Screening And Identification Of Proteins Associated In Children Neuroblastoma

BackgroundNeuroblastoma (NB) is the most common one of malignant solid tumors inchildren, accounting for8%to10%of all childhood tumors, some of the highincidence areas, such as France, Israel and Switzerland, New Zealand, the annualincidence rate of11/1000000(0-15years old), the United States is25/1000000,China and India, reported less than5/1000000. NB originated in sympathetic neuronsof embryonic neural crest cells, small round blue cell morphology, as part of occultoccurrence, and generally no special symptoms, early diagnosis is difficult, and thehigh degree of malignancy, rapid growth, early stage can occur more transfer, andthus diverse clinical manifestations, easy to misdiagnosis and delayed treatment, sothe survival rate is very low. Age, tumor location and extent of tissue differentiationso different biological characteristics and clinical manifestations are very different,and some may be converted into natural healing or benign, but the other children withrefractory partial but very poor prognosis. In the past30years, infantile or early NBprognosis has been significantly improved, but the prognosis of older children are stillvery bad late.There are many factors affecting the prognosis in NB, age and stage is still the mostimportant factor.65%of children with primary tumor in the abdominal cavity, olderchildren in primary adrenal accounted for40%, while only25%in infants. Other common sites of chest and neck. About10%of cases the primary site is not clear.Approximately70%NB onset before age5, after a handful of onset10years of age.NB The current clinical diagnosis of primary tumor suspicious parts of B-, such as CTor MRI, to check the bone marrow metastases must find NB cells, skeletal parts ofsuspicious violations or CT scan or X-ray line radionuclide scanning for tumor biopsyor surgical exploration to obtain tumor tissue pathology examination revealed smallround NB cells can be confirmed. Detection of biological characteristics such as urineVMA homovanillic acid (HVA) were significantly increased. At present, for thetreatment of NB principle more complicated treatment regimens, and continues toexplore and improve in, there is no uniform standard programs and specific measuresfor each patient. Currently identified mainly based INSS COG clinical staging andrisk grouping. Mainly in the surgical treatment with radiation therapy or thebiological treatment, prognosis and tumor staging and more negatively correlated,early diagnosis, early treatment can be seen in children with neuroblastoma tumors, aswell as to improve the efficacy has an important role to improve the prognosis.Serology is currently the easiest non-invasive way of checking the change in serumtumor markers are often older than the performance of imaging. But NB disease, asingle positive rate and specificity index is not high, the JIU each tumor markers canimprove cancer diagnosis accuracy and sensitivity, NES (neuron-specific enolase) iscurrently the first choice for the diagnosis of tumor markers NB material, can becombined with LDH (lactate dehydrogenase), SF (serum ferritin) and other serummarkers, can provide important clinical laboratory based in neuroblastoma tumorstaging, recurrence efficacy evaluation and judgment. At this point we can see,looking for a simple, easy, accurate and early detection methods to detect specificpediatric tumors of the nerve cell tumors in children can get an early diagnosis andthus earlier treatment and long-term survival is now urgent Pediatric Surgeryrequirements.Proteomics is the forefront of the development of medical science, along with thesuccessful completion of the international Human Genome Project, followed byprotein sequence databases have gradually improved, tandem mass spectrometry andproteomics research will combine database searches to identify proteins that can reduce the time and resources consumption, seeking to identify as many proteins,proteomics research to meet the automation and high throughput requirements, andgradually became one of the main methods of human proteome expression profilestudies. Commonly used software SEQUEST and Mascot database search canachieve fast, accurate and effective identification of peptides, proteins and then getthe appropriate information. Proteomics early as1994after the concept was proposedto get a lot of recognition and research. Essentially refers to the large-scale study ofprotein levels through a special method signature. Whether or qualitative changes inthe amount of any disease in any activities of the body will change at the proteinlevel. Therefore, we can change based on the level of protein species to monitorprotein levels in a disease-specific changes in quantity and quality to achieve earlydiagnosis of the disease diagnosis purpose of the system, which is now clinicians andresearchers for the disease, in particular a special important research direction forearly diagnosis of cancer. The finding of a specific protein corresponding to the coreissues of development have become disease research. With the development ofproteomics technology updates, and many disease marker proteins were detected andidentified. Drug targets and disease-specific protein target marker screening methodsapplied proteomics been considered one of the most convenient method effect andthus we can see that the relevant technology is accurate and easy to find feasible NBprovides a means of early detection possible. Proteins at different stages of geneexpression proteomics technology for high-throughput screening to quickly detect theexpression of disease, thereby detecting the diagnostic significance of specificproteins labeled molecules, and these high expression of specific proteins couldprovide the basis for early diagnosis may also provide a target for the treatment ofdisease, is expected to better serve the clinic.Protein fingerprinting technology, also known as surface-enhanced laser desorptionionization time of flight mass spectrometry (MELDI-TOF-MS) technology, the chiprolled chromatography and mass spectrometry techniques, the traditionalchromatographic principles to the design matrix protein chips, enhanced separationability over the biological or chemical principle, to the chip surface using an affinitycapture hydrophobicity, hydrophilicity, and chelating protein or other molecule bonded to the metal to obtain the desired molecular weight of the protein isoelectricpoint, glycosylation important information sites and phosphorylation