SW와 TMV는 국립 보건원에서 NCRR 및 NHLBI 지원합니다. BSK와 HS은 국립 보건원의 NINDS와 NIGMS에 의해 지원됩니다.

실험 절차 실험 절차 (그림 2) 아래 단계 현명한 방식으로 설명하는 네 부분으로 구성되어 있습니다. 1 부 : P2X2 수용체의 C - 말단의 Subcloning, 그리고 표현. 우리는 바인딩하는 뇌 단백질을 식별하는 전체 길이 박테리아 P2X2 수용체의 C - 말단을 표명했습니다. <ol><li> P2X2 수용체 (그림 1)의 C ​​- 말단은 (잔류물 353-472) pGEX 4NT1 (GE 생명 과학)로 복제 및 시퀀싱과 검증, PCR에 의해 증폭되었다. </li><li> 재조합 플라스미드가 E.으로 변화되었다 재조합 단백질의 표현에 대한 대장균 (BL21). </li><li> 플라스미드 P2X2 CT - GST를 포함하는 박테리아 (E. 대장균 BL21)의 글리세롤 주식은 멸균 피펫 팁과 스크랩한 적합 선택적 마커 (50 G / ML 암피실린)와 함께 Luria - Bertani (LB) 미디어 5 ML에 주사했다 37C에서 궤도 통에 하룻밤 incubated. </li><li> 600 nm의에서 광학 밀도가 도착할 때까지 밤새 재배 문화는 적절한 항생제 (50 μg / ML 암피실린)와 LB 미디어 250 ML에 주사하여 37C에서 2-3 시간 250 rpm으로 궤도 통에 incubated되었습니다 ~ 0.6-0.7. </li><li> 문화 IPTG의 1 ㎜로 유도 나아가 단백질 표현을위한 3 시간을 위해 37C에 incubated했다. </li><li> 문화 다음 4C (문화의 1ml이 표현 수준을 분석 저장하고 &#39;유도&#39;로 표시했습니다) 15 분 5천그램에 소용되었다. 뜨는은 폐기되었으며 펠렛은 살린 트리스 - EDTA (에틸렌 다이아 민 테트라 초산) (인트) 버퍼 (10 MM 트리스 - HCL, 150 MM NaCl, EDTA (에틸렌 다이아 민 테트라 초산) 1 ㎜의 산도 8.0) 20 ML에 resuspended되었습니다. </li><li> 정지 50 ML 팔콘 튜브에서 15 분 5,000g에 소용되었다. 뜨는은 폐기되었으며 세미 드라이 펠렛은에 - 70C가 하룻밤 냉동했다. </li><li> - 70C 펠렛은 얼음 차가운 용해 완충액 (2 % (W / V) sarkosyl 15 MG 라이 소 자임, 150 MM NaCl, 트리스 pH7.5 50 MM, 5 MM의 DTT 완벽한 테아제 억제제 정제) 40 ML에 resuspended되었습니다 그리고 정지 30 분 얼음 incubated되었습니다. 이 부화하는 동안, 3 ML 글루타티온 - 세파 로스 4B 비즈는 인산 버퍼 살린 (PBS)과 PBS 다음 20 ML T - PBS (0.1 % (V / V) PBS에서 tritonX - 100) 20 ML로 세탁했다. 비즈는 1 ML PBS에서 resuspended되었습니다. </li><li> 세균 현탁액 액체 질소에 3 번 동결 - 해동 및 sonicated (1S ON / OFF, 얼음 3 분 30 마이크론)와 추가 4퍼센트 30 분 (V / V) 트리톤 X - 100, 10 MM MgSO에 대한 incubated되었습니다 4 얼음 2 MM ATP. </li><li> lysate는 (펠렛 1 뜨는의 ML cytosol과 세포막에있는 단백질의 양을 테스트 SDS 페이지에 저장된에서 샘플) 삭제되었습니다 4C 및 펠릿 15 분 2만그램에 소용되었다. 뜨는은 1 시간 (또는 야간) 4C에 대한 주판으로 incubated했다. </li><li> 구슬은 5 분 500g에 늘인되었으며 뜨는가 (예제는 언바운드 분율 저장되었습니다) 삭제되었습니다. 구슬은 T - PBS 다섯 번 (세척은 세척 컨트롤에 대한 저장된)로 세탁했다. </li><li> 구슬 그런 다음 50 % (W / V에게) 슬러리를 제공하기 위해 PBS에 resuspended 및 사용까지 4C에 냉장고에 저장되었습니다. 단백질 농도는 브래드 분석 (제조 업체의 지침에 따라)에 의해 추정되었다. </li></ol> 2 부 : 전체 두뇌 lysates의 준비 P2X2 수용체가 다량으로 뇌 8 표현하는 것으로 알려져 있습니다. 현재 분석에서 우리는 쥐의 뇌 전체 lysates (그림 3)에서 P2X2 수용체와 상호 작용하는 단백질을 식별하는 모색. <ol><li> 성인 쥐 두뇌는 수확 (동물 UCLA IAACUC 승인 프로토콜에 따라과 실험실 동물의 관리 및 사용에 대한 NIH 가이드에 따라 희생되었다)와 즉시 얼음 차가운 PBS (3 배)을 씻어서했습니다. </li><li> 10 ML (수확 뇌의 ~ 5 배 젖은 무게) 150 MM를 포함하는 얼음 차가운 용해 버퍼의 NaCl, 50 MM 트리스 - HCL, pH를 7.4, 10 MM EDTA (에틸렌 다이아 민 테트라 초산), 1 MM EGTA, 1 MM NaF, 1 ㎜ 셀을 lyse하기 나 3 할머니 4, 1 ㎜ PMSF, 5 류펩틴의 μg / ML, 1 % (V / V) NP - 40, 그리고 프로 테아제 억제제 칵테일의 정제는 수확 두뇌에 추가되었습니다. </li><li> 균질 일관성의 혼합물이 달성되기 전까지는 뇌 조직은 유리 다운스의 균 질기와 얼음 homogenised되었습니다. </li><li> 세포 lysate은 깨지지 세포를 제거하는 5 분 2,000그램에 소용되었다. 