separate LC-MS/MS runs. MS measurements were all performed on a predefined 50e2400 m/z acquisition window. Tandem MS data were processed via Compass Data Analysis v 4.0 SP4 (Bruker Daltonics). Parameters of peak finder were set to S/ N at least 3 and minimum 10 counts intensity. Deconvolution of MS and MS/MS charges between 200 and 2500 m/z, maximum of 5 þ for MS and maximum of 3 þ for MS/MS spectra. All iTRAQ data were searched against the NCBInr database (downloaded at Nov 11th, 2010) with MASCOT (v2.30, Matrix Science, London, UK). The iTRAQ modifications were set to include 4-plex iTRAQ mass shifts (iTRAQ ions were labeled at Lys (K), Tyr (Y) and N-term), phosphorylation of serine and threonine (þ80 Da, Ser(S)/Thr(T)) and oxidation of methionine (þ16 Da, Met(M)) as variable modifications. Mass tolerances for all identifications were set to 0.06 Da for MS and 0.1 Da for MS/MS. Peptide level filters were set to a MASCOT score of 20 and at a p 0.05 (Zhang, Ficarro, Li, & Marto, 2009). The identified iTRAQ peptides (MS/MS) were automatically calculated using WARP-LC 1.2 (Bruker Daltonics).
3. Results and discussion Based on the fragments released from CID, i.e., 4-plex iTRAQ reporter ions at 114.1, 115.1, 116.1 and 117.1, iTRAQ-labeled peptides were counted in WARP-LC® 1.2. The sequence coverage is 53.1% and 27.8% of the identified b-casein at 37 and 50 C, respectively (Tables 1 and 2). The FDR were less than 4%. To improve sequence coverage, sequences of iTRAQ-labeled samples from two parallel experiments were combined (Wright, Chong, Gan, & Pham, 2006). The ratios of 115/114, 116/114 and 117/114 in all 19 peptide fragments, which represented the ratios of concentrations at different sampling intervals, indicated the maximum concentrations were reached at 60 min in the samples hydrolysed with GE at 50 C (Table 2, Fig. 1). In the samples hydrolysed with GE at 37 C (Table 1), two peptides (f1-14, f48-91) reached their maximal concentrations at the beginning of sampling, four peptides (f32-42, f92-100, f92-121, f122-131) reached maximal concentrations at 60 min and the rest 10 peptides were still increasing at 120 min, according to the ratios of 115/114, 116/114 and 117/114 in 16 peptide fragments. None of the peptides identified in the GE hydrolysed bcaseins’ samples matched the reported bioactive sequences in BIOPEP database (Dziuba & Dziuba, 2009). b-Casein f1-14 and f48-91 were only identified in the samples GE hydrolysed at 37 C. These iTRAQ reporter ions 114.1, which represents sampling at 0 min, were maximum among the four iTRAQ reporter ions. The identification indicated that f1-14 might already be cleaved by GE at the beginning of the hydrolysis at 50 C, and f1-14 was also rapidly hydrolysed at 37 C. b-Casein f1-5, RELEE, was identified in both samples hydrolysed with GE at 37 and 50 C. The relative quantitation analysis showed that f1-5 was rising at 37 C from 0 min to 120 min, and the sequence reached maximum concentration in the sample hydrolysed with GE at 50 C for 60 min. A hydrophobic peptide with molecular weight of 5315 Da (Ward, 1998), b-casein f48-91, was observed with low intensity (data not shown) in the sample hydrolysed with GE at 37 C. The cleavage on C-terminal of Asp with GE was already reported as 1000-fold slower than the cleavage on C-terminal of Glu (Breddam & Meldal, 1992). This is a critical factor of the low intensity identification on f48-91 in the sample hydrolysed with GE at 37 C. Interestingly, even with low intensity, the sequence was identified with MASCOT score of 30. Therefore, GE cleavage on both Glu and Asp was further demonstrated.
Please cite this article in press as: Zhu, Y., et al., Quantitative analysis of bovine b-casein hydrolysates obtained using glutamyl endopeptidase, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.04.021
Y.-s. Zhu et al. / LWT - Food Science and Technology xxx (2015) 1e5
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Table 1 iTRAQ-labeled peptide sequences identified upon incubation of b-casein with glutamyl endopeptidase for 0, 15, 60 and 120 min at 37 C. Fragment f1-5 f1-14
Sequence
Ion selected for MSMS (charge)
MASCOT scores.
