KISEP Rhinology Korean J Otolaryngol 2006;49:41-6 프로테오믹스 (Proteomics) 를이용한반전성유두종의특이발현단백질분석 이재용 1 이상학 2 Proteomic Analysis of the Specific Protein Expression in Inverted Papilloma Jae Yong Lee, MD 1 and Sang Hag Lee, MD 2 1 Department of Otorhinolaryngology-Head and Neck Surgery, Soonchunhyang University College of Medicine, Bucheon and 2 Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, Korea ABSTRACT Background and Objectives:Inverted papilloma is a benign tumor of nasal cavity and paranasal sinuses with a propensity for local invasiveness, recurrence, and malignant transformation. Proteomics is a powerful tool for protein analysis, providing valuable information on biochemical processes involved in diseases, monitoring of cellular processes, and characterizing the protein expression levels. We tried to find the proteins that are associated with pathophysiology of the inverted papilloma and mechanisms of the disease by proteomic approach. Materials and Method:Normal nasal mucosa and inverted papilloma tissue was obtained during augmentation rhinoplasty and endoscopic surgery, respectively. Total protein was isolated and separated into numerous spots by two-dimensional electrophoresis. Twenty four protein spots that were only detected in inverted papilloma were selected and subsequently analyzed with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Results:About 700 protein spots were detected. Selected spots were analyzed, and various proteins were identified. These include T-cell receptor beta chains, Ca 2+ binding proteins, caltractin, calneuron, ras-related proteins, a rab-2b oncogene family, chloride intracellular channel proteins, tumor protein D53, and tumor necrosis factor precursors. Conclusion:We identified the proteins expressed in the inverted papilloma with proteomic approach. These proteins may help us in understanding the mechanisms of pathogenesis of inverted papilloma, and may be used as possible tumor markers. (Korean J Otolaryngol 2006;49:41-6) KEY WORDS:Proteomics Inverted papilloma. - 41
반전성유두종의특이발현단백질분석 42 - - - - - Korean J Otolaryngol 2006;49:41-6
이재용외 Table 1. Identified proteins that were only expressed in the inverted papilloma tissue. Protein name, sequence coverage rates, number of matched peptides, accession code, isoelectric point and molecular weight are listed Spot No. Protein name Sequence coverage rates No. of matched peptides Accession code pim.wt 01 T-cell receptor beta chain 43 11 4261641 4.68503 02 Apolipoprotein C-II precursor 96 15 4704650 4.92677 03 Ca 2+ binding protein 33 10 4.310445 04 Myosin regulatory light chain 26 08 P10916 4.918789 05 Caltractin 28 09 Q12798 4.819570 06 Calneuron 1 16 13 Q9BXU9 4.824837 07 Ras-related protein RAP-2b 38 15 P17964 4.720505 08 RAP-2b, member of ras oncogene family 38 06 13386338 4.720505 09 Sodium channel beta-3 subunit precursor 18 07 Q9NY72 4.724703 10 Chloride intracellular channel protein 2 23 08 3121853 5.327813 11 Interleukin-1 alpha precursor 20 07 P01583 5.030607 12 G protein signaling regulator 19 26 06 5031705 5.424636 13 Tumor protein D53 29 06 Q16890 5.522449 14 Ras-related protein RAP-1b 28 07 P09526 5.620825 15 Immunoglobulin lambda light