지수성장기 Helicobacter pylori 26695의 주요 단백체 절대정량과 전사물의 상대정량에 관한 연구
Absolute Quantity of Major Proteome Components and Relative Quantity of Their Transcripts of Helicobacter pylori 26695 in the Exponential Growth
단백체 전사물 nano-UPLC-MS/MS;
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Abstract H. pylori, gram negative spiral and capnophilic bacteria, colonizes exclusively gastric mucosa of human. H. pylori has infected to a half of worldwide population causing various gastroduodenal disorder of mankind like chronic active gastritis, gastric atrophy, and peptic ulcer as well as gastric cancers. However, most of persons infected with H. pylori do not progress to serious diseases. H. pylori-host relationship, therefore, has been exploited to be clear for understanding of bacterial roles on pathological changes of gastric mucosa. Global expression of H. pylori genome has to be investigated for providing the grounding knowleges to understand comprehensively the bacterial adaptation on various environments including host. Here, quantification of proteome and transcriptome components of exponentially-growing H. pylori 26695 was attempted to identify genes being associated with bacterial growth. Absolute quantities of proteomes were obtained by nano-UPLC-MSE. Quantification of transcripts was done by using microarray and RNA sequencing. H. pylori 26695 was grown in the thin-layer liquid culture for 18 h up to an early expinential phase and then harvested, lysed, and subjected to tube gel digestion of trypsin. The clarified peptide solution was separated and analyzed by nano-UPLC-MSE. Protein mixture of Gpb, Adh, Eno and BSA was used as an internal reference standard. Protein quantity of 10 ug was subjected to label-free quantitative proteomic analysis using nano-UPLC-MSE. As a result, proteins of 449 genes could be identified with 16 cycles. The molar quantities of proteins were ranged from 8.07 fmole (HP1533) to 2,296.88 fmole (HP1205) with a mean±standard deviation(STD) of 89.49±171.70 fmole. Protein quantites were ranged from 0.126 ng (HP1311) to 1,078.22 ng (HP0072). Total molar quantity of 449 proteins was 10.134 ug which was nearly the same amount applied for the analysis. Proteins showing high quantity were EF, UreA, AhpC, UreB, GroEL, NapA, FldA, Cat, Sod, Omp2, Omp9, Omp20, Omp28, TagD, FrpB, AND TrxA. Urease consist of identical mole of UreA and UreB. Quantities of UreA and UreB were 731,06 fmole and 1749.11 fmole, respectively, respectively, demonstrating that a large amount of nonfunctional UreB might exist in the bacterial cells. The absolute quantitative information will help to analyze biological and pathological role of H. pylori proteins. Quantification of H.pylori transcripts were analyzed by the microarray techniques and RNA sequencing. With the microarray techniques the ratio of the activity of each gene in total RNA to that in genomic DNA was obtained. Of H. pylori ORFS, 1,527 genes could be analyzed by using H. pylori 26695 microarray of Mycroarray. The mean±STD of 1,527 genes was 1.164±1.201 with a coefficient of variance of 9.4% in which the highest ratio was 30.656 (HP0784) and the lowest was 0.205 (HP0942). Relative copy number of transcripts were obtained by fold-values of the ratios to the lowest ratio value. Of 449 gens idengified quantified by nano-UPLC-MSE, 445 genes have their relative copy number of transcripts so that the correlation between absolute concentrations of proteins and the copy numbers of transcripts could be analyzed. The correlation coefficient (r2) between absolute concentration of proteins and relative copy numbers of correspinding transcripts was 0.0235. Nolk (HP0045) showed the lowest expression of protein per transcript whereas TufB (HP1205) did the highest. Data of RNA sequencing converted to produce the average number of transcript reads of each ORFs annotated in whole genome of H. pylori 26695. Of H. pylori ORFS, 1,397 genes could be analyzed by using the NGS techniques. The mean±STD of 1,397 genes was 99.93±302.3 reads in which the highest was 4,896 (HP0320) reads and lowest was 1.4 (HP0442). Of 449 gens identified quantified by nano-UPLC-MSE, 428 genes had the numbers of their transcript reads so that the correlation between absolute concentrations of proteins and the numbers of transcript reads of each genes could be analyzed. The correlation coefficient (r2) between absolute concentrations of proteins and the read numbers of correspinding transcripts was 0.237. HP1457 showed the lowest concentration of protein per a transcript read whereas VacB (HP1248) did the highest. Conclusively, the absolute quantity of proteomes and the number of transcripts of H. pylori in an exponental growth phase were comprehensively analyzed to provide the grounding knowleges of global expression of H. pylori genome in this study. These data will be used for investstigation of genomic expression, network of gene expression, hypothetical genes, and bacterial adaptation to environments.