Optimization of ultrasound-assisted extraction of protein from egg white using response surface methodology (RSM) and its proteomic study by MALDI-TOF-MS

Abstract

The presented work proposes an approach based on proteomic techniques to identify proteins in egg white. Response surface methodology (RSM) with Box–Behnken design (BBD) was used to optimize the ultrasound-assisted extraction process of proteins from the egg white. The effects of extraction parameters, including trifluoroacetic acid (TFA) concentration, ultrasonic power and ultrasonic time on the extraction process of proteins were evaluated. The optimized extraction parameters were trifluoroacetic acid (TFA) concentration, 0.53% (v/v); ultrasonic power, 50% and ultrasonic time, 40 min. The effects of three factors (trypsin volume, pH of tryptic digestion and incubation time) on the tryptic digestion process of extracted proteins were investigated. The results showed that the optimized tryptic digestion conditions were as follows: volume of 10 μg mL⁻¹ trypsin, 8 μL; pH, 7.0 and incubation time, 16 h. Under the optimized conditions, the peptide mixture was analysed using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The methodology was successfully applied to free-range and barn-raised chicken egg white for protein identification. The results showed that 23 proteins and 29 proteins were identified in free-range chicken and barn-raised chicken egg white, respectively. Among them, the collagen peptides were found in egg white for the first time.

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1. Introduction

Chicken egg is an inexpensive source of high quality protein in the human diet, which is composed of water (75%), proteins (12%), and lipids (12%), as well as carbohydrates etc. egg white, a high-viscosity solution, in particular, has been used as a raw ingredient for decades in the food industry.^1,2 Egg polyfunctionality in food systems is correlated, to a high extent, with its chemical composition and more specifically with its protein content. Besides that, some of the minor components could be interesting for nonfood applications, such as health uses.³

Many researches have been conducted to identify the novel proteins and their isoforms in egg white. Egg proteins were previously studied using classical biochemical techniques such as chromatographic and electrophoretic separations, together with Edman sequence analysis. Up to 1989, only 13 proteins were usually referenced in egg white, some of which were not even fully characterized. It is difficult to identify the proteins of egg white because of their different molecular weights and pI values, different concentration levels, diversified polymorphic forms as well as their complex matrices. With the advent of new ionization techniques of matrix-assisted laser desorption/ionization (MALDI), MS has become a powerful tool in structural characterization of large biomolecules,^4–6 and it has high selectivity and sensitivity, tolerance toward contaminants and provides fast analysis. Proteomic methodologies for identification of proteins by MS can provide reliable identification of the different proteins in comparison with other methods and have gained more attention during recent years.⁷ Guérin-Dubiard et al.,⁸ via 2-D electrophoresis and ESI LC-MS/MS, identified 16 proteins in egg white in which Tenp and VMO-1 were detected for the first time. Raikos et al.,⁹ via SDS-PAGE and 2-D PAGE, combined with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), identified five proteins, namely ovalbumin, ovotransferrin, clusterin, activin receptor IIA and the hypothetical protein FLJ10305. A major breakthrough came with the work of Mann¹⁰ who Using 1-D PAGE and LC-MS/MS and MS³ identified 78 proteins in chicken egg white, 54 of which were identified in egg white for the first time. D'Ambrosio et al.¹¹ have doubled the number of proteins (to 148) found in egg white by exploiting a peptide ligand libraries technology. Omana et al.¹² determined the changes of proteins in egg white from white-shell eggs during storage to further understand the biochemical basis of egg white thinning. Egg white thinning is regarded as a sign of loss of quality that leads to staleness.¹³ Some researches compared free-range chicken egg varieties and barn-raised chicken egg varieties in xanthophyll composition,¹⁴ whipping capacity and foam consistency,¹⁵ levels of PCDD/Fs, PCBs and PBDEs,¹⁶ and so on. Proteins are the main components in egg white except water, however, to date, many of the proteins located in eggs from different housing systems remain uncharacterised, if not unknown. The main objective of the present study was to evaluate the proteins in egg white from free-range and barn-raised chicken.

The key steps in proteomic studies by MS methods usually involve extraction of proteins in complex mixtures. H. C. Lee et al.¹⁷ optimized extraction conditions of protein and starch from lentils via single-factor experiment which showed no significant difference was observed for extracted protein at the various extraction pH and temperature. Response surface methodology (RSM), an effective statistical technique for optimizing complex extraction processes, can reduce the number of experimental trials and evaluate the mutual interactions between multiple parameters. Box–Behnken design (BBD) is an RSM model that examines the relationships between several explanatory variables and one or more response variables.^18–20 In this current study, we have used MALDI-TOF-MS in an attempt to identify the proteins in the free-range chicken and barn-raised chicken egg white. Methods for response surface methodology (RSM) with Box–Behnken design (BBD) have been used to optimize the conditions of ultrasound-assisted extraction of protein from egg white. Under the optimized conditions, the peptide mass fingerprints (PMF) of the proteins of free-range chicken and barn-raised chicken egg white were determined by MALDI-TOF-MS.

