Identification and purification of a novel fish allergen from largemouth bass (Micropterus salmoides)

Figures & data

Table 1. Description of fish-allergic subjects.

Subjects	Code	Sex/age (years)	Allergic history	Symptoms	Fish Allergy IgE (IU/mL)
Z.Y.	P1	M/32	FA	Urticaria, vomiting, diarrhoea	27.6
T.R.Y	P2	F/2	FA, EA, MIA,PA, WA	Vomiting, diarrhoea	1.3
W.C.B	P3	F/31	FA, MAA	Asthma	2.4
Y.L.	P4	M/28	FA	Urticaria, asthma	3.3
Z.R.P	P5	M/3	FA, MIA	Asthma	0.7
C.W.H	P6	M/44	FA	Urticaria	2.1
C.D.L	P7	M/24	FA	Urticaria, diarrhoea	2.4
G.N	P8	F/24	FA	Urticaria	2.1
Z.Y.B	P9	F/26	FA, LA	Diarrhea, cramps	1.5
T.H	P10	F/31	FA, MIA	Urticaria, vomiting	0.6
C.Y.S	P11	M/49	FA, EA	Asthma	2.9
Y.Q.L	P12	M/24	FA	Vomiting, diarrhoea	3.7
C.Y.F	P13	M/19	FA	Vomiting	7.1
H.Z.P	P14	M/21	FA	Urticaria, vomiting	9.8
Y.X.L	P15	M/24	FA, MAA	Asthma	1.0

FA: fish allergy; PA: peanut allergy; WA: wheat allergy; LA: legume allergy; MAA: mango allergy; EA: egg allergy; MIA: milk allergy

Table 2. Database search parameters.

Parameter	Parameter selected	Parameter	Parameter selected
Type of search	MS/MS	Fragment mass tolerance	±0.6 Da
Ion search enzyme	Trypsin	Max missed cleavages	2
Fixed modifications	Carboxymethyl (C)	Protein mass	Unrestricted
Variable modifications	Oxidation (M)	Peptide charge state	1 +
Mass values	Monoisotopic	Number of queries	94
Peptide mass tolerance	±100 ppm	Taxonomy

Figure 1. SDS-PAGE and western blot protein profile of the largemouth bass extract. M: molecular weight marker; 1: largemouth bass proteins profiled with Coomassie blue stain; 2: visualisation of proteins transferred to nitrocellulose with Ponceau; 3: western blots incubated with pooled fish-allergic serum; 4–18: western blots incubated with 15 individual sera from patients with fish allergy; 19: negative control serum.

Figure 2. 2DE and IgE immunoblot analysis of largemouth bass allergens. (A) Coomassie blue-stained 2D gel (the prominent 17-kDa and 27-kDa proteins separated into distinct spots are shown by the arrows); (B) IgE immunoblot showing the positive reaction with the 17-kDa and 27-kDa protein spots. 2DE profiles were transferred onto a nitrocellulose membrane and probed with pooled sera from P1–15.

Figure 3. Peptide mass fingerprinting (PMF) of the 17-kDa protein of largemouth bass.

Table 3. Summary report of the top five proteins matched by the Mascot search.

	Accession	Mass	Score	Description
1	gi∣295792242	17206	324	nucleosidediphosphate kinase B [Epinepheluscoioides]
2	gi∣158705971	14368	168	nucleosidediphosphate kinase B
3	gi∣18859071	19558	109	nucleosidediphosphate kinase 3 [Daniorerio]
4	gi∣318206757	17427	109	nucleosidediphosphate kinase 3 [Ictaluruspunctatus]
5	gi∣224069619		74	similar to nucleoside-diphosphate kinase 3 [Taeniopygiaguttata]

Table 4. Effective peptides from MS compared with data in NCBInr.

Observed	Mr (expt)	Mr (calc)	Missed sequence	Sequence
1330.7501	1329.7428	1329.7405	0	R.TFIAVKPDGVQR.G
2096.0796	2095.0723	2095.0482	0	K.YMSSGPILAMVWEGQSIVK.L
1803.8806	1802.8733	1802.8655	0	R.MMLGETNPADSKPGSIR.G

Figure 4. Amino acid sequence alignment of species of nucleoside diphosphate kinase B. Species names and their corresponding GenBank accession numbers are as follows: Epinephelus coioides, ADG29125.1; Gillichthys mirabilis, AAG13336.1; Anoplopoma fimbria, ACQ58455.1; Scyliorhinus torazame, AAD08900.1; shaded amino acids marked are conserved in the four types of NDPK B.

Figure 5. Chromatographic purification of NDPK-B from largemouth bass. (A) DEAE-Sepharose CL-6B chromatography; (B) Sephacryl S-200 gel filtration purification. The numbers at the top of each lane correspond to the fraction number. Target protein fractions under the bars were pooled.