뜨는은 그대로 organelles 및 멤브레인 집계를 제거하는 60 분 2만9천g에서 다시 수집 소용되었다. </li><li> 즉시 원심 분리 후 뜨는은 깨끗한 튜브로 이송되었다. 전체 뇌 lysate의 단백질 농도는 브래드 포드 분석에 의해 측정되었다 (보통 ~ 15 MG / ML). </li><li> lysate는 aliquoted과에서 - 70C 저장되었습니다. </li></ol> 파트 3 : P2X2 C - 꼬리 관련 단백질의 분리 전체 뇌 lysates에서 P2X2 CT - GST의 바인딩 파트너를 식별하려면 분석 아래로 끌어가 CT - G를 사용하여 수행되었습니다ST 미끼로 글루타티온 세파 로스 4B 비즈를 고정. 컨트롤, 구슬로 바운드 혼자 GST를 사용했습니다. <ol><li> 브레인 lysate는 4C에서 1 시간에 대한 GST 비즈와 precleared했다. 펠렛은 폐기되었으며 뜨는은 추가 분석을 위해 사용되었다. </li><li> GST와 P2X2 글루타티온 세파 로스 4 B 비즈에 바운드 CT - GST (10 μg)의 동등한 양의 용해 버퍼에 4C에서 회전, 쥐의 뇌 전체 lysate에서 precleared solubilised 단백질 100g과 함께 하룻밤 incubated되었습니다. </li><li> 비즈 5 분 1천g에 늘인되었으며 뜨는가 삭제되었습니다. </li><li> 구슬은 용해 버퍼와 한번 세탁하고 수정 Laemli 버퍼에 삶은되었습니다 [4 % (W / V) SDS, 10 % (V / V) 2 - 메르 캅 토 에탄올, 2 % (V / V) 글리세롤, 0.004 % (W / V) bromophenol 청색과 0.125 M TrisCl 산도 6.8] 5 분. </li><li> 샘플은 5 분와 10 % SDS - PAGE (그림 4)로 구분하여 뜨는 위해 5,0​​00그램에 소용되었다. 젤은 SYPRO 루비 단백질 얼룩 젤 (제조 업체의 지시에 따라)과 자외선을 시각 물들일되었습니다. P2X2 CT - 관련 단백질은 미끼로 GST - NULL을 사용하여 복구 사람과 비교했다. 네 생물 학적는 (4 머리과 네 개의 독립적인 끌어 오기 때로는 IE) 수행되었습니다 복제합니다. </li></ol> 4 부 : 단백질의 식별. 겔 전기 영동에 의해 분리된 단백질은 젤에서 excised 및 질량 분석법 12 확인되었습니다 참고 :. 프로세스 전반에 걸쳐 먼지의 부재는 각질 오염을 줄이기 위해 중요합니다. 실험자는 항상, 헤어 네트와 장갑을 얼굴 마스크를 착용하고 장갑을 사용하지 않고 관심의 겔 영역을 터치하지 않습니다. <ol><li> 아래 P2X2 당겨에서 발견한 모든 보이는 단백질 밴드는 깨끗한 면도날로 excised하고 (그림 4) 절단되었습니다. GST - NULL에 젤의 해당 영역 레인 풀다운도의 수준 아래의 단백질의 존재를 확보하기 위해 밴드 얼룩 (GST - NULL 미끼 특정 밴드도 excised 및 식별되었다)에 관계없이 동일한 방식으로 excised되었습니다 얼룩의 감도는보고되지 않았습니다. </li><li> 겔 조각은 200 50 % 50 MM의 탄산수 소 암모늄의 μl (V / V) acetonitrile (ACN)과 RT 15 분 vortexed 튜브 incubated되었습니다. 이 세척 단계를 반복합니다. </li><li> 겔 조각 200 μl acetonitrile (겔 조각이 수축 및 색상의 불투명 될한다)를 추가하여 탈수 있었다. </li><li> Acetontrile 제거하고 젤 조각 ~ 10 분 speedvac에서 건조되었다. </li><li> 겔 조각 그런 다음 조각을 담가에 신선한 10 MM MM DTT/10 트리스 (2 - 카르복시 - 에틸) 충분한 볼륨의 phosphine 하이드로 클로라이드 (TCEP) 30 μl와 incubated했다. 이것은 56C 물을 욕조에 30 분 남았는데. </li><li> 다음 DTT 솔루션 100 μl 500 50 % acetonitrile 솔루션 MM의 탄산수 소 암모늄 및 ammounium의 중탄산염로 씻은 겔 슬라이스로 대체되었습니다. </li><li> 세탁 솔루션은 aspirated과 acetonitrile 200 μl가 젤을 탈수에 추가되었습니다. </li><li> Acetonitrile 그런 다음 제거 100 μl 신선한 100 MM의 iodoacetamide 솔루션 무료 sulfhydryls을 alkylate에 추가되었습니다. 이 단계는 어둠 속에서 실온에서 25 분 진행. </li><li> Iodoacetamide 솔루션은 제거하고 vortexing하면서 겔 조각은 2 분 50 % acetontrile (산도 8.0) 50 MM의 탄산수 소 암모늄 200 μl로 세탁했다. 이 세척 단계가 반복되었다. </li><li> 세척 용액을 제거한 후 겔 조각 그러면 ~ 10 분 대한 speedvac에 의해 제거되었습니다 200 μl acetonitrile와 incubated했다. </li><li> 이 단계에서는 겔 조각은 트립신의 소화를 위해 준비가되어 있습니다. 