Differential Proteomics Analysis Of The Fluoride-resistant Strain Of The Streptococcus Mutans By Label-free Quantitation

Caries is a kind of bacteria-infected disease of the tooth hard tissue, which is also the most common disease occurred in oral cavity. The streptococcus mutans is the main pathogenic bacteria of caries. Fluoride is extensively used for caries preventive. Its effect of cariogenic bacteria is among important mechanisms for the caries preventive of fluoride. For the past few years, concentration of fluoride is increased and interval is shorten in order to improve and maintain high fluorin level, then its possible result is appearance of fluoride-resistant strain. From those patients who had used high concentration of fluoride sodium gel for rampant caries prevention, researchers had successfully insolated the fluoride-resistant Strain of streptococcus mutans. Then, some other investigaters had also insolated the fluoride-resistant strain of streptococcus mutans from Xerostomia patients. These researches suggested that the fluoride-resistant strain of streptococcus mutans could live in some special people. The fluoride-resistant strain of streptococcus mutans has stronger cariogenic ability, include its acidogenic ability and acid resistance. The Genome and proteome have been changed, but idiographic gene and protein are unknown. The fluoride-resistant strain of streptococcus mutans has not been separated in normal people now, but the success of inducement of fluoride-resistant strain in vitro and using the fluoride for long-term suggest the possibility of appearance of fluoride-resistant strain in normao people. The fluoride-resistant strain of streptococcus mutans could reduce the anti-caries effect of fluoride and are more difficult to be suppressed, which had given us a new question in this area focussed by many scholars. So the research about the fluoride-resistant strain of streptococcus mutans is very important, for the using fluoride reasonably and looking for new drug to prevent the caries.Our prophase research about the acid-proof-related genes of ffh, fhs, dgk and dltc from the fluoride-resistant strain of the S.M. showed that these genes have some mutations to different extent compared with that from the parental strain, but we are still short of the data about their encoded proteins. About 2% disease is correlative with gene sequence, but 98% disease is closely correlative with expression of protein.Genes are the carriers of hereditary information, and their encoded products, the proteins, are the executors of biological activities. The rsearch about protein will clarity the mechanism which the life changes in physiological or pathological condition. The behave of protein when protein performs physiologic function is variform and dynamic, but genome is basily unchanged. The same cell, tissue and organ express the protein is incompletely alike at different physiologic condition, development stage and environmental influence. The cell proteome is different at pathologic state and normal physiologic condition. These are studied by differential proteomics.Differential proteomics is used to compare the dynamic and changed the whole of proteome at different space and time, analyze the different proteome in expressing quantity, between protein expression level and modified state differences, study differential protein and its function, protein research technique is very difficult because of diversity and variability of the protein itself, and complexity of the conformation of the protein. Proteomics success depends-largely on its level of technical methods. Differential proteomics does not require to capture all protein, which focuses on meaningful differences to identify proteins, and thus has a very high technically achievable.Of proteins in different states, different environments, under different processing conditions to detect changes in expression, which requires use of quantitative proteomics technology. Quantitative techniques are the best parts of the whole proteomics, this quantitative detection of protein in the cells usually do not have the absolute content, but simply to quantify their relative content. Quantitative Proteomics is a subject which precisely quantitate and identify all proteins of a gene group or all proteins of a complex mixture within the system, which marks the proteomics study has been from simple qualitation to precise quantitation direction.There are two main quantitative methods of proteomics research. One is based on the traditional two-dimensional gel electrophoresis and staining, the other is based on mass spectrometry techniques, including labeling quantitation and label-free quantitation. The first method due to two-dimensional gel electrophoresis own limitations, can not achieve absolute separation of proteins, can not effectively detect proteins with extreme isoelectric points, molecular weight too big and too little, low abundance and membrane proteins, so it will be gradually instead by label-free quantitation of proteomics technology based on mass spectrometry.Seen from the principle, the main strategy of labeling quantitation is that proteins or peptides of compared samples are respectively labeled by light and heavy isotope, so that they have certain differences in the molecular weight, at the same time digested peptides are analyzed by mass spectrometry, according to the strength of isotopic peaks in pairs on the MS relative quantitation is in progress. The principle of label-free quantitation is the study sample is not labeled and digested products of the sample are respectively analyzed by mass spectrometry, then peptides and proteins are relatively quantitated according the peak areas or the total numbers of peptides.Label-free quantitation does not need expensive isotope tags as internal standard, so the experiment consumes lowly. The operation of the sample with label-free quantitation is least, so the sample is the most close to its original state. Label-free quantitation overcomes the defect of labeling quantitation to quantitate the multiple samples, so it has been praised highly by many scientists in the quantitative proteomics,and has been more and more widely applied. Now there are already a series of analysis software about label-free quantitation including the DeCyder MSTM by GE company used in this study, with good quantitative accuracy and credibility. The principle of the DeCyder MSTM is the mass spectrometry data has been translated from spectral peaks form intothe direct-viewing similar bidirectional gelatin maps, on the map each dot represents a peptide, not protein. Then compares the intensity of the corresponding peptide from different samples, relatively quantitates the proteins corresponded by peptide.We studied the differences of protein expression between the fluoride-resistant strain of the streptococcus mutans (UA159) and parent strains with label-free quantitation and the DeCyder MSTM by GE company. We found and identified 67 kinds of evident differential proteins, including 24 proteins up-regulation and 43 proteins down-regulation. These differential proteins included some virulence factors for caused caries, the two related adhesion is glucosyltransferase and Major cell-surface adhesin Pac, the one related Producing acid is L-lactate dehydrogenase, the four related acid-resistant is F-ATPase beta-subunit, Ffh, molecular chaperone DnaK and chaperonin GroEL. We analyzed the differential proteins of biological processes, molecular function and cellular localization with Gene Ontology, and we constructed the data base of differential proteins for the fluoride-resistant strain of the streptococcus mutans (UA159) and parent strains.The innovation of our research was that it is the first time to analyse the differential proteomics of the fluoride-resistant strain and parental strain(UA159) of the streptococcus mutans with the technique of label-free quantitation. On basis of the DeCyder MSTN of GE company and related information searched from the NCBI protein data base of the streptococcus, we found 67 kinds evident differential protein.we have preliminarily constructed the differential proteomics data base of the fluoride-resistant strain and parental strain of the streptococcus mutans. The results could provide the basis for analysing the functions of proteins encoded by caries-related genes from the fluoride-resistant strain and parental strain of the streptococcus mutans.