2. Experimental section

2.1. Reagents and materials

All of chemicals used in the current study were purchased as the analytical reagent grade. Deionized water was used throughout whole process.

Sequencing grade trypsin was purchased from Promega (Madison, WI, USA). α-Cyano-4-hydroxycinnamic acid (CHCA), dithiothreitol (DTT) was purchased from Merck (Darmstadt, Germany). Iodoacetamide (IAA) was purchased from Amresco (Ohio, USA). Trifluoroacetic acid (TFA) was purchased from Aladin (Shanghai, China). Urea and ammonium hydrogen carbonate were purchased from Xi'an Chemical Reagent Plant (Xi'an, China).

Free range eggs were obtained from the countryside of Weinan city, Shaanxi province, China. Barn-raised eggs were obtained from the local supermarket of Xi'an.

2.2. Sample preparation

Free range eggs and barn-raised eggs, both less than 5 days after being laid, were used. They were both stored at 4 °C. The egg white was drawn from eggs with small glass sucker (0.4 mm diameter) and collected. The egg white was spread-out as thin film across several microscope glass slides and evaporated at ambient temperature (25 °C). Every effort was made to prevent contamination of samples during the sampling process.¹¹

2.3. Protein extraction

The proteins were extracted from the samples (40 mg) using 0.6 mL of 0.6% (v/v) TFA. The resulting extracts were subjected to an ultrasonic bath treatment for 45 min. The mixtures were then centrifuged 15 min at 4000×g and the supernatant was collected.

The protein concentration of the supernatant (soluble protein extracts) was determined using Bradford's method.^21,22

2.4. Experimental design for protein extraction

To further optimize the extraction conditions, a three-variable-three-level BBD was employed in this experiment.^23–25 The three independent variables were TFA concentration (v/v), ultrasonic power (%) and ultrasonic time (min), respectively. Each variable was set at three levels. A total of 15 experiments were designed according to BBD. Each experiment was performed in triplicate and the average protein concentration (mg mL⁻¹) was taken as the response.

2.5. Enzymatic digestion

The procedure used for the hydrolysis of the extracted proteins in the current study has been described in detail elsewhere in the literature^26,27 with some improvement here. In brief, the 150 μL of protein solution was denatured in 0.5 mL of 40 mM NH₄HCO₃ (pH 7.8) by urea at a final concentration of 8 mol L⁻¹. Then, it was reduced with 12 mM dithiothreitol (final concentration) and incubated at 50 °C for 3 h. The alkylation was achieved with iodoacetamide at 300 mM final concentration, and the reaction was carried out in the dark at room temperature for 30 min. This sample was dialyzed against deionized water and phosphate buffer solution (PBS, pH 7.0) overnight. Finally, 8 μL of 10 μg mL⁻¹ trypsin was added to the prepared 50 μL of protein solution and the mixture was incubated at 37 °C for 16 h. The solution was then stored at −20 °C until further analysis.

2.6. MALDI-TOF-MS and database search

The MW (molecular weights) of peptides was measured using an AutoflexIII Smartbean MALDI-TOF mass spectrometer (Bruker, Germany) equipped with a standard nitrogen laser (λ 337 nm) in linear mode. Each experiment started with recalibration of the mass spectrometer with a commercial standard of peptide mixture (Pepmix, Bruker). A 1 μL aliquot of the peptide mixture was mixed in a 1 : 1 ratio (v/v) with the matrix (CHCA) solution (10 mg mL⁻¹ in ACN : TFA 0.1%, 50 : 50). The resulting mixture was spotted on the stainless steel MALDI target and was leaved for air drying at room temperature before MALDI-TOF-MS analysis. At least 300 laser shots were collected for each spectrum.

All of the searches of the PMF spectra were obtained against SwissProt non-redundant database (SwissProt_2013.6.27) and carried out using MS-Fit search engine, accessible at http://prospector.ucsf.edu/.²⁷

3. Results and discussion

3.1. Single-factor optimization of protein extraction condition

It is important for identification of proteins to select the appropriate methods and conditions of protein extraction in the experiment. The extraction of protein using concentrated HCl, NaOH, or NH₃ has been proposed,^28–30 but these reagents often lead to selective degradation of amino acids, which affects the identification of protein. TFA is shown to be a promising extraction agent in the identification of protein.^26,31 Compared to other methods,^32,33 TFA-ultrasound-assisted extraction can provide the total proteins, it has such advantages as simple and rapid operation, good repeatability, low consumption chemical reagents. It can minimize the sample loss and avoid the risk of sample contaminant result from in the digestion or derivatisation. In this current work, TFA-ultrasound-assisted extraction was selected to extract protein from chicken egg white. The effects of TFA concentration, ultrasonic power and ultrasonic time on ultrasound-assisted extraction of protein from egg white were investigated (Fig. 1). As shown in Fig. 1A, protein concentration increased with TFA concentration increased until 0.6% (v/v), and began to decrease. A certain concentration of TFA can dissolve protein well but high concentration of TFA maybe lead to protein denaturation. The effects of ultrasonic power (Fig. 1B) and ultrasonic time (Fig. 1C) showed the similar fluctuation trend to TFA concentration. Protein concentration increased accompanying increases of the two variables. When ultrasonic power and ultrasonic time increased respectively over 70% and 45 min, protein concentration reduced as a result of hydrophilic interactions of some globulin proteins and protein denaturation or hydrolysate.

