| Purpose:1. Establishment the methods of removing high abundancein proteins of nasopharyngeal carcinoma and enrichment of small molecules of protein / peptide .2. Fixing the best multiples of Acetonitrile, and establish the different protein expression profile with nasopharyngeal carcinoma and normal human serum .3. Separate the protein of below 10kDa, analysis and identify the possible biomarker of nasopharyngeal carcinoma .Methods:1. Using different multiples of Acetonitrile (1.5:1,1.2:1) to optimize the method of high abundance proteins removal and polypeptide enrichment in NPC serum proteome .2. Using SELDI-TOF-MS technology, with a CM10 ProteinChip to detect proteins, and the results were compared with those for non-acetonitrile-treated NPC sera. We used these approaches to better understand the protein profiles of acetonitrile-treated and untreated sera from NPC patients and provide a new method for collecting and identifying serum proteins showing abnormal upregulation or downregulation in a specific disease setting.3. Using Tricine-SDS-PAGE electrophoresis, SELDI-TOF-MS and MALDI-TOF-MS/MS identified these gel, in search of the related serum markers of NPC proteins.Results:1. Acetonitrile precipitation could remove mostly of high abundance proteins while retaining more of the small-molecule protein or polypeptide of 10kDa, Tricine-SDS-PAGE can separate well serum small protein or polypeptide, the bands of 10kDa protein are clear..2. Using SELDI-TOF-MS technology, with a CM10 ProteinChip. Under standard conditions, protein peaks with mass/charge (m/z) values ranging from 2000 to 20000, 99 protein or polypeptide peaks were detected in untreated sera from NPC patients, with a detection rate of 18.1% for proteins with MW of 6000–10,000 Da, 11.1% for proteins with MW >10,000 Da, and 5.1% for proteins at a peak density of >5. In contrast, 80 peaks were detected in acetonitrile-treated sera from NPC patients, with a detection rate of 26.2% for proteins with MW of 6000–10,000 Da, 37.5% for proteins with MW >10,000 Da, and 13.8% for proteins at a peak density of >5. Comparison of acetonitrile-treated sera between NPC patients and healthy controls showed that CM10 detected 13 differentially expressed protein peaks at P<0.05 and mean>SD, of which five (5638.8, 8480.3, 8579.0, 8705.1, and 13772.8 Da) were upregulated, and eight were downregulated.The MWs of the upregulated protein peaks (5633.1, 8563.9, 8691.8, and 13738.6 Da) found in the protein profiles of untreated NPC sera were similar to those of acetonitrile-treated NPC sera (5638.8, 8579.0, 8705.1, and 13772.8 Da). Specifically, the patterns for two protein peaks (8563.9 and 8691.8 Da) in untreated NPC sera were the same as those in acetonitrile-treated NPC sera (8579.0 and 8705.1 Da), although the peak intensity was 5-7 times higher in the latter. Because the equipment and methodology were the same in all tests, these four upregulated proteins obtained from acetonitrile and untreated NPC sera can be regarded as the same proteins/polypeptide.3. SELDI-TOF-MS and MALDI-TOF-MS/MS identified the first gel that was 8705.1Da maybe the protein of ApoC-III.Conclusions:1. Protein peaks with mass/charge (m/z) values ranging from 2000 to 20,000, there are differences in the protein profiles of acetonitrile-treated and untreated NPC sera. Of note, a large proportion of the proteins from untreated NPC sera were <6000 Da, while the detection rate of protein peaks >6000 Da was higher in acetonitrile-treated NPC sera, accounting for over half of all protein peaks detected (26.2+37.5%). Most upregulated proteins were found in both the acetonitrile-treated and untreated sera from NPC patients, and the peak value densities increased.2. Acetonitrile is effective in removing most of the high-abundance macromolecular proteins from serum samples of patients with NPC. Acetonitrile treatment can be applied in serum proteomic research and may facilitate detection and identification of important differentially expressed proteins. |
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