젤 조각은 30 μl 트립신 용액 (20 μL / NG 작업 농도)에 incubated 잘 부푼 수 젤류 10 분 얼음에 배치했다. 초과 트립신은 제거하고 그들은 37C에서 12~16시간 위해 진행하는 허용했다 소화에 걸쳐 포장되어 보관 50 MM의 탄산수 소 암모늄 30 μl와 중첩 겔 입자를 rehydrated했다. </li><li> 5 % 다섯 μl (V / V) 개미의 산성은 트립신과 체포 소화를 비활성화에 추가됩니다. </li><li> 튜브는 ~ 15 분 vortexed 후 깨끗하고 표시 0.5 ML의 약병으로 전달했던 튜브의 바닥에 액체를 가지고 간단히 centrifuged했다. </li><li> 그들은 단지 간단한 원심 분리로 구분하여, 10-15 분 두 vortexing 다음 적용 너무나도 50 % acetonitrile에 0.1 %의 향후 30 μl (V / V) 개미의 산성은 겔 조각에 추가되었습니다. 이 단계는 단계 사이의 산성 - acetonitrile 개미의 교체, 한 번 반복했다. </li><li> 최종 액체는 하나의 약병에 제거하고 모든 추출 솔루션을 결합했다. 이 볼륨은 speedvac에서 ~ 30 UL로 축소되었다. 샘플은 현재 대량 분석법에 의해 반대로 위상 나노 액체 크로마 토그래피 및 단백질 식별을 위해 준비했습니다. 이 특정 예제에서,이 단계는 ThermoF에서 Orbi - 트랩 탠덤 질량 분석기에서 수행됩니다다른 모델 및 제조 업체의 악기가 쉽게이 시점에 설명된 방식으로 결합 수 있지만 isher (높은 질량 정확성, 단백질 검출을위한 빠른 듀티 사이클 일반적으로 강력한 운영 기능 선택). </li><li> 대량 spectrometric 분석에 대한 자세한 내용 및 기준은 이제 간단히 소개하는 부분이있다. 모든 펩티드는 반대 위상 나노 LC (Eksigent)와 펩티드가 3 μl / 분 유량에서 C18 리버스 위상 칼럼에 로드된되었다로 구분되었다. 모바일 위상 0.1 % 개미 산성과 물에 2 % ACN했습니다, 모바일 위상 B는 0.1 % 개미 산성과 ACN 20 %의 물이되었다. 펩티드는 220 NL / 분 5 분 이상 95 % B로 다음 90 분 이상 50 % B로 5 % B에서 선형 그라디언트를 사용 유량에있는 기둥에서 eluted, 그리고 마지막 5 분 지속적인 95 % B를 유지했다 . 스펙트럼은 MS / MS 검사에 대한 MS의 검색 및 LTQ 사용되는 Orbi - 트랩과 함께 데이터 종속 모드로 인수되었습니다. 펩티드는 Bioworks 소프트웨어 패키지에 통합된 SEQUEST 알고리즘을 사용하여 쥐의 IPI 데이터베이스 v.3.53에 대한 스펙트럼을 검색하여 확인되었다. 각 펩티드는 다음 기준을 충족 : XCorr ≥ 2 (+1), ≥ 3 (+2), ≥ 4 (3), 그리고 DeltaCN&gt; 0.1. 단백질 식별에 사용되는 모든 스펙트럼은 수동 검사 및 채 2 펩티드가 확인되었을 때 어떤 단백질 허용되지 않았습니다. 단 단백질은 GST - NULL 끌어 오기 다운즈 동안 네 개의 P2X2 끌어 오기 양지 각각 결석 다음 현재는 추가 분석을 위해 고려되었다. </li></ol><img src="/files/ftp_upload/1178/1178fig1.jpg" alt="그림 1" /> 그림 1. P2X2 수용체의 subunit의 도식 표현. A.이 만화는 P2X2 수용체의 subunit의 토폴로지를 보여줍니다. cytosolic 도메인 N과 C 테르 미니 (Termini)로 구성되어 있습니다. P2X2 수용체 (적색)의 C - 말단은 분석 풀다운을위한 미끼로 사용되었습니다. 본 연구에서 사용되는 P2X2 수용체의 C - 말단의 B. 아미노산 서열은. <img src="/files/ftp_upload/1178/1178fig2.jpg" alt="" /> 그림 2. 플로우 차트와 표현에 사용되는 프로토콜의 시간 라인, 정화는 풀다운 및 단백질의 식별. 우리는 시간 라인 프로토콜의 개요를 보여줍니다. 성인 쥐 두뇌 lysate은 즉시 assays 풀다운을 위해 사용하기 전에 신선 준비했습니다. <img src="/files/ftp_upload/1178/1178fig3.jpg" alt="" /> 그림 3. 쥐의 두뇌에 P2X2 C - 터미 너스 네트워크의 이진 proteomic 분석의 도식 표현. GST 비즈에 바운드 GST 단백질과 융합 P2X2 수용체의 C - 말단은 assays 풀다운을 위해 사용되었다. 브레인 lysate 준비되었고 단백질은 재조합 단백질 고정화와 incubated했다. 언바운드 분율은 용해 버퍼로 세탁했다. 단백질은 질량 분광법으로 확인되었습니다. <img src="/files/ftp_upload/1178/Fig%204%20Singh%20et%20al.jpg" alt="" /> 그림 4. P2X2 수용체의 C - 말단의 바인딩 파트너의 식별. GST (파란색 상자)와 융합 수용체의 C - 말단과 상호 작용하는 단백질 putative의 스펙트럼을 보여주는 Sypro 스테인드 젤. 혼자 혼자 GST (노란색 상자)과 글루타티온 세파 로스 비즈에 대한 컨트롤 차선도 표시됩니다. 화살표 대량 분석법에 의해 더 분석을위한 excised되었습니다 독특한 밴드의 예를 나타냅니다.