Fig. 1 Effects of extraction parameters on protein extraction process. ((A) TFA concentration, %; (B) ultrasonic power, %; (C) ultrasonic time, min).

Proteins in egg white contain a significant amount of polar and hydrophilic amino acid, such as glutamine/glutamic acid, lysine and arginine which contribute to favorable hydrophilic interactions between protein molecules and water via hydrogen bonds and electrostatic interactions.³⁴ Hydrophilic interactions may also be partly responsible for the single-factor experiment results.

3.2. Response surface optimization

In order to systemically investigate the effects of extraction parameters on the concentration of protein extracted from egg white and their interactions, RSM employing BBD was used to optimize ultrasound-assisted extraction. The range and center point values of three independent variables were based on the results of the single-factor experiment. The results from 12 experimental runs and three central point runs (TFA concentration, 0.5%; ultrasonic power, 70% and ultrasonic time, 45 min) using BBD are presented in Table 1. By applying multiple regression analyses on the experimental data, the predicted response variables can be obtained by the following second-order polynomial equation: Y = 15.91 + 1.92A − 1.16B − 0.90C + 2.21AB + 0.53AC + 0.97BC − 3.70A² − 2.32B² − 0.85C², where A, B, and C are in terms of coded factors of the test variables, namely TFA concentration, ultrasonic power and ultrasonic time.

Table 1 BBD matrix and the response values for observed protein concentration

Run	Independent variable			Response Y: C (Pr), mg mL^−1
	A: C (TFA), %	B: t, min	C: W, %
1	0.50 (0)	60.00 (+1)	40.00 (−1)	11.92
2	0.50 (0)	45.00 (0)	70.00 (0)	15.34
3	0.20 (−1)	60.00 (+1)	70.00 (0)	3.91
4	0.80 (+1)	45.00 (0)	100.00 (+1)	12.65
5	0.50 (0)	30.00 (−1)	40.00 (−1)	15.41
6	0.50 (0)	60.00 (+1)	100.00 (+1)	12.00
7	0.50 (0)	45.00 (0)	70.00 (0)	16.17
8	0.20 (−1)	45.00 (0)	100.00 (+1)	8.32
9	0.50 (0)	30.00 (−1)	100.00 (+1)	11.62
10	0.20 (−1)	30.00 (−1)	70.00 (0)	11.44
11	0.20 (−1)	45.00 (0)	40.00 (−1)	11.13
12	0.80 (+1)	45.00 (0)	40.00 (−1)	13.33
13	0.80 (+1)	30.00 (−1)	70.00 (0)	11.44
14	0.80 (+1)	60.00 (+1)	70.00 (0)	12.76
15	0.50 (0)	45.00 (0)	70.00 (0)	16.24

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A summary of analysis of variance (ANOVA) for the BBD experimental results is shown in Table 2. The smaller the P-values (p < 0.05) are, the bigger significance of the corresponding coefficient, which is implied that the response model was more suitable for reflecting the expected optimization. It can be also seen from Table 2 that F-value for the lack of fit was not significant (p > 0.05) relative to the pure error, confirming the validity of the model. In this case, A, B, C, AB, BC, A² and B² are significant model terms. So TFA concentration, ultrasonic power and ultrasonic time were important factors in the extraction process.

Table 2 ANOVA for the experimental results of the BBD

Parameters	Sum of squares	df	Mean square	F-Value	p-Value
Model	138.06	9	15.34	32.33	0.0007
A	29.61	1	29.61	62.40	0.0005
B	10.84	1	10.84	22.84	0.0050
C	6.46	1	6.46	13.62	0.0141
AB	19.58	1	19.58	41.26	0.0014
AC	1.13	1	1.13	2.39	0.1830
BC	3.75	1	3.75	7.90	0.0375
A^2	50.60	1	50.60	106.62	0.0001
B^2	19.92	1	19.92	41.98	0.0013
C^2	2.69	1	2.69	5.67	0.0631
Residual	2.37	5	0.47	—	—
Lack of fit	1.87	3	0.62	2.49	0.2993
Pure error	0.50	2	0.25	—	—

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The three-dimensional (3D) response surface and the contour plots (Fig. 2) were obtained using Design-Expert 7.0.0 software. They provide a means of visualizing the relationship between the responses and experimental levels of each variable and the type of interactions between the two test variables. As shown in Fig. 2, the mutual interaction between TFA concentration and ultrasonic power (Fig. 2B) was found to be more significant than the two others. It showed that the response was affected significantly by TFA concentration. It can be concluded that optimal extraction conditions of protein from egg white were following ranges of the examined variables: TFA concentration, 0.35–0.65% (v/v), ultrasonic power, 55–85% and ultrasonic time, 37.5–52.5 min. The optimum values of the test variables were TFA concentration, 0.53% (v/v); ultrasonic power, 49.41% and ultrasonic time, 39.89 min. Under these conditions, the maximum protein concentration was predicted as 16.5257 mg mL⁻¹. Taking convenience into account, the optimum experimental parameters were modified as follows: TFA concentration, 0.53% (v/v), ultrasonic power, 50% and ultrasonic time, 40 min. To compare the predicted results with experimental values, rechecking was performed using modified optimal conditions. The result showed that experimental value (16.56 mg mL⁻¹, n = 3) and predicted results (16.5257 mg mL⁻¹) were not significant (p > 0.05).

