이 작품은 암 대책, 5U24CA126477 - 04에 대한 임상 Proteomic 기술에 의해 지원되었다.

1) 렉틴과 - 복합 POROS 열 준비 <ol><li> 1.2 - 1.1 단계 동안 POROS - AL 비즈의 흡입을 방지하기 위해 마스크를 쓰세요. POROS 비즈의 원하는 금액 (100 MG beads/300 μl 최종 볼륨)을 무게와 깨끗한 eppendorf 튜브에 전송할 수 있습니다. </li><li> 1 ML 인산 버퍼 호수 (PBS)의 추가로 구슬을 씻으십시오. 3 분 최대 속도 microcentrifuge에서 원심 분리하여 펠렛 비즈. 뜨는을 제거하고 세척을 반복합니다. </li><li> unconjugated 렉틴과의 원하는 금액 (1-4 MG / 200 μl 구슬)을 무게와 깨끗한 eppendorf 튜브에 전송할 수 있습니다. 5 형태로 PBS를 추가 - 20 MG / ML 솔루션. 이 솔루션의 보호 25 μl. </li><li> POROS 비즈에 남아있는 렉틴 솔루션을 전송합니다. 50 mm의 최종 농도 나트륨 cyanoborohydride를 추가합니다. 락커에 튜브를 삽입하고 실온에서 하룻밤 반응. (참고 :. 나트륨 cyanoborohydride은 독성이며 퓸 후드에서 처리되어야합니다 오염된 폐기물은 적절히 폐기해야합니다.) </li><li> 단계 1.2로 펠렛 POROS 비즈. 뜨는을 제거하고 사후 활용 솔루션으로 저장합니다. </li><li> 1 ML 담금질 버퍼 (1 M 트리스 - CL, 산도 7.4)으로 구슬을 씻으십시오. 단계 1.2에서 안전하게 같은 펠렛 비즈가 뜨는 폐기합니다. </li><li> 1 ML 담금질 버퍼로 POROS 비즈에 남아있는 반응 사이트를 차단합니다. 50 mm의 최종 농도 나트륨 cyanoborohydride를 추가합니다. 락커에 튜브를 삽입하고 30 분 실온에서 알을 품다. </li><li> 펠렛 단계 1.2로 구슬과 뜨는 폐기. </li><li> 1 ML 1 M NaCl과 구슬을 씻으십시오. 펠렛 비즈와 뜨는 폐기. 오 세척 총 네 번 반복합니다. 구슬 이제 팩을 준비가되었습니다. </li><li> POROS 비즈로 복합했습니다 단백질의 양을 확인하려면 사전 복합 및 사후 복합 렉틴 솔루션에 단백질 농도 분석을 수행합니다. 농도의 차이는 구슬로 복합했습니다 단백질의 금액입니다. 비드 볼륨 당 복합 단백질의 금액은 구슬에 렉틴의 농도 같습니다. 대상 렉틴과의 농도는 2 -20 MG / ML 사이입니다. </li></ol> 2) 피크 칼럼에 렉틴 - 복합 POROS 비즈 포장 <ol><li> 포장 시스템 (설명이 다음)를 조립하고 금속 링 스탠드에 지원합니다. 포장 시스템은 바닥에서 위로, 압력 제한기, 최종 칼럼 커플러 1, 프릿, 열 (2 X 50mm), 최종 열 커플러 2 열 커넥터, 최종 칼럼 커플러 3 열 (4.5 X 50mm)로 구성되어 있습니다 최종 칼럼 커플러 4 및 최종 피팅. 상단 열은 포장의 저수지 역할을합니다. </li><li> 버퍼의 원하는 볼륨의 POROS 비즈를 Resuspend (10 MM 트리스 버퍼, 산도 7.4, 150 MM 나트륨 염화물, 10 MM의 염화칼슘, 10 MM 마그네슘 염화물). 400 μl 버퍼 A.에 resuspend 하나의 열로 표시 (~ 200 μl 침대 볼륨)을 포장하는 경우 </li><li> 상단 열 (저수지)에 복합 POROS 비즈를 전송합니다. 버퍼가 칼럼의 맨 위에 도달 때까지 저수지에 버퍼 추가합니다. 부드럽게 컬럼에서 공기 방울을 피하기 위해 노력하고, 컬럼의 상단에 최종 피팅을 넣으십시오. HPLC 시스템 상단 열의 최종 피팅에 연결합니다. </li><li> 시스템을 통해 버퍼를 흐르는하여 열을 팩. 0.5 ML / 분 유량을 시작합니다. 4 ML / 분 또는 최대 압력 (3000 PSI) 중 하나에 도달할 때까지 0.5 ML / 분 각 분에 의해 유량을 향상시킬 수 있습니다. 버퍼의 적어도 35 ML이가 열이 통과하기 전까지 가능한 최대 유량에서 계속합니다. </li><li> 펌프를 끄고 &lt;20 PSI 드롭으로 컬럼에 압력을 수 있습니다. </li><li> 부드럽게, 상단부터, 포장 시스템을 분해. 