초파리는 유전학, 발달 생물학, 행동 유전학, 노화 연구에 이르기까지 수많은 연구에 이용 되어져 왔다. 최근 초파리 전체 유전자의 염기서열이 보고되었으며, 유전자의 기능 분석과 발현 단백질에 대한 연구가 진행되고 있다. 본 연구에서는 야생형 초파리에서 추출한 단백질을 이차원 전기영동을 통해 400여개 이상의 단백질 스폿으로 분리하였으며, 각 스폿을 적출하여 트립신으로 처리하여 얻어진 펩타이드 단편을 MALDI-TOF-MS를 이용한 펩타이드 지문 검색으로 질량을 측정하였다. 측정된 질량을 초파리 데이터 베이스를 이용하여 분리한 단백질을 동정함으로써 59개의 유전자에서 발현되는 65개의 단백질 스폿을 동정하였다. 이러한 결과는 향후의 발생단계, 외부 자극, 노화 등에 관련되는 특이적 단백질 연구의 기초 자료로 활용될 수 있을 것이다.
초파리는 유전학, 발달 생물학, 행동 유전학, 노화 연구에 이르기까지 수많은 연구에 이용 되어져 왔다. 최근 초파리 전체 유전자의 염기서열이 보고되었으며, 유전자의 기능 분석과 발현 단백질에 대한 연구가 진행되고 있다. 본 연구에서는 야생형 초파리에서 추출한 단백질을 이차원 전기영동을 통해 400여개 이상의 단백질 스폿으로 분리하였으며, 각 스폿을 적출하여 트립신으로 처리하여 얻어진 펩타이드 단편을 MALDI-TOF-MS를 이용한 펩타이드 지문 검색으로 질량을 측정하였다. 측정된 질량을 초파리 데이터 베이스를 이용하여 분리한 단백질을 동정함으로써 59개의 유전자에서 발현되는 65개의 단백질 스폿을 동정하였다. 이러한 결과는 향후의 발생단계, 외부 자극, 노화 등에 관련되는 특이적 단백질 연구의 기초 자료로 활용될 수 있을 것이다.
With the completely discovery of the Drosophila genome sequence, the next great challenge is to extract its biological information by systematic expression and to perform functional analysis of the gene. Here we reported a proteome analysis of D. melanogaster with two-dimensional electrophoresis (2-...
With the completely discovery of the Drosophila genome sequence, the next great challenge is to extract its biological information by systematic expression and to perform functional analysis of the gene. Here we reported a proteome analysis of D. melanogaster with two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The cell extracts of D. melanogaster, $200{\mu}g$ were resolved to more than 400 silver-stained spots by 2-DE. The most abundant protein spots were ranged from 4.0-7.5 of pI and from 15-90 kDa of molecular weight. The excised spots were destained and in-gel digested by trypsin. The masses of the resulting peptide mixtures were measured by MALDI-TOF-MS. Identified proteins were compared with measured peptide mass and a dynamic peptide searching database which is accessible via the internet. The results revealed that identified proteins were produced by 59 genes derived from 65 protein spots.
With the completely discovery of the Drosophila genome sequence, the next great challenge is to extract its biological information by systematic expression and to perform functional analysis of the gene. Here we reported a proteome analysis of D. melanogaster with two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The cell extracts of D. melanogaster, $200{\mu}g$ were resolved to more than 400 silver-stained spots by 2-DE. The most abundant protein spots were ranged from 4.0-7.5 of pI and from 15-90 kDa of molecular weight. The excised spots were destained and in-gel digested by trypsin. The masses of the resulting peptide mixtures were measured by MALDI-TOF-MS. Identified proteins were compared with measured peptide mass and a dynamic peptide searching database which is accessible via the internet. The results revealed that identified proteins were produced by 59 genes derived from 65 protein spots.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
The aim of this study was to create a protein map of adult D. melanogaster that can serve as a reference map for future studies on Drosophila proteins, such as development study through comparison with embryo, disease, response of insecticide or drug etc. Therefore, we will continuously study differential expression of proteins expressed during different development stages.
제안 방법
0. IEF for the treated gel according to preset method of the protein IEF Cell was performed by slightly modified conditions composed of the conditioning step, voltage ramping, and final focusing. The first step was to remove salt ions and charged contaminants at 10, 000 V for 3 h.
melanogaster proteome was separated by 2-DE used 3—10 IPG (inunobilized pH gradient) strip, and abundant protein spots were excised, destained, and then followed by in-gel digestion with trypsin. The masses of peptide mixtures were measured by MALDI-TOF-MS and measured mass data applied to search the protein sequence database for DROSOPHILA MELNOGASTER using the ProteinProspector package program (UCSF). The result of proteome analysis exhibited that D.
대상 데이터
Proteins (200㎍) were separated by IEF using 17㎝ IPG strips (pH 3~10) and subsequently on 12% SDS-PAGE gel; detection by silver staining. The original gel size was 18×20×0.12㎝. X-axis indicates pl values of 3~10, and Y-axis marks protein molecular weight by kDa.