Fig. 2 Response surface plots of the protein concentration affected by TFA concentration (v/v), ultrasonic time (min) and ultrasonic power (%).

3.3. Effects of parameters in the process of hydrolysis

To establish an adequate system for enzymatic hydrolysis of the protein sample, the effects of tryptic aliquot, pH and incubation time were investigated. The experiment results were consistent with the reported condition of enzymatic hydrolysis.³⁵ The extracted proteins were incubated in the presence of different aliquot (4, 6, 8, 10 μL) of 10 μg mL⁻¹ trypsin and PBS (pH 7.0, 7.5, 8.0, 8.5) prior to mass spectrometric analysis. The effect of incubation time (10, 13, 16, 19, 22 h) was also investigated (Table 3). The results show that the number of peptides increased from 7 to 17 with the trypsin aliquot increasing from 4 μL to 8 μL and decreased to 9 with the trypsin aliquot increasing to 10 μL. 8 μL of 10 μg mL⁻¹ trypsin was selected in the process of hydrolysis. A few peptides generated as result of partly enzymatic action in the presence of insufficient trypin. The trypsin may be degraded by itself when excessive trypin was added, which affects the identification of protein. The number of peptides decreased from 19 to 9 with the pH increasing from 7.0 to 8.0 and changed slightly with the pH increasing from 8.0 to 8.5. This showed that the trypin has higher activity with a pH value of 7.0 in the tryptic hydrolysis of the proteins. Prolonged incubation time from 10 h to 16 h, the number of peptides increased from 9 to 14 and then began to change slightly, so the suitable incubation time was selected at 16 h.

Table 3 Effects of trypsin volume, incubation time and tryptic digestion pH on tryptic hydrolysis of proteins

Parameters			Number of peptides
V (trypsin), μL	pH	t, h	No. (V)	No. (pH)	No. (t)
4	7.0	10	7	19	9
6	7.5	13	14	7	8
8	8.0	16	17	9	14
10	8.5	19	9	9	14
—	—	22	—	—	12

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3.4. Identification of protein

Since all the biological liquids previously described in reality bathe a number of tissues in the human organism, one would expect to find plenty of proteins, especially novel proteins. PMF approach²⁹ for protein identification implies that each protein subjected to tryptic hydrolysis give a unique set of peptides which represent fingerprint of the protein. Fig. 3 displays the mass spectra of the hydrolyzed protein extracted from the egg white. The different proteins identified in the free-range chicken egg white and barn-raised chicken egg white were summarized in Tables 4 and 5. 23 proteins for free-range chicken egg white and 29 proteins for barn-raised chicken egg white were identified respectively. Among them, 16 proteins were identified in both two egg white varieties including well-known proteins such as ovalbumin, ovostransferrin, lysozyme C and ovostatin, which were already identified by D'Ambrosio et al.¹¹ Collagen peptides were identified for the first time. 6 collagen peptides were detected in free-range chicken egg white, namely collagen alpha-3 (VI) chain, collagen alpha-1 (XII) chain, collagen alpha-2 (I) chain (Fragments), 72 kDa type IV collagenase, collagen alpha-3 (IX) chain and collagen alpha-1(VIII) chain. 9 collagen peptides were detected in barn-raised chicken egg white, namely collagen alpha-3 (VI) chain, collagen alpha-1 (XII) chain, collagen alpha-2 (I) chain (Fragments), 72 kDa type IV collagenase, collagen alpha-1 (III) chain (Fragments), collagen alpha-2 (VI) chain, collagen alpha-2 (IX) chain, collagen alpha-1 (I) chain and collagen alpha-1 (XIV) chain.

Fig. 3 MALDI-TOF-MS spectrum of the hydrolyzed protein extracted from the egg white in free-range chicken eggs (A) and barn-raised chicken eggs (B).