sites and so on.The laser radiation pulses of the core pool analyte desorption, charged ions areformed, depending on the mass to charge ratio of ions in the instrument field varyingduration of the flight, thereby rendering the spectrum, processed by the computer toform an analog spectrum direct display of various molecular weight and content ofthe sample protein. The spectrum of disease group and the control group werecompared, to find and capture the relevant disease-specific proteins CiphergenProteinChip software or the application of new tools Ciphergen Biosystems analysissoftware packages from all aspects of the control of the chip reader to facilitate thecollection and analysis of data, with automatic reading multiple data comparison, youcan choose the spectrum display and so on. Application software can easily getprotein chip experimental data, as long as the required input parameters, the systemcan automatically collect data and display the spectrum of the detected object. Inorder to make full use of the data collection, analysis software provides a variety ofdisplay, including a scan is used to describe the molecular weight of the proteinspectra, clearly understandable and analog bar graph gel electrophoresis samplespectra. Three-dimensional display mode can be simultaneously evaluated in severalsmall spectral changes of the correlation peak, and development of drug effects on thestudy of disease are particularly suitable. The research method has many advantages,its high sensitivity, simple, fast, high-throughput, small sample volume, through acombination of LC-MS/MS, SELDI-TOF-MS and other new technologies haveidentified many tumors such as breast, liver, pancreatic cancer and othertumor-specific proteins in serum.This study is the application MELDI detect cases of children with neuroblastomain children with normal serum and serum proteins, screening high specificity proteinexpression and application of mass spectrometry technologies to filter out specificproteins were identified. Application of proteomics in several important technicalbasis, including SELDI, MALDI, SPE, HPLC and LC-MS series used by childrenwith neuroblastoma cases serum, SIRS serum and serum of normal childrenqualitative and quantitative comparative analysis of proteins, screening and identification of serum proteins neuroblastoma tumor cells expressed high specificity.ObjectiveScreening and identification of children with neuroblastoma serum specific proteinsto build a more perfect early diagnosis of serum protein fingerprint models.Materials and MethodsSerum samples collected and processed: Collected in our hospital from2011to2013before surgery in children with neuroblastoma tumor serum of30patients, including18males and12females, aged3to13years, mean6.0+0.5years old, did not receivepreoperative chemotherapy, radiotherapy, etc. treatment. After surgery and tissuebiopsy and pathological diagnosis of neuroblastoma, the relevant results are two ormore pathologists confirmed. Normal serum collected30cases of children withneuroblastoma in children of age and sex-matched. Morning fasting serum samples atroom temperature for0.5-1h,3000r/min centrifugation15min, the supernatant wasthen placed in-80℃refrigerator.Method: First, a good deal after a weak positive specimens factor (WCX) beads,handling a sample, add matrix protein chip board point, editing the film readingprocedures, the highest molecular weight set30000Da, optimum range to2000Da~20000Da, tune set optimal laser intensity and the best detection sensitivity, and set theparameters. The chip board into the mass spectrometer using MELDI-TOF-MSplatform to collect screening results. Through analysis software package, to give eachsample m/z peaks, consider the difference is less than0.3%of a class. After thepreliminary results, the m/z peak data between different groups Wilcoxon rank sumtest, P value is smaller, the greater the intensity of the expression of differences, twodifferent samples of the more meaningful. Resulting in tumor group and the normalgroup, the difference was a group of m/z peak, range+0.3%, and then found in thetumor and normal children serum differences in serum proteins. Collected serumtumor2ml, diluted with deionized water to10ml. The peristaltic pump is connected toa tangential flow ultrafilter good, and out of the tube into the sample, forming a loop.EP collection tube into another tube, collecting the filtrate. The filtrate points to10EPtubes, each tube about1ml, spout cover film, leaving small hole into fully frozen at-80℃. After fully solidified, into the freeze dryer full lyophilization. The powder is diluted with deionized water to250ul. After solid phase extraction (SPE) by30%,50%,70%,100%organic phase concentration packaging, and make a record, afterdrying in a vacuum drying apparatus to10ul, adding Sample Buffer5ul, boiled15min,centrifugal5000r/min, the supernatant was collected. After the treated samplesseparated by gel electrophoresis, the gel corresponding to a molecular weight cut-axis,of the type fitted to the EP tube, after washing, bleaching, drying, and finally addingtrypsin digestion of the target protein.10000r/min extracted supernatant wascentrifuged10-15min. Spotting to protein chip board, put MALDI-TOF/TOF, laserbombardment, collect the corresponding peptide by Mascot search software, andSwissProt database connection, comparison, matching, and then get the target protein.ResultsMS data neuroblastoma pediatric serum and normal group after the initial screeningand filtration statistical analysis P values less than0.01m/z peak of11, significantlydifferent from any combination of protein peaks, using SVM screened forecastyouden index value (the difference between the positive rate and negative rate of) thehighest combined model, weed out the m/z5920protein markers located in theneuroblastoma group expression (intensity of6180.6±2328.0), normal children weresignificantly low expression (intensity of419.1±493.0), the difference wasstatistically significant (P <0.01).Serum samples were isolated target protein, purified and digested usingMALDI-TOF/TOF platform peptide mixture was subjected to testing. m/z5920peak is identified as a protein Apo C-III (Apolipoprotein C-III).ConclusionM/z peak at5920proteins were identified as Apo C-III, and the current commonlyused in clinical NES, LDH, SF, etc. associated with tumor markers can build a morecomprehensive model of serum protein fingerprint, early diagnosis of neuroblastomatumor cells, surgical results and prognostic monitoring is important.