| Objective: Magnetic bead purification for the analysis of proteins in body blood serum facilitates the identification of potential new biomarkers with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The aim of our study was to establish a standard proteome fractionation technique for proteomic pattern analysis. To screen a suitable magnetic bead from three kinds of magnetic beads IMAC-Cu, MB-WCX, MB-HIC C8, and use MB-WCX to search biomarkers and evaluate their diagnostic value by comparing different patients′serum protein. To provide a new method of finding biomarkers for breast cancer, while establish the diagnostic model of breast cancer and provide a simple way for the early diagnosis.Methods: 1 The influence of freeze-thaw cycles and the ratio of sample to matrix were evaluated by comparison of their mass spectrum.2 Serum sample′s protein spectrum was detected by MALDI-TOF MS after serum samples were purified by MB-WCX magnetic bead. In order to evaluate the reproducibility of MALDI-TOF MS at different spots, nine proteins in different mass ranges were selected and their CV% was calculated.3 Before large scale patients′serum testing, three kinds of magnetic bead (IMAC-Cu, MB-WCX, MB-HIC C8)were compared about their peak number, peak area and peak intensity of mass spectrum.4 The MB-WCX was selected and used to detect breast cancer patients′and healthy controls′blood serum. After separation and purification by magnetic bead MB-WCX, their mass spectrums were detected by MALDI-TOF MS. During the process, flexControlMS3.0, ClinProToolsTM2.1 and flexAnalysis3.0 software were used in instrumentation control, data analysis and mass spectrum collection. Results: 1 More freeze-thaw cycles had more influence on mass spectrum, especially in small range proteins, so the samples should be operated within 3 cycles in order to get good results.2 Reproducibility of MALDI-TOF test in this study was quite satisfactory; its CV% was within 9.88%-30.07%.3 After comparison, MB-WCX was better than IMAC-Cu and MB-HIC C8 magnetic bead.4 There were 15 main protein peaks were detected whose molecular mass 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 Two protein peaks were of significant difference whose molecular mass were 4964.52Da and 7765.28Da. These two proteins were up-regulated in breast cancer patients, and could be seen as potential breast cancer biomarkers.6 Breast cancer serum diagnostic model were built up and correct rate validation were both more than 80% according to QC, GA and SNN methods.Conclusion: 1 As MALDI-TOF MS is a high-tech method in proteomics analysis, quality control of operating sequence and reduce freeze-thaw cycles in whole procedure is very important.2 MB-WCX was better than IMAC-Cu and MB-HIC C8 magnetic bead in protein purification.3 There was significant difference between the group of breast cancer patients and healthy controls.
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