Table 4 MS-Fit search results of egg white in free-range eggs^a

Accession no.	Protein name	Mowse score	^#pep ^#mat %mat	Cov %	MS-digest index no.	Peptide MW m/z		Position		M. cut	Sequence
						Exp.	Theor.	Start	End
a Asterisk indicates proteins detected in both two egg whites. Proteins detected only in free-range chicken egg white are indicated with (#) sign. Proteins detected for the first time in free-range chicken egg white are indicated with section mark (§).
P49702	ADP-ribosylation factor 5*	217	5/4/18	32.8	19 411	1678.6980	1678.9325	128	142	0	(K)QDMPNAMVVSELTDK(L)
P08250	Apolipoprotein A-I*	226	5/5/23	18.2	17 522	1048.7157	1049.2193	241	249	0	(R)LTPYAENLK(N)
Q90593	78 kDa glucose-regulated protein^#	36.4	5/4/18	8.1	155 514	1537.9069	1537.8265	137	150	0	(K)TFAPEEISAMVLTK(M)
P15505	Glycinedehydrogenase^#	551	9/7/32	15.2	142 275	1413.8618	1413.6381	15	28	1	(R)GAPRHLRPAAGGPR(R)
Q02391	Golgi apparatus protein 1^#	59.2	10/7/32	10.1	157 307	2552.1632	2551.9534	829	848	1	(K)LQETEMMDPELDYTLMRVC(Carbamidomethyl)K(Q)
P09987	Histone H1*	298	4/4/18	26.6	159 972	1654.8826	1654.8469	1	18	0	(−)MSETAPVAAPAVSAPGAK(A)
P00698	Lysozyme C^#	964	5/5/23	36.7	217 798	2129.7899	2129.7823	1	19	1	(−)MRSLLILVLC(Carbamidomethyl)FLPLAALGK(V)
Q8AXY6	Muscle, skeletal receptor tyrosine protein kinase^#	217	6/5/23	10.6	247 982	1654.8826	1654.9153	297	311	1	(K)AAATISVSEWSKLYK(G)
P01012	Ovalbumin^#	1942	8/5/23	29.5	277 728	2326.6172	2326.7314	1	20	1	(−)MGSIGAASMEFC(Carbamidomethyl)FDVFKELK(V)
P01014	Ovalbumin-related protein Y^#	299	4/4/18	11.1	277 727	994.5084	994.2516	278	285	1	(K)SMKVYLPR(M)
						1413.8618	1413.6289	373	384	0	(R)YNPTNAILFFGR(Y)
Q98UI9	Mucin-5B^#	3881	8/6/27	6.1	243 903	1859.7428	1860.1960	1870	1887	0	(K)GVC(Carbamidomethyl)VSEGVEFKPGAVVPK(S)
P20740	Ovostatin^#	80.0	9/8/36	9.1	277 762	994.5084	994.2486	1382	1390	0	(K)MLSGFVPVK(S)
						1413.8618	1413.6646	921	931	0	(R)EETQNFLIC(Carbamidomethyl)MK(D)
						1537.9069	1537.8509	421	433	1	(K)IFDPELSLKALYK(T)
P02789	Ovotransferrin^#	26 938	8/8/36	16.5	463 788	994.5084	994.1017	633	641	1	(K)RFGVNGSEK(S)
Q91348	6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase^#	30.4	5/5/23	15.3	120 743	1583.4363	1583.7700	361	373	1	(R)YPKGESYEDLVQR(L)
P26446	Poly[ADP-ribose] polymerase 1*	238	9/8/36	12.7	282 338	1883.2303	1883.8336	282	298	0	(R)VADGMAFGALLPC(Carbamidomethyl)EEC (Carbamidomethyl)K(G)
						2129.7899	2130.4211	484	502	1	(K)TEHQEVAVDGKC(Carbamidomethyl)SKPANMK(S)
P19121	Serum albumin*	96.9	8/7/32	18.2	12 314	994.5084	994.2296	434	441	1	(K)SILIRYTK(K)
						1558.9205	1558.8455	443	456	0	(K)MPQVPTDLLLETGK(K)
P10039	Tenascin^#	1689	9/7/32	7.9	452 759	1262.5332	1262.5074	195	204	1	(R)NC(Carbamidomethyl)LNRGLC(Carbamidomethyl)VR(G)
						1262.5332	1262.4135	858	868	0	(K)EVFVTDLDAPR(N)
P15989	Collagen alpha-3(VI) chain^#,§	2.30 × 10^7	30/18/82	14.2	66 803	1537.9069	1537.7645	560	573	1	(K)SMDAVEQAAAEMKR(N)
						1545.6119	1545.7431	3030	3043	1	(K)SQPKVTYTGTFSTK(T)
						2326.6172	2326.6628	800	818	0	(K)MVYFMDDFSDLTTLPQELK(K)
P13944	Collagen alpha-1(XII) chain^#,§	1.30 × 10^6	25/13/59	11.8	69 538	1348.3614	1348.4725	2228	2238	1	(R)WSPHRSATSYR(L)
						1545.6119	1545.7027	766	779	1	(R)QVTVSANERSTTLR(N)
P02467	Collagen alpha-2(I) chain^#,§	206	6/4/18	8.5	66 671	1654.8826	1654.8999	412	430	1	(R)AGVMGPAGNRGASGPVGAK(G)
Q90611	72 kDa type IV collagenase^#,§	165	7/6/27	11.2	232 153	1545.6119	1545.6274	313	324	1	(R)WC(Carbamidomethyl)GTTEDYDRDK(K)
P32017	Collagen alpha-3(IX) chain*^,§	66.9	4/4/18	10.2	66 896	2326.6172	2325.6024	203	227	1	(K)EGEKGSPGPPGPPGIPGSVGLQGPR(G)
Q7LZR2	Collagen alpha-1(VIII) chain*^,§	40.9	4/4/18	8.6	66 870	1348.3614	1347.5229	414	428	0	(K)GEGGIVGPQGPPGPK(G)