Wild type D. melanogaster and medium was purchased with sangmulnara (http/ /www.biozoa.co.kr). Flies were reared on culture tube in a 12 h/12 h light/dark photoperiod at 25℃ and 50% humidity.
이론/모형
Protein concentrations were determined by the Bradford method[4]z using protein assay reagent (Bio-Rad) diluted five-fold in distilled water. Reagent (990 μl)was mixed with 10 p] protein sample.
성능/효과
The masses of peptide mixtures were measured by MALDI-TOF-MS and measured mass data applied to search the protein sequence database for DROSOPHILA MELNOGASTER using the ProteinProspector package program (UCSF). The result of proteome analysis exhibited that D. melanogaster proteins were identified by 59 genes expressed to 65 protein spots, including transport proteins, enzymes and structural proteins.
참고문헌 (23)
Adams, M. D., S. E. Celniker, R. A. Holt, et al. 2000. The genome sequence of Drosophila melanogaster. Science 287, 2185-2195
Baggerman G., A. Cerstiaens, A. De Loof, and J. Schoofs. 2002. Peptidornics of the larval Drosophila melanogaster central nervous system. J. BioI. Chem. 277, 40368-40374
Bossie, C. A. and M. M. Sanders. 1993. A cDNA from Drosophila melanogaster encodes a lamin C-like intermediate filament protein. J. Cell Sci. 104, 1263-1272
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254
Ericsson, C., Z. Petho, and H. Mehlin. 1997. An on-line two-dimensional polyacrylamide gel electrophoresis protein database of adults Drosophila melanogaster. Electrophoresis 18, 484-490
Gong, I., M. Purl, M. Unlu, M. Young, K. Robertson, S. Viswanathan, A. Krishnaswamy, S. R. Dowd, and J. S. Minden. 2004. Drosophila ventral furrow morphogenesis: a proteomic analysis. Development 131, 643-656
Gorg, A., W. Postel, S. Gunther, J. Weser, J. R. Strahler, S. M. Hanash, J. Somerlat, and R. Kuick. 1988. Approach to stationary two-dimensional pattern: influence of focusing time and immobilinel carrier ampholytes concentrations. Electrophoresis 9, 37-46
Heukeshoven, J. and R. Dernick. 1988. Improved silver staining procedure for fast staining in PhastSystem Development Unit. J. Staining of sodium dodecyl sulfate gels. Electrophoresis 9, 28-32
Langen, H., C. Gray, D. Roder, J. F. Juranville, B. Takacs, and M. Fountoulakis. 1997. From genome to proteome: protein map of Haemophilus injluenzae. Electrophoresis 18, 1184-1192
Messina, A, M. Neri, F. Perosa, C. Caggese, M. Marino, R. Caizzi, and V. De Pinto. 1996. Cloning and chromosomal localization of a cDNA encoding a mitochondrial porin from Drosophila melanogaster. FEBS Letter. 384, 9-13
O'Connell, K. J. and J. T. Stults. 1997. Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests. Electrophoresis 18, 349-359
Ordway, R. W., J. Pallanck, and B. Ganetzky. 1994. Neurally expressed Drosophila encoding homologs for the NSF and SNAP secretory proteins. Proc. Natl. Acad. Sci. USA. 91, 5715-5719
Quadroni, M. and P. James. 1999. Proteomics and automation. Electrophoresis 20, 664-677
Shevchenko, A, M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric sequencing of proteins from silverstained polyacrylamide gels. Anal. Chem. 68, 850-858
Talluri, S., A Bhatt, and D. P. Smith. 1995. Identification of a Drosophila G protein a subunit (dGqa-3) expressed in chemosensory cells and central neurons. Proc. Natl. Acad. Sci. USA. 92, 11475-11479
Theurkauf, W. E., H. Baum, J. Bo, and P. C. Wensink. 1986. Tissue-specific and constitutive alpha-tubulin genes of Drosophila melanogaster code for structurally distinct proteins. Proc. Natl. Acad. Sci. USA. 83, 8477-8481
Vierstraete, E., A Cerstiaens, G. Baggerman, G. Van den Bergh, A De Loof, and J. Schoofs. 2003. Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins. Biochem. Biophys. Res. Commun. 304, 831-838
Vierstraete, E., P. Verleyen, G. Baggerman, W. D'Hertog, G. Van den Bergh, J. Arckens, A De Loof, and J. Schoofs. 2004. A proteomic approach for the analysis of instantly released wound and immune proteins in Drosophila rnelanogaster hemolymph. Proc. Natl. Acad. Sci. USA. 101, 470-475
Weekes, J., C. H. Wheeler, J. X. Yan, J. Weil, T. Eschenhagen, G. Scholtysik, and M. J. Dunn. 1999. Bovine dilated cardiomyopathy: proteomic analysis of an animal model of human dilated cardiomyopathy. Electrophoresis 20, 898-906
Wilkins, M. R., C. Pasquali, R. D. Appel, K. au, O. Golaz, J. C. Sanchez, J. X. Yan, A A Gooley, G. Hughes, J. Humphery-Smith, K. J. Williams, and D. F. Hochstrasser. 1996. From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Biotechnology 14, 61-65
※ AI-Helper는 부적절한 답변을 할 수 있습니다.