115/114
116/114
RELEE-L
410.2201 (2)
23.3
1.0200
1.2651
117/114 1.4454
RELEELNVPGEIVE-S
885.4737 (2)
44.25
0.6524
0.7983
0.3047 14.0303
f6-11
E-LNVPGE-I
772.4321 (1)
21.84
2.5152
9.0000
f22-31
E-SITRINKKIE-K
745.4688 (2)
23.69
2.6184
4.8289
5.3947
f22-31
E-SITRINKKIE-K
817.5199 (2)
29.07
1.8739
5.0000
11.2941
f32-37
E-KFQS*EE-Q
568.2674 (2)
21.33
2.3045
6.9701
11.1537
f32-42
E-KFQS*EEQQQTE-D
875.4004 (2)
66.5
0.7544
4.6140
0.01
f32-44
E-KFQS*EEQQQTEDE-L
665.2925 (3)
30.22
1.7123
2.8082
3.4110
f48-91
D-KIHPFAQTQSLVYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPE-V
1063.9928 (5)
30.62
0.5550
0.7450
0.4500
f92-100
E-VMGVSKVKE-A
632.8805 (2)
30.62
2.4455
4.0091
3.6182
f92-100
E-VMGVSKVKE-A
704.9315 (2)
49.37
1.6727
5.3673
9.5908
f92-121
E-VMoGVSKVKEAMAPKHKEMoPFPKYPVEPFTE-S
1086.8438 (4)
39.27
1.2870
3.7685
2.2500
f101-121
E-AMAPKHKEMPFPKYPVEPFTE-S
1017.5492 (3)
52.39
0.9545
2.1894
3.7576
f101-121
E-AMAPKHKEMoPFPKYPVEPFTE-S
767.4124 (4)
39.25
1.3333
4.3977
6.1520
f101-121
E-AMAPKHKEMPFPKYPVEPFTE-S
799.4392 (4)
36.09
1.5630
4.3810
4.5294
f122-131
E-SQSLTLTDVE-N
618.8256 (2)
40.78
1.6095
3.0714
2.3939
Note: S* represents phosphorylated serine; the amino acid residue with dash line means it is an iTRAQ labeled amino acid residues; Mo represents oxidised methionine. 114, 115, 116 and 117 represent the iTRAQ ions' intensity labeled on samples digested at 0, 15, 60 and 120 min respectively. The 115/114, 116/114 and 117/114 represent the ratios of iTRAQ ions intensity labeled on samples digested at 15, 60 and 120 min (in relation to 0 min) respectively.
Table 2 iTRAQ-labeled peptide sequences identified upon incubation of b-casein with glutamyl endopeptidase for 0, 15, 60 and 120 min at 50 C. Sequence
Ion selected for MSMS (charge)
MASCOT scores.
115/114
RELEE-L
410.2201 (2)
20.51
0.6693
116/114 2.7511
117/114 0.6347
E-SITRINKKIE-K
745.4688 (2)
29.34
1.1280
12.1611
2.1943
f1-5 f22-31 f22-31
E-SITRINKKIE-K
817.5199 (2)
57.68
1.0683
7.6211
4.3851
f32-42
E-KFQS*EEQQQTE-D
875.4004 (2)
40.78
0.0100
4.2586
0.6897
f32-44
E-KFQS*EEQQQTEDE-L
997.4351 (2)
71.09
1.9796
4.4694
2.4490
f92-100
E-VMGVSKVKE-A
560.8295 (2)
21.32
0.5862
3.6207
1.6034
f92-100
E-VMoGVSKVKE-A
568.8269 (2)
29.11
0.0100
16.8077
2.3462
f92-100
E-VMGVSKVKE-A
632.8805 (2)
35.87
0.5976
8.2948
1.3068
f92-100
E-VMoGVSKVKE-A
640.8779 (2)
33.07
4.0678
29.9322
7.4576
f92-100
E-VMGVSKVKE-A
704.9315 (2)
38.22
0.7550
7.0860
2.2890
f92-100
E-VMoGVSKVKE-A
712.9290 (2)
41.97
1.5040
18.0887
4.9718
f92-121
E-VMoGVSKVKEAMoAPKHKEMoPFPKYPVEPFTE-S
1050.8183 (4)
25.91
1.7250
4.9375
0.0100
f92-121
E-VMoGVSKVKEAMoAPKHKEMoPFPKYPVEPFTE-S
1086.8438 (4)
29.35
0.6637
2.4159
0.2389
f101-121
E-AMAPKHKEMPFPKYPVEPFTE-S
1017.5492 (3)
62.43
1.1653
6.4959
1.2562
f101-121
E-AMAPKHKEMoPFPKYPVEPFTE-S
767.4124 (4)
31.08
1.4203
11.7953
2.5647
f101-121
E-AMoAPKHKEMoPFPKYPVEPFTE-S
771.4111 (4)
20.47
1.5339
14.7119
5.5847
f101-121
E-AMAPKHKEMPFPKYPVEPFTE-S
799.4392 (4)
34.26
0.7783
5.2544
1.1436
f122-131
E-SQSLTLTDVE-N
618.8256 (2)
62.61
0.7001
2.7142
0.9740
*
o
Note: S represents phosphorylated serine; the amino acid residue with dash line means it is an iTRAQ labeled amino acid residues; M represents oxidised methionine. 114, 115, 116 and 117 represent the iTRAQ ions' intensity labeled on samples digested at 0, 15, 60 and 120 min respectively. The 115/114, 116/114 and 117/114 represent the ratios of iTRAQ ions intensity labeled on samples digested at 15, 60 and 120 min (in relation to 0 min) respectively.