<ol>
	<li>Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion channels of excitable membranes. , (2001).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors+and+ATP+signalling+at+synapses.">Molecular physiology of P2X receptors and ATP signalling at synapses.</a> Nat Rev Neurosci. 2, 165-174 (2001).</li><li>Egan, T. M., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+calcium+ions+to+P2X+channel+responses.">Contribution of calcium ions to P2X channel responses.</a> J Neurosci. 24, 3413-3420 (2004).</li><li>Burnashev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+permeability+of+ligand-gated+channels.">Calcium permeability of ligand-gated channels.</a> Cell Calcium. 24, 325-332 (1998).</li><li>Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling.">Calcium signaling.</a> Cell. 131, 1047-1058 (2007).</li><li>Levitan, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+protein+complexes+associated+with+neuronal+ion+channels.">Signaling protein complexes associated with neuronal ion channels.</a> Nat Neurosci. 9, 305-310 (2006).</li><li>Khakh, B. S., North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X+receptors+as+cell-surface+ATP+sensors+in+health+and+disease.">P2X receptors as cell-surface ATP sensors in health and disease.</a> Nature. 442, 527-532 (2006).</li><li>Roger, S., Pelegrin, P., Surprenant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+of+P2X7+receptor+currents+and+membrane+blebbing+via+constitutive+and+dynamic+calmodulin+binding.">Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding.</a> J Neurosci. 28, 6393-6401 (2008).</li><li>North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors.">Molecular physiology of P2X receptors.</a> Physiol Rev. 82, 1013-1067 (2002).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+union+of+pharmacology.+XXIV.+Current+status+of+the+nomenclature+and+properties+of+P2X+receptors+and+their+subunits..">International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits..</a> Pharmacol Rev. 53, 107-118 (2001).</li><li>Young, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+shape,+architecture+and+size+of+P2X4+receptors+determined+using+fluorescence+resonance+energy+transfer+and+electron+microscopy.">Molecular shape, architecture and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.</a> J Biol Chem. 283, 26241-26251 (2008).</li><li>Edmondson, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+epsilon+signaling+complexes+include+metabolism-+and+transcription/translation-related+proteins:+complimentary+separation+techniques+with+LC/MS/MS.">Protein kinase C epsilon signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS.</a> Mol Cell Proteomics. 1, 421-433 (2002).</li><li>Evans, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthosteric+and+allosteric+binding+sites+of+P2X+receptors.">Orthosteric and allosteric binding sites of P2X receptors.</a> Eur Biophys J. 38, 319-327 (2009).</li></ol>