최종 칼럼 커플러 2에 도달하면, 조심스럽게 제거합니다. 일부 포장 재료 칼럼의 상단에서 압출해야합니다. 면도날 또는 유사 날카로운 가장자리로 부드럽게 열 상단과 심지어는 구슬 표면을두고, 압출 구슬을 닦아. 이렇게하면서 비즈에 압력을 적용하지 마십시오. </li><li> 반지 스탠드에서 포장 열을 공격 중지. 새로운 최종 커플러에 새 프릿을 놓고 오픈 (이전 TOP) 열 끝에있는 프릿 / 끝 커플러와 이것을 연결하는 포장 컬럼을 넘기다. </li><li> 적절하게 열을 라벨. 이제 사용할 수 있습니다. 최대 6 개월까지 사용하지 않을 때, 컬럼은 4 0.02 % 나트륨 azide ° C로 PBS에 저장되어있을 수 있습니다 때. </li></ol> 3) 프로그램 HPLC 방법 <ol><li> 귀하의 HPLC 프로그래밍의 내용은 제조 업체의 소프트웨어의 구체적인 내용에 따라 달라집니다. 우리는 Michrom 패러다임 MG4 HPLC를 사용합니다. 화면의 상단에이 컴퓨터에, 방법은 건설되고있는 &quot;설정&quot;탭 아래에 액세스할 수. 다음 방법을 프로그램 : <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 시간 </td><td> 흐름 속도 (μl / 분) </td><td> % </td><td> %의 B </td></tr><tr><td> 0시 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 9시 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 9시 1분 </td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13시 50분 </td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13시 51분 </td><td> 3000 </td><td> 100 </td><td> 0 </td></tr><tr><td> 19시 50분 </td><td> 3000 </td><td> 100 </td><td> 0 </td></tr></tbody></table> 10 MM 트리스 버퍼, 산도 7.4, 150 MM 나트륨 염화물, 10 MM의 염화칼슘은 10 MM 마그네슘 염화물 : 버퍼 버퍼 B : 0.5 M 아세트산의 산성 UV 검출기는 0시에서 19시 50분에 280 nm의에서 읽을 프로그래밍해야합니다. . 50 AU - Y 축이 낮은 흡광도 레벨, 예를 들어, 0을 감지하는 조정해야합니다. </li><li> autosampler 사용 가능한, 프로그램 분수 수집에 대해 다음 일정있다면. 크로마 토그래피를 수행할 때 표시 달리, 손으로 분수를 수집합니다. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 분수 </td><td> 시료 주입에서 컬렉션에 지연 </td><td> 수집 기간 (분) </td></tr><tr><td> 흐름 -을 통해 </td><td> 2.8 </td><td> 4.1 </td></tr><tr><td> 바운드 </td><td> 9 </td><td> 2.6 </td></tr></tbody></table></li></ol> 4) HPLC에 대한 샘플을 준비 <ol><li> 이 프로토콜에서 사용하기 전에, 인간의 플라즈마는 MARS 칼럼 (; 산타 클라라, CA 애질런트)를 사용하여 14 가장 풍부한 단백질의 소진해야합니다. 소진 플라즈마, 인간 락토페린 및 효모 invertase 개별적으로 트립신 - 소화하고 이전 1 설명된대로 desalted해야합니다. </li><li> 렉틴과 특정 락토페린 표준을 준비하려면, 제 3의 방법을 사용하여 AAL 또는 SNA 열 중 50 개 이상의 UG 트립신 - 소화 락토페린에서 크로마 토그래피를 수행합니다. 바운드 분수는 렉틴 특정 락토페린의 glycopeptides 포함됩니다. 6 부에서와 부품 4.4로 샘플 및 탈염하다를 무력화. 