There are five phosphorylated serines in b-casein. However, only phosphorylated Ser35 was identified in the samples hydrolysed with GE at 37 and 50 C, i.e., f32-42 and f32-44 of b-casein. The identification indicates that phosphorylated peptides were less detectable from the non-phosphorylated peptides with iTRAQlabels. Interestingly, although the phosphorylated peptides with iTRAQ-labels were less detectable in the identification, there is no difference in digesting rates from the ratios of 115/114, 116/114 and 117/114 (representing the ratios of iTRAQ ions intensity labeled on samples enzymatically hydrolysed at 15, 60 and 120 min (in relation to 0 min) respectively) between phosphorylated peptides and non-phosphorylated peptides (Tables 1 and 2), which indicated that phosphorylation would not be affected by the iTRAQ-labeling procedure.
Oxidation of Met was a common reaction during heat treatment of proteins and peptides (Li, Schoneich, & Borchardt, 1995). Zhu et al. (Zhu & FitzGerald, 2010) reported that pH-dependent heat effect on methionine oxidation was observed. From Tables 1 and 2, temperature effect was also observed on the oxidation of Met. Three Met containing sequences, i.e., triply oxidised f92-121, singly oxidised f92-100 and singly/doubly oxidised f101-121, were identified in the samples hydrolysed with GE at 50 C. Singly oxidised f101-121 and doubly oxidised f92-121 were identified in the samples hydrolysed with GE at 37 C. In the same procedure of the sample treatment, more Met were oxidised in the samples GE hydrolysed at higher temperature, i.e., 50 C. From the previous report, the Met of f92-100 was identified as non-oxidised. However, three different iTRAQ-labeled sequences of singly oxidised f92-100
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Y.-s. Zhu et al. / LWT - Food Science and Technology xxx (2015) 1e5
Fig. 1. The fragment ion spectra and the sequence with identified b and y ions of iTRAQ-labeled f77-85 in the b-casein hydrolysate sample digested by glutimyl endopeptidase at 50 C with the quantification of the iTRAQ reporter ions.
Fig. 2. The iTRAQ reporter ions of iTRAQ-labeled b-casein f185-209 in the samples digested with GE at 37 (A) and 50 (B) C identified in BioTools.
were identified in the samples hydrolysed with GE at 50 C, which suggests that the procedure of the iTRAQ-labeling introduced oxidation to Met. The C-terminal peptides from f132 to f209 contain only single glutamyl residue (E195) and single aspartic acid residue (D184). None peptides in the range was identified with iTRAQ-labels in all samples hydrolysed with GE at 37 and 50 C in WARP-LC® (Bruker Daltonics). However, iTRAQ-labeled f185-209 was identified in BioTools (Bruker Daltonics). The MASCOT scores were 32 and 18 for the sequence in the sample hydrolysed at 37 and 50 C, respectively. Interestingly, the intensity results of iTRAQ reporter ions reached maximum at 115 and 116 in the MSMS data of sequences in the sample hydrolysed at 37 and 50 C, respectively (Fig. 2). The results indicate that the f185-209 was hydrolysed at 37 C quicker than hydrolysed at 50 C. It might be a reason that the sequence f185-209 was hydrolysed faster with GE in lower temperature i.e., 37 C. Partially and fully iTRAQ-labeled peptides were identified in the samples of GE hydrolysed both at 37 and 50 C, i.e., f22-31, f92-100 and f101-121 of b-casein. The result demonstrated that incubation for 1 h at room temperature may not a suitable duration for the iTRAQ-labeling process, which is suggested in the iTRAQ application protocol (Applied Biosystems). The study on incubation time should be further investigated to provide an optimised incubation time to fully labeling peptides.
4. Conclusion Relative quantification analysis indicates detail hydrolysing process of b-casein with GE at 37 and 50 C. Incubation temperature was important in enhancing the hydrolysis rate, e.g., iTRAQ-labeled f1-14 and f48-91 were not identified in the sample hydrolysed with GE at 50 C. The facts that GE cleaves on Asp/Glu and the rate of Asp cleavage was very slow, were further demonstrated with iTRAQ analysis. High temperature during hydrolysation, i.e., at 50 C, could generate more oxidised Met. In the iTRAQ-labeling procedure of samples, oxidised Met was generated as well. The quantification methodology herein provides an opportunity to specifically study the release procedure of bioactive peptides from industry of food protein hydrolysate. Acknowledgements This study was part of Irish Food and Health Research Alliance project which is financially supported by the Higher Education Authority under the Programme for Research in Third Level Institutions (cycle 4) as part of the National Development Plan 2007e2013, Ireland. This work was funded under the National Development Plan (2006e2010), through the Food Institutional Research Measure, administered by the Irish Department of Agriculture and Food. And it was part of A Project Funded by the Priority
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Please cite this article in press as: Zhu, Y., et al., Quantitative analysis of bovine b-casein hydrolysates obtained using glutamyl endopeptidase, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.04.021