이온 채널은 필수적인 멤브레인 단백질의 주요 클래스입니다. 그들은 선택적으로 플라즈마 막 걸쳐 자신의 전기 그라디언트 아래 이온의 움직임을 허용 물이 가득 모공이 포함되어 있습니다. 개방과 폐쇄 상태 사이의 이온 채널 게이트. 게이팅 단계 P2X의 리간드 게이 티드 이온 채널의 경우에는 송신기 (예 : ATP)에 의해 트리거됩니다, 또는 그것은 다른 단백질과 상호 작용에 의해 규제 수 있습니?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetonitrile</td>
<td>Reagent</td>
<td>JT Baker</td>
<td>9829-02</td>
<td>&nbsp;</td>
<tr>
<td>Acrylamide</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0156</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1507-01</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Bicarbonate</td>
<td>Reagent</td>
<td>Fluka</td>
<td>09830</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Persulphate (APS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3678</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine Triphosphate (ATP)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>Bradford reagent</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>B-392</td>
<td>&nbsp;</td>
<tr>
<td>Commassie blue R-250</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-24972</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotritol (DTT)</td>
<td>Reagent</td>
<td>EMD</td>
<td>3860</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1474-01</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene Glycol tetraacetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E8145</td>
<td>&nbsp;</td>
<tr>
<td>Formic acid</td>
<td>Reagent</td>
<td>EMD</td>
<td>11670-1</td>
<td>&nbsp;</td>
<tr>
<td>Glutathione Sepharose 4B beads</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-5132-01</td>
<td>&nbsp;</td>
<tr>
<td>Hydrochloric acid (HCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H1758</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>15502</td>
<td>&nbsp;</td>
<tr>
<td>Iodoacetamide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I1149</td>
<td>&nbsp;</td>
<tr>
<td>Luria-Bertani (LB) Media</td>
<td>Reagent</td>
<td>EMD</td>
<td>1.00547.5007</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L8511</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>Reagent</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Sulphate (MgSO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride (NaCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Flouride (NaF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7920</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Orthovanadate (Na3VO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S6508</td>
<td>&nbsp;</td>
<tr>
<td>Nonidet P40</td>
<td>Reagent</td>
<td>Fluka</td>
<td>74385</td>
<td>&nbsp;</td>
<tr>
<td>Phenylmethanesulphonylfluoride (PMSF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor tablet</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Protein standard </td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0305</td>
<td>&nbsp;</td>
<tr>
<td>Sarkosyl</td>
<td>Reagent</td>
<td>Acros</td>
<td>61207</td>
<td>&nbsp;</td>
<tr>
<td>Screw top vial</td>
<td>Tool</td>
<td>Agilent Technologies</td>
<td>5182-0866</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4509</td>
<td>&nbsp;</td>
<tr>
<td>SYPRO&#174; Ruby protein gel stain</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>170-3125</td>
<td>&nbsp;</td>
<tr>
<td>N,N,N',N'-Tetramethylethylenediamine (TEMED)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9281</td>
<td>&nbsp;</td>
<tr>
<td>Tris base</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5927</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>Reagent</td>
<td>Burdick&Jackson</td>
<td>365-4</td>
<td>&nbsp;</td>
<tr>
<td>LTQ-Orbitrap tandem mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nano Liquid Chromatography System</td>
<td>Tool</td>
<td>Eksigent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B-Mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