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Table 5 MS-Fit search results of egg white in barn-raised eggs^a

Accession no.	Protein name	Mowse score	^#pep ^#mat %mat	Cov. %	MS-digest index no.	Peptide MW m/z		Position		M. cut	Sequence
						Exp.	Theor.	Start	End
a Asterisk indicates proteins detected in both two egg whites. Proteins detected only in barn-raised chicken egg white are indicated with (#) sign. Proteins detected for the first time in barn-raised chicken egg white are indicated with section mark (§).
O57579	Aminopeptidase N*	4315	6/6/18	10.2	14 844	1524.7059	1524.7304	669	682	0	(R)AHNVNVTLALNTTR(F)
Q90839	Dickkopf-related protein 3*	23 274	6/6/18	24.6	96 229	1535.7096	1535.6363	59	72	0	(R)NAVQEMEAEEEGAK(K)
Q90593	78 kDa glucose-regulated protein^#	327 721	10/9/26	18.4	155 514	1535.7096	1535.7539	122	136	0	(K)AKPHIQVDVGGGQTK(T)
						1567.8431	1567.7493	59	72	0	(R)ITPSYVAFTPEGER(L)
						1678.0392	1678.8069	80	94	0	(K)NQLTSNPENTVFDAK(R)
						2150.2119	2150.3176	305	322	0	(R)IEIESFFEGEDFSETLTR(A)
P15505	Glycine dehydrogenase^#	5832	8/7/21	12.6	142 275	1559.6078	1559.7738	633	648	0	(R)SAHGTNPASAQMAGMK(I)
Q02391	Golgi apparatus protein 1^#	537	15/12/35	15.1	157 307	1599.6078	1599.8790	324	337	1	(K)LIAQDYKVSYSLAK(S)
P01875	Ig mu chain C region*	15 940	5/5/15	13.5	183 863	1535.7096	1335.7168	141	150	1	(R)RRPTEVTWYK(N)
P00698	Lysozyme C^#	610	5/4/12	36.1	217 798	2171.7799	2171.8199	1	19	1	(−)MRSLLILVLC(Carbamidomethyl)FLPLAALGK(V)
Q8AXY6	Muscle, skeletal receptor tyrosine protein kinase^#	3279	8/7/21	14.1	247 982	2248.0482	2248.0339	827	845	1	(R)NMYSADYYKANENDAIPIR(W)
Q5ZJH2	Nicalin*	3977	7/6/18	15.5	254 388	1414.7226	1414.6605	316	328	0	(R)GNSLHLHVSKPPK(E)
						2130.4930	2130.4835	83	98	1	(R)C(Carbamidomethyl)VMMRLVDFSYEQYQK(A)
P01012	Ovalbumin^#	180 084	11/8/24	29.8	277 728	2285.9692	2285.7061	201	219	0	(R)VTEQESKPVQMMYQIGLFR(V)
P01014	Ovalbumin-related protein Y^#	895	4/4/12	14.4	277 727	995.1070	994.2516	278	285	1	(K)SMKVYLPR(M)
Q98UI9	Mucin-5B^#	1.46 × 10^7	16/14/41	11.3	243 903	1678.0392	1677.8629	941	955	1	(R)IQEIATDPGAEKNYK(V)
						2150.2119	2150.4951	85	103	0	(K)NSHLIYFTVTTDGVILEVK(E)
P20740	Ovostatin^#	514 326	16/11/32	13.8	277 762	1711.7647	1711.9209	762	776	0	(K)ASVSYTIPDTITEWK(A)
P02789	Ovotransferrin^#	2.85 × 10^7	10/10/29	25.2	463 788	1535.7096	1535.8002	141	154	0	(R)SAGWNIPIGTLLHR(G)
						1678.0392	1677.9748	540	553	0	(K)YFGYTGALRC(Carbamidomethyl)LVEK(G)
Q91348	6-phosphofructo-2-Kinase/fructose-2,6-bisphosphatase^#	2220	9/9/26	28.3	120 743	1567.8431	1567.8438	1	16	0	(−)MAAVASGQLTQNPLQK(V)
Q05199	Pro-neuregulin-1, membrane-bound isoform*	1464	6/6/18	16.9	264 094	1535.7096	1535.8139	147	159	0	(K)AFC(Carbamidomethyl)VNGGEC(Carbamidomethyl) YMVK(D)
						2308.7218	2308.5198	118	140	0	(K)ASVIITDTNATSTSTTGTSHLTK(C)
Q8JGM4	Sulfhydryl oxidase 1*	827	4/4/12	8.7	322 688	1265.4434	1265.4637	147	158	0	(R)IAHPTATVADLR(R)
P10039	Tenascin^#	431 586	13/12/35	11.4	452 759	1678.0392	1677.8659	872	885	1	(K)RVSQTDNSITLEWK(N)
P41366	Vitelline membrane outer layer protein 1*	4167	5/5/15	31.7	489 488	2171.7799	2171.6473	3	21	0	(K)VLTPAALILLFFFYTVDAR(T)
P47990	Xanthine dehydrogenase/oxidase*	30 977	14/11/32	18.0	493 835	2552.9333	2552.9467	45	67	1	(K)LGC(Carbamidomethyl)GEGGC(Carbamidomethyl) GAC(Carbamidomethyl)TVMISKYDPFQK(K)
P15989	Collagen alpha-3(VI) chain^#,§	2.62 × 10^10	37/25/74	18.1	66 803	1524.7059	1524.8177	705	717	1	(K)SDIIQRLGQLRPK(G)
						2328.4408	2328.6027	2670	2690	0	(R)VAVLQQAPYDHETNSSFPPVK(T)
P13944	Collagen alpha-1(XII) chain^#,§	1.42 × 10^9	40/22/65	19.3	69 538	2552.9333	2552.8264	2475	2495	1	(K)IASKPSERHVFIVDDFDAFEK(I)
P12105	Collagen alpha-1(III) chain*^,§	108 409	13/11/32	19.5	66 727	1265.4434	1265.4854	1120	1129	1	(K)KHVWFGESMK(G)
						1883.2478	1883.1492	572	591	1	(R)GQPGVMGFPGPKGNEGAPGK(N)
P15988	Collagen alpha-2(VI) chain*^,§	52 993	11/10/29	17.4	66 800	1369.3730	1369.4858	475	489	0	(R)GPTGAVGEPGNIGSR(G)
						1524.7059	1524.6808	676	688	1	(K)LDDERINSLSSFK(E)
P02467	Collagen alpha-2(I) chain^#,§	10 288	7/7/21	10.0	66 671	1414.7226	1414.6112	1285	1297	0	(K)AVILQGSNDVELR(A)
						1678.0392	1677.8252	180	197	0	(R)GHNGLDGLTGQPGAPGTK(G)
P12108	Collagen alpha-2(IX) chain*^,§	6462	10/8/24	13.3	66 893	1265.4434	1265.3735	265	277	1	(R)EGPKGPPGDPGEK(G)
P32018	Collagen alpha-1(XIV) chain*^,§	14 5424	16/13/38	12.1	60 688	1524.7059	1524.7961	497	511	1	(R)GLLGERGVPGMPGQR(G)
						1567.8431	1567.8316	530	543	0	(K)MMQEQLAEVAVSAK(R)
Q90611	72 kDa type IV collagenase^#,§	1434	10/7/21	19.2	232 153	1546.5090	1546.6218	313	324	1	(R)WC(Carbamidomethyl)GTTEDYDRDK(K)
						1678.0392	1677.8819	637	649	1	(K)DQYYLQMEDKSLK(I)
P02457	Collagen alpha-1(I) chain*^,§	18 638	12/9/26	14.0	66 660	1265.4434	1265.3521	1086	1097	1	(K)GETGEQGDRGMK(G)
						1559.6078	1559.7731	753	770	0	(R)GLTGPIGPPGPAGAPGDK(G)
						1711.7647	1711.8182	276	293	0	(K)GEPGSPGENGAPGQMGPR(G)

