Serum samples were selected from 10 cases of children with BWT (BWT group), 20 cases of children with UWT (UWT group) and 20 cases of healthy children (control group). All of the children were sampled after overnight fasting. The 3 males and 2 females in the BWT group, aged 7–48 months (average 34.20±12.41 months), were confirmed as BWT pathologically. Six males and 4 females in the UWT group, aged 4–56 months (average 41.20±10.12 months), were confirmed as UWT pathologically. The 5 males and 5 females in the control group were aged 4–74 months (average of 34.53±15.52 months). According to WT staging criteria, BWT was in stage V and UWT in stages I–IV. The serum samples standing at 25°C for 1–2 h were centrifuged for 20 min at 2,500 × g, and the supernatant was placed in Eppendorf tubes and preserved at −80°C.

The present study was approved by the Ethics Committee of Zhengzhou University (Zhejiang, China). Written informed consent was obtained from the patient's relatives and/or guardians.

Urea, NaNC, dithiothreitol, acetonitrile, 3-[(3-cholamidopropyl) dimethylammonio)-1-propanesulfonate, trifluoroacetic acid, sinapinic acid and trypsin were purchased from Sigma (St. Louis, MO, USA). Spectra Multicolor Low Range Protein Ladder and SPD SpeedVac were purchased from Thermo Fisher Scientific (Waltham, MA, USA). PBSII SELDI-TOF MS and the WCX2 protein chip were purchased from Ciphergen Biosystems Inc. (Fremont, CA, USA). Ammonium persulfate, 2X/4X Laemmli Sample Buffer, 10X Tris/Glycine/SDS, TEMED, Powerpac Universal and Mini-PROTEAN Tetra vertical electrophoresis system were purchased from Bio-Rad (Berkeley, CA, USA). Matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) was purchased from Kratos Analytica Inc. (Spring Valley, NY, USA). Uhraflex III MALDI-TOF/TOF MS were purchased from Bruker Corp. (Bremen, Germany).

Sera taken from the three groups were thawed on ice-bath for 30–60 min and centrifuged at 8,000 × g. A total of 5 µl of serum was taken and added to 10 µl U9 serum conditioning fluid, mixed fully at 600 times/min for total of 30 min on shaker at 4°C. The serum conditioned with U9 was diluted with binding buffer to 200 µl, and incubated on a shaker at 4°C 600 times/min for 2 min and WCX2 protein chip was applied (1). All-in-One standard protein chip with known molecular weight was used to correct the error of SELDI MS system to <0.1%, and then the protein chip well connected with protein was put into the mass spectrometer for detection (2,3). Ciphergen Biosystems Inc. (Version 3.1) was used to correct the data collected, to homogenize the total ionic strength and molecular weight, and Biomarker wizard and ZUCI protein chip data analysis system were used for de-noising of the signal of original data with non-sampling discrete wavelet transform approach. A mark of signal strength was adjusted according to the three peak values marked and appeared in all selected spectra. The baseline of spectrum was corrected based on the monotonous and aligned minimum curve and quality, and peaks with signal-to-noise ratio >2 were filtered. In the cluster analysis, the minimum threshold was set to 10%, and peaks with proton number/charge number (m/z) difference <0.3% in each sample were clustered as a category (4).

Gene electrophoresis was performed using a protein marker (1.7–40 kDa) in the first lane, and serum samples of the tumor and control groups were added in other lanes (5 µl in each lane) at 90 V. The spacer gel was removed and dyed using Coomassie blue coloring agent overnight, and then de-colored for 1.5 h. The de-coloring liquid was discarded to continue decolorizing for 2.5 h, by comparing with a protein ladder. The corresponding target protein bands were incised in aseptic condition, chopped and preserved for protein identification (5).

The purified protein fluid at different periods of time were, respectively, pulled into an integrated centrifuging enrichment system (SPD SpeedVac) and freeze-dried to 20 µl. Freeze-dried proteins (1.5 µl) were mixed with 1.5 µl CHCA, respectively, and cytochrome C (molecular weight of 12,361.96) + CHCA and insulin (molecular weight of 5,734.51) + CHCA were used to correct the samples. The target board was placed in MALDI-TOF MS for detection and peaks of the target protein were tracked again.

The protein containing the target protein was added with ultrapure water (40 µl) and 4 µl 0.1 mol DTT solution of certain concentration was placed in 37°C warm water for 1 h. The 1.6 µl IAM was added into the mixture and placed in the dark for 1 h, and 1.6 µl of 1 mol DTT solution, 150 µl of 0.1 mol NH₄HCO₃ (ammonium bicarbonate) and 2 µl trypsin were added successively, and placed in 37°C warm water overnight (6–8 h).

Trypsin samples were digested and the substrate CCA was mixed and applied to the target sample for natural drying. The protein chip was placed into MALDI-TOF/TOF MS to detect the peptide fragment of enzymolysis, and Mascot search software (Matrix Science, Ltd., London, UK) was used to retrieve the proteins that may match with the identified peptides and amino acids in the SwissProt database (6).

First, SDS-PAGE was used to extract and separate total protein from the serum sample. Antigen-antibody immunological reaction was carried out after semi-dry trarsmembrane, GAPDH was used as internal reference. The chemiluminescence, developing and photographic fixing operation were carried out, and negative film was retained. After scanning negative film, Image pro plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) was used to measure the gray value of each negative protein band of the film, and the gray values were divided into the BWT, UWT and healthy control groups, and the gray values were statistically assessed.

After noise filtering and clustering analysis, the Wilcoxon rank sum test was conducted against the m/z peaks selected, and the t-test was conducted against the data of each group. P<0.01 was considered statistically significant.

SELDI-TOF MS was applied to screen the serum samples of each group, to obtain a series of data of protein expression intensity, and then the Wilcoxon rank sum test (α=0.01) was conducted against data of the BWT, UWT and healthy control groups, to screen the m/z peaks of differential expression protein. Fourteen differential protein peaks were selected from the BWT and healthy control groups, 8 of which were of high level expression in the BWT group and 4 were high level expression in the healthy control group. Ten differential protein peaks were selected from the BWT and UWT groups, 8 of which were of high level expression in the BWT group and 2 of high level expression in the UWT group. A total of 13 differential protein peaks were selected from the UWT and healthy control group, 11 of which were of high level expression in the BWT group and 2 of high level expression in the UWT group. By comparing the differential protein peaks of the above groups, the difference of the 5,648 kDa m/z differential expression proteins selected between the BWT group and healthy control group was of statistical significance (P<0.01) (Table I), and the difference between the BWT and UWT groups was of statistical significance (P<0.01) (Table II), and the difference between UWT group and the normal group was of statistical significance (P<0.01) (Table III). The protein expression in the BWT, UWT and healthy control group were 100, 200 and 300, respectively.

Peptide fragments of m/z of the 5,648 kDa or protein samples were digested by enzymolysis, respectively, and peptide fragments were detected with the 2D-LC-LTQ-MS system (Fig. 1). According to the results of importing peptide fragments to SEQUEST retrieval program and retrieving in Bioworks database, the peptides were apolipoprotein C-III (APO C-III) with a coverage rate of 57.22% (Table IV).

A total of 18 patients were randomly selected to carry out western blot analysis, including 6 samples from the BWT group, 6 samples from the UWT group, and 6 samples from the control group. Target protein APO C-III appeared in all the bands, statistical analysis on the band gray value of target protein in the 3 groups of sera. The result indicated that the differences between the APO C-III gray values of the APO and control groups, and the differences between the APO C-III gray values of the BWT and UWT groups have statistical significance (Figs. 2 and 3).