리간드 - 게이 티드 이온 채널은 신경계 1 시냅스 커뮤니케이 션을 기초. 포유 동물에서 리간드 - 문이 채널의 세 가족이 있습니다 cys 루프, 글루 탐 산염 - 게이 티드과 P2X 수용체 채널 2. 각각의 경우에 송신기의 바인딩은 이온들이 전기 그라디언트를 흘러이를 통해 기공의 개통에 이르게한다. 많은 리간드 - 문이 채널은 또한 하류 채널 개방 시간을 초과할 수 있습니다 역할 5 (예 : 유전자 규정)을 신호가 칼슘 이온 3, 4에 투과 수 있습니다. 따라서 리간드 - 문이 채널 몇 밀리초에서 일에 이르기까지 광범위한 시간 단위 이상의 신호 수 있습니다. 그것이 자신이 단백질에 의해 규제하는 방법 리간드 - 게이 티드 이온 채널을 이해하기 위해 필요하며, 어떻게 이러한 단백질이 중요한 역할을 감안할 때하면 신호를 조정할 수 있습니다. 최근 연구 많은 경우 전부는 아니 채널이 단백질 신호 단지 6 일부가 될 수있다하는 것이 좋습니다. 이 문서에서 우리는 P2X2 수용체 cytosolic 도메인의 C - 말단 부분에 바인딩 단백질을 식별하는 방법에 대해 설명합니다. P2X 수용체는 ATP - 게이 티드 양이온 채널이 일곱 subunits (P2X1 - P2X7)로 이루어져 있습니다. P2X 수용체 널리들이 흥분성의 시냅스 전송 및 신경 전달 물질 릴리스 7 presynaptic 촉진을 중재 두뇌에 표현됩니다. P2X 수용체는 고르기가 아닌 고르기 세포에서 발견의 연결 신호, 염증과 심장 혈관 기능 8 키 역할을 중재하고 있습니다. P2X2 수용체는 신경계 9 풍부하고 본 연구의 초점입니다. 각 P2X의 subunit는 세포외 지역 7 세포내 N과 C 테르 (그림 1A) 7로 구분이 멤브레인 스패닝 세그먼트를 (TM1 &amp; TM2) 소유로 생각됩니다. P2X의 subunits 10 (P2X1 - P2X7)는 아미노산 수준 11시 30~50% 시퀀스 상동을 보여줍니다. P2X 수용체가 ionotropic 수용체 중에서 가장 단순한 stoichiometry는 단지 세 subunits를 포함합니다. P2X2의 C - 말단 120 아미노산 (그림의 1B)로 구성되어 있으며, P2X2 수용체는 신호 단지의 일부가 될 수있는 가설을 지원하는 여러 단백질 도킹 합의 사이트가 포함됩니다. 몇 가지 기능이 P2X2 수용체 9 C - 말단에 기인하고 있지만 그러나, 연구는 분자 파트너 설명도 없어 그 전체 길이 C - 말단을 통해이 단백질의 세포 내쪽으로 몇 가지. 이 방법은 종이에 우리는 전체 길이 P2X2 수용체의 C - 말단과 상호 작용하는 단백질을 식별할 수있는 proteomic 방법을 설명합니다.

