Mud crabs (Scylla paramamosain), approximately eight months old and female, were purchased from a local market before tissue samples were collected. Crude extracts were prepared as previously described20. Briefly, approximately 5 g of raw muscle tissues in the crab claws were homogenized in 20 mL of phosphate-buffered saline (PBS) extraction buffer and incubated overnight at 4°C on a constant temperature shaker. Subsequently, the extracts were centrifuged at 5000 × g for 20 minutes at 4°C. The supernatant was transferred to 100K Omega Microsep Advance Centrifugal Devices (Pall, USA - MCP100C41) for protein purification and centrifuged at 5000 × g for 20 minutes at 4°C. The filtered supernatant containing the retained bioactive compounds was preserved at -80°C. Protein concentrations were measured using Pierce™ Bradford Plus Protein Assay Kits (Thermo Fisher Scientific, Massachusetts, USA).

Food allergens were subjected to 10-12% SDS-PAGE, followed by both Coomassie blue staining and electrotransfer into PVDF membranes. SDS-PAGE gels were then incubated in destain solution (10% methanol + 10% acetic acid). The SDS-PAGE gel containing the proteins was placed on a glass plate illuminated by a light box, and a razor blade was used to excise an individual band from the gel and place it into a microcentrifuge tube.

Patient sera (1:10 dilution) were used for immunoblotting, followed by the addition of HRP-conjugated anti-human IgG (ThermoFisher Scientific, USA). Immunocomplexes were visualized with 1-Step™ TMB-Blotting Substrate Solution (Thermo Fisher Scientific).

The protein bands on the SDS-PAGE gel were sliced into small pieces and subjected to destain for 30 minutes until the gel particles became clear. Gel pieces were dehydrated by adding 100 µL of 100% acetonitrile (ACN). Then, ACN was removed and the gel pieces were left to air-dry for 5-10 minutes. The gel pieces were then washed with a 50:50 mixture of 100 mM NH₄HCO₃ and 50% ACN for 10 minutes, which was then discarded. The samples were resuspended in 10 mM DTT in 100 mM NH₄HCO₃ for 45 minutes for reduction, followed by incubation with 50 µL of 50 mM iodoacetamide in 100 mM NH₄HCO₃ and kept in the dark for 30 minutes. The gel pieces were washed as described above. Afterwards, gel pieces were rehydrated on ice with 10 ng/µL trypsin in 50 mM NH₄HCO₃ with 10% ACN at 4°C for 30 minutes. Fifty mM NH₄HCO₃ was added to cover the gel pieces, and the tubes were placed in a 30°C water bath overnight. To terminate the reaction, 30 µL of 1% formic acid was added and centrifuged at 13,000 rpm for 5 minutes to collect the peptide-rich first supernatant in a labeled Eppendorf tube. The gel pieces were incubated with 50% ACN and 5% formic acid for 45 minutes. The tubes were subjected to sonication, followed by centrifugation at 13,000 rpm for another 5 minutes, and the second supernatant was collected in a labeled Eppendorf tube. Later, gel pieces were incubated with 90% ACN with 5% formic acid for 5 minutes. The final supernatant was collected in a labeled Eppendorf tube. Finally, all supernatants were dried via SpeedVac and desalted with a Ziptip C18 column. The pipette was set to 10 µL and attached to the Ziptip column. To prepare the ZipTip for peptide binding, the column was conditioned with 3 washes of 100% acetonitrile and 3 washes of 0.1% formic acid. The sample was loaded onto the column by pipetting the protein digest up and down 10 times. The ZipTip was washed with 6 washes of 0.1% formic acid. The peptides were extracted from the ZipTip by pipetting the elution buffer 10 times in a 4 µL solution containing 60% acetonitrile/0.1% formic acid. The organic phase of the elution buffer eluted the peptides from the resin into the buffer. The sample was subsequently desalted and concentrated in a 4 µL solution.

