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Establishment and Analysis of Serum Protein Fingerprint Patterns for Human Breast Cancer by MALDI-TOF MS

Author: ZhaoHongYuan
Tutor: AiJun；DanBaoEn
School: Hebei Medical University
Course: Oncology
Keywords: Proteomics Matrix-assisted laser desorption ionization time-of-flight mass spectrometry MB-WCX Breast Cancer Diagnostic model
CLC: R737.9
Type: Master's thesis
Year: 2010
Downloads: 88
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Abstract

Purpose: the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (matrix-assisted laser desorption effect / ionization time-of-flight mass spectrometry, MALDI-TOF MS) to detect serum samples polypeptide expression profiling, analyze repeatability of clinprot experimental methods and the serum different number of freeze-thaw on the experimental results, in order to establish the MALDI-TOF MS detection method for a follow-up test; comparative analysis weak cation exchange beads (MB-WCX), copper chelating beads (MB-IMAC Cu ), the hydrophobic C8 Beads (MB-HIC C8) spectral acquisition differences in the serum protein peptide separation and enrichment, MB-WCX select the separation of proteins ability to detect breast cancer and serum samples of healthy control group, a high-pass the amount analysis software for data processing, filter out the differences between breast cancer and the healthy control group peptide mass-to-charge ratio peaks, and to establish the diagnosis of breast cancer model, model validation to confirm the diagnosis model has a high degree of sensitivity and specificity, looking for clinical breast cancer serum tumor markers provide a theoretical basis. Methods: 1 MB-WCX separation and purification of the serum samples after freezing and thawing 1,3,5 compare the protein profiles detected by MALDI-TOF MS in order to determine the impact of freezing and thawing of the number of experimental results. 2 MB-WCX serum sample protein separation and purification, MALDI-TOF MS detection, select the proteins of different molecular weight range of 9 repeated measurements compared between groups, and the coefficient of variation in the group in order to examine the reproducibility. 3 Comparative MB-WCX, the MB-iMac Cu, MB-HIC C8 average peaks amount, the average peak area and the average peak intensity of the other parameters to select the isolated protein preferably a bead for purification of a large number of serum samples . 4 Select the separation of proteins better MB-WCX detect serum samples of breast cancer and the healthy control group, and then detected by MALDI-TOF MS protein spectra and comparative analysis of the different groups of patients. Instrument operation, data analysis and image acquisition in the course of the study were used flexControlMS3.0, ClinProToolsTM2.1 flexAnalysis 3.0 software. The effect found differences in protein recognition rate prediction ability and verify the accuracy of, or differences in protein composition after diagnosis to guide clinical application. Results: 1 study found that the number of freezing and thawing of the sample in less than 3 times do not affect the collection of the mass peaks, and there were no significant differences (P GT; 0.05), freezing and thawing the more the number, the greater the impact on the low molecular weight protein or polypeptide. 2 repeatability studies have shown that the coefficient of variation of the standard range of 10.67% to 29.59%, the coefficient of variation of the mixed samples range from 9.88% to 30.07%, good repeatability. 3 is the same as the sample average out the peak amount of MB-WCX treatment and average peak area were significantly better than the MB-IMAC-Cu MB-HIC C8 (P lt; 0.05). 4 Application MB-WCX MALDI-TOF MS detected 15 differentially expressed protein peaks molecular weights were 4964.52Da, 7765.28Da, 3261.95Da 1621.2Da 3192.55Da 7008.09Da 2288.99Da 1741.74Da 3225.04Da , 4363.01Da, 6910.35Da, 7631.46Da, 3935.34Da, 8140.66Da, 4054.5Da. 5 Molecular Weight differences of the two protein peaks of 4964.52Da and 7765.28Da most significantly upregulated in breast cancer patients, it is possible to become a potential early diagnosis of breast cancer markers. 6 distinct proteins to establish the diagnosis of breast cancer model after application QC (Quick classification algorithm), GA (genetic algorithm) and SNN (neural network algorithm) model to verify the model to distinguish between the sensitivity and specificity of breast cancer patients and healthy subjects are higher than 80%. Conclusion: 1 MALDI-TOF MS as a high-sensitivity research platform strict rules to reduce the freezing and thawing can reduce the number of variations, improve the repeatability of the test results and credibility. 2 MB-WCX protein separation is better than MB-IMAC-Cu and MB-HIC C8; MALDI-TOF MS technology can create a high degree of sensitivity and specificity for the diagnosis of breast cancer model.

Establishment and Analysis of Serum Protein Fingerprint Patterns for Human Breast Cancer by MALDI-TOF MS

Abstract

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