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

우리는 ATP - 문이 P2X2 수용체의 전체 길이 C 터미 너스에 바인딩 뇌 단백질을 식별하는 간단한 프로토콜을 설명합니다. 모든 P2X 수용체에 대한 확장 및이 방법의 체계적인 응용 프로그램은 P2X 수용체 신호의 더 나은 이해로 이어질 것으로 예상된다.

P2X2 리간드 - 게이 티드 양이온 채널과 상호 작용하는 단백질을 확인하는 Proteomics

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

proteomics-to-identify-proteins-interacting-with-p2x2-ligand-gated

Research

JoVE Journal

Biology

14.6K 조회수.  David Geffen School of Medicine, University of California, Los Angeles. 우리는 ATP - 문이 P2X2 수용체의 전체 길이 C 터미 너스에 바인딩 뇌 단백질을 식별하는 간단한 프로토콜을 설명합니다. 모든 P2X 수용체에 대한 확장 및이 방법의 체계적인 응용 프로그램은 P2X 수용체 신호의 더 나은 이해로 이어질 것으로 예상된다.

비디오: Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

연구

생물학

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR - 클립 - RNA 단백질의 바인딩 Transcriptome 전체 바인딩 사이트를 식별하는 방법

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Coregulated 진스 잠재적인 규제 모티브를 식별하기 위해 범위를 사용하여

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

펩타이드 면역 친 화성 농축을 사용하여 단백질의 양을 정함은 질량 분광법과 함께

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Lipidic Bicelle 방법을 사용하여 막 단백질의 높은 처리량 결정

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

성장 팩터 Progranulin와 상호 작용하는 단백질을 식별하기 위해 수정된 효모 - 2 하이브리드 시스템

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

포유류의 핵 수용체 잔류를 확인하기 위해 효모 두 하이브리드 시스템을 반전하는 리간드 및 / 또는 길항제와 상호 작용

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

종합 달팽이 프로테옴을 확인하는 상향식 (bottom-up)과 샷건 단백질 체학 (Proteomics)

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

세포 표면 단백질 체학 (Proteomics)에 대한 글리코 펩타이드 캡처

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.