Tryptic peptides were separated and sequenced by nanoscale liquid chromatography on a nanoAcquity system (Waters, Milford, MA, USA) and MS/MS on an Orbitrap Elite hybrid mass spectrometer (Thermo Electron, Bremen, Germany). For peptide separation, a 25 cm long and 75 μm ID- C18 BEH column (Waters, Milford, MA) packed with 1.7 μm particles with a pore size of 130 Å was used at a flow rate of 300 nL/min and a column temperature of 35°C. Peptides dissolved in Solvent A (0.1% formic acid in water) were loaded into the column and eluted by a 120-minute segmented gradient from 5 to 35% solvent B (acetonitrile with 0.1% formic acid). The peptides were sequenced by the tandem MS/MS method. Initially, the mass spectrometer scanned precursor ions in orbitrap mode (m/z 350−1600, 120,000 resolution at m/z 400, and an automatic gain control (AGC) target at 10^6). The most intense ions were then isolated for CID MS/MS fragmentation and detection in the linear ion trap (AGC target at 10,000). The mass spectrometer was operated using data-dependent scans of MS spectra, which excluded previously selected ions for 60 seconds. Single and unrecognized charge ions were also excluded for MS/MS fragmentation.

Raw MS/MS data were first processed using PEAKS 6 software (Bioinformatics Solutions, Canada). The proteome database in PEAKS was taken from the UniProt Knowledgebase (UniProtKB), accessed in June 2023. Protein ID was identified by searching against the database with known parameters for protein modifications [variable oxidation (M) and fixed carbamidomethylation (C)]. Accepted peptides in protein ID were limited to two missed cleavages, and thresholds for parent mass inaccuracy and precursor mass tolerance were chosen at 10 ppm and 0.6 Da. The false detection rate (FDR) was set at 1%, and identified proteins must have had at least two unique peptides.

The normality of the data was checked with the Shapiro–Wilk test. For continuous variables, data were presented as median ± standard error (SE), and comparisons between the two groups were performed using the Mann–Whitney U test. For categorical variables, data were displayed as N (%), and Fisher's exact test was used to compare the two groups. Correlation was calculated by Spearman's rank correlation. Data analysis was performed using the statistical software packages IBM SPSS 20.0 (Armonk, NY, USA), and JASP (Version 0.18.3) [Computer software]. Graphs were prepared using GraphPad Prism 8 (San Diego, CA, USA). A P-value of less than 0.05 was considered significant.

Table 1

Characteristics	All (N=17)	Crab allergic group (N=12)	Crab tolerant group (N=5)	P-values
Age (*)	29 ± 1 (*)	29 ± 1	27 ± 2	0.79
Sex (female, %)	11 (66.11%)	7 (58.33%)	4 (80%)	0.24
Comorbid fish/mollusk allergy	0	0	0	NA
Total IgE (*)	2411 ± 869.65	2432 ± 772.26	886.15 ± 3056.74	0.48
sIgE to seafood mix (*)	0.35 ± 3.11	0 ± 4.02	0.56 ± 3.02	0.48
sIgE to crab (*)	2.05 ± 5.05	3.40 ± 6.19	0.48 ± 8.86	0.55
Wheal diameter of Der P SPT (*)	7 ± 1.3	8 ± 1.5	5 ± 2.6	0.35
SPT to Der P (+) (*)	16 (88.89%)	12 (100%)	4 (80%)	1
Wheal diameter of Der F SPT	6 ± 1.7	7 ± 2	4 ± 3	0.20
SPT to Der F (+)	15 (83.33%)	12 (100%)	3 (60%)	0.43
Wheal diameter of crab SPT	4 ± 0.5	4 ± 0.5	3.5 ± 1.1	0.79
SPT to crab (+)	15 (83.33%)	12 (100%)	3 (60%)	0.43
Co-sensitization to Der P and crab	15 (83.33%)	12 (100%)	3 (60%)	0.43
Co-sensitization to Der F and crab	15 (83.33%)	12 (100%)	3 (60%)	0.43

Categorical data were shown as N (%). P values between groups were calculated by Fisher’s exact test. (*) Continuous data were shown as median ± SE. P-values between groups were calculated by Mann-Whitney U test.

Table 2

Band No.	MW (kDa) observed	MW (kDa) Predicted	Protein identification	Organism	Accession No.	Coverage of protein sequence
1	20-25	21.9	Sarcoplasmic calcium-binding protein	Scylla paramamosain	I2DDG2|SCP_SCYPA	59
2	25-35	26.7	Triosephosphate isomerase	Macaca mulatta	P15426|TPIS_MACMU	14
3	35-50	40	Arginine kinase	Scylla paramamosain	H6VGI3|KARG0_SCYPA	86
4	50-70	40	Arginine kinase	Scylla paramamosain	C9EIP1|KARG_SCYSE	32
5	70-100	75	Hemocyanin	Scylla serrata	C9EIP1|KARG_SCYSE	12