| Serum proteomics is an important branch of proteomics, duing to serum proteins have unique properties, the important physiological function and important significance, so serum proteomics attract people's attentions. This topic aimed to establish the technology to deplete high abundance proteins from serum, and develop high performance liquid chromatographic fingerprinting, then, we studied its preliminary application in lung cancer.This experiment developed a centrifugal ultrafiltration method to depletes high abundance and high molecular weight proteins from serum in serum proteomics research. four microliters of human serum was diluted by the addition of 16 microliters of 20%(v/v) acetonitrile, and aliquots of serum ultrafiltered applied onto centrifugal filters with molecular weight cutoff of 100,000,50,000,30,000 and 10,000 respectively. The sample was centrifuged at 8,000g about 15 min, then the filtrate was lyophilized to dryness and resuspended with 100μl ultrapure water.the resuspended filtrate was analyzed by Tricine-SDS-PAGE method and the binary gradient high performance liquid chromatography (HPLC). The electrophoresis analysis showed that comparing with molecular weight cutoff of 100KDa, the bands of the the high abundance and high molecular weight proteins of the filtrate of 50KDa,30KDa,10KDa narrow significantly, and when the molecular weight cutoff decrease from 100KDa to 10KDa the molecular weight of the retained proteins became lower and lower, after centrifuging by centrifugal filters. HPLC results showed that using centrifugal filters with molecular weight cutoff of 50,000 separation, the low abundance proteins were seperated completely,and the number of the peaks was moderate. Repeatability (n=6) results showed that the RSD of relative retention time and relative peak area of the low abundance proteins were<0.45% and 5.49% respectively. The centrifugal ultrafiltration method offers a simple method to deplete high abundance and high molecular weight proteins and enrichment of low abundance and low molecular weight serum proteins.The experiment established and validated acetonitrile precipitation method to deplete albumin from serum. The albumin is precipitated by mixing delipidated serum, water, acetonitrile at ratio of 1:2:4.5 (v/v/v), The removal effects and repeatability were analyzed by One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). We analyzed the gel images of serum samples from 4 healthy controls and 4 non-small-cell lung carcinoma patients before and after treatment. In the last, we handle the serum from a non-small cell lung cancer patients and a normal with the centrifugal ultrafiltration and acetonitrile precipitation, and the handled serum was analysed by HPLC. The results showed that the average concentration of album in the serum decreased from 50.65±2.82 g/L to 2.50±0.71 g/L, the number of album of total proteins decreased from 65.1% to 33.3%, there were significant statistical differences between them (P< 0.01). Compared with the gel images of the original serum with the same amount, the result showed that band of album narrows significantly, thereby the band with low abundance proteins can be seen. Finally, comparing the HPLC spectrum of lung cancer and healthy people, the results revealed a good correlation between both methods. The ACN-depletion strategy offers an economical, simple method to remove human serum albumin, and the effection was confirmed by centrifugal ultrafiltration method. It lie foundation for further studying and exploiting the serum proteomics.The experiment further explored the method to develop high performance liquid chromatographic fingerprinting. We studied mobile phase, column temperature, detection wavelength, and the internal standard peak, finally discussed the repeatability, stability, precision of the method. Under the analytical condition, the retention time of the internal standard peak was about 23 minutes and the peak shape was clear and symmetrical. The internal standard peak was separated completely from other proteins in the sample. We screened 11 peaks from the chromatogram spectra of the control group. Using the relative peak area as a parameter to analyse the repeatability, stability, precision of the method. The relative standard deviations were <5.66%,<4.79%,<2.80% respectively.The experiment further verified the difference of spectra among non-small cell lung cancer patients, pneumonia patients and healthy control group in bulk, samples. We selected 41 cases of gender and age-matched healthy controls,14 cases of pneumonia patients,50 cases of non-small cell lung cancer patients, application of acetonitrile precipitation to remove albumin and other HPA from serum, and separated the low molecular weight LPA using RP-HPLC. Corrected by internal standard, the results showed that the relative retention time between groups had no statistically significant difference. and there was no statistically significant difference between health control group and the pneumonia patients among the eleven peaks in common, but relative peak area of both of them had statistically significant difference comparing with non-small cell lung cancer patients in peak 4, peak 5, peak 6, p <0.001. This experiment provided a basis for screening out the special high performance liquid chromatographic fingerprint of non-small cell lung cancer patients. |
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