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The main nutritional characteristics of the two chicken varieties may also be partly responsible for their deferent proteins in egg white. These differences were most probably caused by differences in the intake of feedstuffs of the free-range and barn-raised chicken. The food of free-range chicken can come from a variety of different sources, including their feed, worms, insects, grass, herbs and soil. Barn-raised chickens, however, are fed on a mixture of known feedstuffs prepared from corn, soybean meal, animal-products and synthetic additive.^36,37 Thus, the protein from the animal-products in the mixture of known feedstuffs, such as protein feeds, which are the food products resulting from the hydrolysis of animal tissues such as bones and skin, could be found in the egg white of the barn-raised chickens. Compared to free-range chicken egg white, more big molecular mass proteins exist in the egg white from barn-raised chicken when the feedstuff which is rich in proteins were part absorbed by chickens and the unabsorbed big molecular mass proteins may be accumulated in its tissues. This reason may lead to more species of proteins in egg white from barn-raised chicken than from free-range chicken.

Some other proteins reported previously may be hidden and failed to detect in this current study probably because the fractionation techniques were not adopted.

4. Conclusion

In the present report, PMF approach of identification of protein in egg white were established and successfully applied to identify the proteins in egg white from free-range and barn-raised chicken by MALDI-TOF-MS. Extraction process of protein were optimized using RSM method with BBD. Besides that, enzymatic hydrolysis conditions were optimized, especially that the effect of pH on tryptic hydrolysis of proteins was successfully investigated via dialyzing against PBS of different pH. The experimental results show that the species of proteins in barn-raised chicken egg white was more than in free-range chicken egg white. The collagen peptides were identified in both two egg whites for the first time. The knowledge of the protein composition of egg white may contribute to the food industry and health care. Further detailed study will be necessary to characterize these novel proteins and to determine their possible function.