chain 19 06 2765427 5.725235 16 Proteasome activator complex subunit 1 17 08 Q06323 5.828723 17 MHC class I antigen 23 10 8925742 6.121095 18 Tumor necrosis factor precursor 18 08 P01375 6.225645 19 Interleukin-6 precursor 34 09 P05231 6.223718 20 Ras-related protein Rab-33B 47 09 Q9H082 6.725718 21 Immunoglobulin heavy chain variable region 36 15 9663229 6.010045 22 Alcohol sulfotransferase 18 11 Q06520 5.733780 23 Dual specificity mitogen-activated protein kinase 3 24 09 1170964 6.236172 24 Serologically defined colon cancer antigen 1 17 08 18088535 6.040439 pi : isoelectric point, MWt : molecular weight - - - - 43
반전성 유두종의 특이 발현 단백질 분석 MWt (Da) 데, 일반적으로 5개 이상의 일치하는 펩타이드와 15% 이 200,000 상의 염기서열차지율을 가진 단백질을 선택하게 된다. 본 연 97,400 구결과 동정된 단백질은 이에 합당한 조건을 가지고 있었다. 66,200 조직을 효소처리하여 분석하면 아직까지 밝혀지지 않거나 명 45,000 tical protein 또는 unmanned protein product라 칭하게 명되지 않은 단백질들도 동정될 수 있는데 이는 hypothe되며 본 연구에서는 이러한 단백질들은 동정되지 않았다. 31,000 고 찰 21,500 14,400 프로테옴(proteome)이란 단어는 어원적으로 단백질체라 6,500 고 풀이될 수 있으나 실제적으로는 게놈(gemone)의 상대 어로서 프로테오믹스는 이러한 protein과 genomics의 합 성어라 할 수 있다.10-12) 이 단어는 1995년 이탈리아 Siena pi 4 7 에서 개최된 이차원적 전기영동학회(2-dimensional elec- Fig. 1. Two-dimensional electrophoresis gel of normal human nasal mucosa. The proteins from the inferior turbinate mucosa were extracted and separated on ph 4to 7 IPG strips, followed by 12% polyacrylamide gel electrophoresis. The gel was stained with silver nitrate. Above 4.5 kda region, protein spots were clouded making a protein band, and below this many scattered protein spots can be detected. trophoresis meeting)에서 Marc Wilkins에 의해 처음 사 용된 이후 보편화되었다. 게놈, 즉 유전체는 각 종(species) 마다 동일하며 정적인 성질을 띄는 반면, 단백질은 어떠한 개체에 주어진 환경에 따라 능동적으로 발현되며 해당 유 전자 없이도 특정 단백질이 발현되거나 유전자의 존재에도 MWt (Da) 200,000 불구하고 단백질이 발현되지 않을 수 있다. 더욱이 유전자 전이후변형이나 단백질간의 상호작용에 의해 전혀 새로운 단 97,400 백질의 형성도 가능하기 때문에 유전체에 대한 연구에서 단 66,200 백질체로의 연구로 관심이 전환된 이유가 여기에 있다고 할 수 있다.13) 프로테오믹스는 크게 이차원적 전기영동과 질량 45,000 분석기를 통한 단백질동정으로 구성되며, 이 중 이차원적 전 기영동은 조직이나 체액내의 단백질을 분리하고 그 발현정 24 8 7 9 10 6 4 5 11 12 도를 알 수 있는 방법으로서 프로테오믹스의 근간을 이루 23 22 31,000 16 13 15 21,500 19 17 14,400 18 14 3 6,500 MS 외에 좀더 정확한 단백질의 동정이 가능한 기계들의 발 전에 의존하여 프로테오믹스는 최근 급성장하고 있다.17)18) 저자들은 프로테오믹스를 이용하여 정상비점막과 반전 성 유두종에서 발현되는 단백질을 알아보고 이러한 단백질 2 1 20 을 비교검토함으로써 가능한 종양표지자의 발견과 함께 반 21 전성 유두종 발생에 관여하는 병태생리학적 기전을 이해하 pi 4 는 기술이다.5-10) 또한 염색기술의 발달과 MALDI-TOF 7 고자 하였다. MALDI-TOF MS를 이용하여 반전성 유두 Fig. 2. Two-dimensional electrophoresis gel of inverted papilloma. Gross pattern of protein spot expression is similar to that of the normal nasal mucosa. Using melanie III software, we detected protein spots that were only expressed in the inverted papilloma. Marked number shows the selected protein spots. 종에서만 특이적으로 발현되는 단백질반점들을 분석하여 동 는 경향을 보이고 있다. 동정된 단백질에 대한 신뢰도는 일 chain, MHC class I antigen, immunoglobulin lambda light 치하는 펩타이드의 수(number of matched peptides)와 염 chain, immunoglobulin heavy chain variable region 등 기서열차지율(sequence coverage rates)에 의해 결정되는 면역조절에 관여하는 단백질, ras-related protein RAP- 44 정된 단백질들은 Table 1과 같다. 동정된 단백질들에 대하 여 살펴보면 caltractin, calneuron, Ca2+ binding protein 등 칼슘조절에 관여하는 단백질들과 T-cell receptor beta Korean J Otolaryngol 2006;49:41-6
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