References

1. Y. Mine, Trends Food Sci. Technol., 1995, 6, 225–232.
2. J. Kovacs-Nolan, M. Phillips and Y. Mine, J. Agric. Food Chem., 2005, 53, 8421–8431.
3. L. Stevens, Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol., 1991, 100, 1–9.
4. L. J. Zimmerman, G. R. Wernke, R. M. Caprioli and D. C. Liebler, J. Proteome Res., 2005, 4, 1672–1680.
5. Y. Hioki, R. Tanimura, S. Iwamoto and K. Tanaka, Anal. Chem., 2014, 86, 2549–2558.
6. M. T. M. Blaze, B. Aydin, R. P. Carlson and L. Hanley, Analyst, 2012, 137, 5018–5025.
7. N. Bergman and J. Bergquist, Analyst, 2014, 1–16.
8. C. Guérin-Dubiard, M. Pasco, D. Mollé, C. Désert, T. Croguennec and F. Nau, J. Agric. Food Chem., 2006, 54, 3901–3910.
9. V. Raikos, R. Hansen, L. Campbell and S. R. Euston, Food Chem., 2006, 99, 702–710.
10. K. Mann, Proteomics, 2007, 7, 3558–3568.
11. C. D. Ambrosio, S. Arena, A. Scaloni, L. Guerrier, E. Boschetti, M. E. Mendieta, A. Citterio and P. G. Righetti, J. Proteome Res., 2008, 7, 3461–3474.
12. D. A. Omana, Y. Liang, N. N. V. Kav and J. Wu, Proteomics, 2011, 11, 144–153.
13. F. G. Silversides and K. Budgell, Poult. Sci., 2004, 83, 1619–1623.
14. J. Schlatterer and D. E. Breithaupt, J. Agric. Food Chem., 2006, 54, 2267–2273.
15. A. Hidalgo, M. Rossi, F. Clerici and S. Ratti, Food Chem., 2008, 106, 1031–1038.
16. M. Roszko, K. Szymczyk and R. Jędrzejczak, Sci. Total Environ., 2014, 487, 279–289.
17. H. C. Lee, A. K. Htoon and J. L. Paterson, Food Chem., 2007, 102, 551–559.
18. C. L. Ye and C. J. Jiang, Carbohydr. Polym., 2011, 84, 495–502.
19. E. Dorta, M. Gloria Lobo and M. Gonzalez, Food Bioprocess Technol., 2013, 6, 1067–1081.
20. B. Du, F. M. Zhu and B. J. Xu, J. Cereal Sci., 2014, 59, 95–100.
21. M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
22. N. Carlsson, A. Borde, S. Wölfel, B. Åkerman and A. Larsson, Anal. Biochem., 2011, 411, 116–121.
23. Q. Z. Jin, X. Q. Zou, L. Shan, X. G. Wang and A. Y. Qiu, J. Agric. Food Chem., 2009, 58, 155–160.
24. R. Minjares-Fuentes, A. Femenia, M. C. Garau, L. A. Meza-Velazquez, S. Simal and C. Rossello, Carbohydr. Polym., 2014, 106, 179–189.
25. Y. Liu, G. L. Gong, J. Zhang, S. Y. Jia, F. Li, Y. Y. Wang and S. H. Wu, Carbohydr. Polym., 2014, 110, 278–284.
26. C. Tokarski, E. Martin, C. Rolando and C. Cren-Olivé, Anal. Chem., 2006, 78, 1494–1502.
27. Y. Y. Li, P. Hao, S. L. Zhang and Y. X. Li, Mol. Cell. Proteomics, 2011, 10, M110–M5785.
28. R. Hynek, S. Kuckova, J. Hradilova and M. Kodicek, Rapid Commun. Mass Spectrom., 2004, 18, 1896–1900.
29. I. D. van der Werf, C. D. Calvano, F. Palmisano and L. Sabbatini, Anal. Chim. Acta, 2012, 718, 1–10.
30. G. Gautier and M. P. Colombini, Talanta, 2007, 73, 95–102.
31. H. T. Yan, J. J. An, T. Zhou and Y. H. Li, Chin. Sci. Bull., 2013, 58, 2932–2937.
32. C. Guérin-Dubiard, M. Pasco, A. Hietanen, A. Q. Bosque, F. Nau and T. Croguennec, J. Chromatogr. A, 2005, 1090, 58–67.
33. J. Wang, Y. Liang, D. A. Omana, N. N. V. Kav and J. P. Wu, J. Agric. Food Chem., 2012, 60, 272–282.
34. M. Jarpa-Parra, F. Bamdad, Y. Wang, Z. Tian, F. Temelli, J. Han and L. Chen, LWT--Food Sci. Technol., 2014, 57, 461–469.
35. A. Shevchenko, H. Tomas, J. Havliš, J. V. Olsen and M. Mann, Nat. Protoc., 2007, 1, 2856–2860.
36. L. D. Coletta, A. L. Pereira, A. A. D. Coelho, V. J. M. Savino, J. F. M. Menten, E. Correr, L. C. Franca and L. A. Martinelli, Food Chem., 2012, 131, 155–160.
37. E. N. Sossidou, A. Dal Bosco, H. A. Elson and C. M. G. A. Fontes, World's Poult. Sci. J., 2011, 67, 47–58.

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