결과 예제를 사용할 준비가 하나 ML 버퍼 A에서 샘플을 Resuspend. </li><li> 세 실험이 수행됩니다 복제합니다. 세 사람 샘플을 준비하려면 복제, 30 μl MARS - 고갈, 트립신 - 소화, 플라즈마 동일한 (PE)에 트립신 - 소화 invertase 3 pmol 및 렉틴 특정 락토페린 (중 AAL - 락 또는 SNA - LAC) 1 μl를 추가합니다. PE는 플라즈마 처리 (MARS -되었고, 트립신 소화)의 주어진 금액이 파생된하는 non-processed/intact/original 플라즈마의 볼륨으로 정의됩니다. 330 μl의 최종 볼륨에 도달하기 위해 샘플을 버퍼에 추가합니다. </li><li> eluate를 수집 샘플 튜브에, 중화 버퍼 (1 M 트리스 산도 8.0)를 추가합니다. 번호와 함께 일하는 튜브의 볼륨은 autosampler의 제약에 따라 달라집니다. MG4 패러다임 들어, 유리병은 흐름을 통해 두가 eluate를 수집 수집합니다. 그것은 약 1.5 ML 트리스 버퍼 eluate 1 ML를 무력화하기 위해 필요합니다. 대상 무력화 산도는 7.0-8.0이며 산도 표시기 용지로 테스트 테스트 최종 산도. </li></ol> 5) 크로마 토그래피를 수행 <ol><li> 에 따라 모든 공백 및 예제 실행은 제 1 부에 설명된 방법을 사용합니다. 열 및 버퍼는 사용하는 동안 실온에서 수 있습니다. 버퍼가 상온에서 저장하는 동안 열이 4 ° C에 저장됩니다. </li><li> HPLC 시스템 중 AAL 또는 SNA의 렉틴 칼럼을 첨부 두 빈 메소드를 (단지 버퍼 주사)를 실행합니다. 렉틴과 열이 렉틴 특정 락토페린 (2 부)에 해당 있는지 확인합니다. </li><li> 7 HPLC의 실행의 총, 각 주입 샘플 사이에 빈 주사, 세 플라즈마 샘플을 크로마토 그래프. 빈 실행에서 분수가 수집 필요가 없습니다. </li></ol> 6) 탈염하다는 분수를 수집 각각의 분율은 다음과 같이 1 1 ML 워터스 오아시스 HLB SPE 카트리지를 사용하는 경우 : <ol><li> 진공 매니폴드에 필요한 카트리지의 개수를 연결합니다. </li><li> 1% 개미 산성 1 ML 80 % ACN (- 20 lnHg를 매니폴드에 진공 게이지 5를 참조한다) 각 카트리지를 젖었어. </li><li> 1.5 ML 0.1 %의 개미의 산성 (- 20 lnHg 매니폴드에서 진공 게이지 5를 참조한다)와 카트리지를 평형 </li><li> 한 카트리지 (. - 2.5 lnHg을하고, 유량이 1 ML / 분 넘지 않아야 매니폴드에서 진공 게이지 2를 참조한다)에 한 샘플의 전체 볼륨을로드 </li><li> 3 X 1 ML 0.1 %의 개미의 산성 (- 20 lnHg 매니폴드에서 진공 게이지 5를 참조한다)와 카트리지를 씻으십시오 </li><li> 로 Elute 펩티드 / glycopeptides은 0.1 % 개미 산성 1 X 1 ML 80 % acetonitrile로 eppendorf 튜브를 표시. 단일 수집 튜브 각 카트리지에 사용해야합니다 - (2.5 lnHg을하고, 유량은 1 ML / 분 넘지 않아야 매니폴드에서 진공 게이지 2를 참조한다.). </li><li> 각 수집 튜브 60 μl 0.5 M의 탄산수 소 암모늄을 추가하여 eluate를 무력화. 대상 neutralized 산도는 7.0-8.0입니다, 산도 표시기 용지와 시험 최종 산도. </li><li> ~ 2 시간 @ 35도 ° C. (예, 써모 사반트 SpeedVac) 진공 원심 분리기에 그들을 실행하여 eluted 펩티드 / ~ 50 μL에 glycopeptides을 집중 </li></ol> 7) PNGase F - 소화 <ol><li> 그것을 보장하기 위해 테스트 샘플 산도 7.0 사이 - 8.0. 필요한 경우 산도를 증가 50 MM의 탄산수 소 암모늄을 추가합니다. 최종 샘플 량은 50-100 μl 사이까지&gt; 100 μl, speedvac 예제입니다.한다면 </li><li> 37 ° C 야간 각 샘플 튜브와 부화 0.5 μl (250 U) 글리세롤 무료 PNGase의 F를 추가합니다. </li></ol> 8) 우편 팁 샘플 <ol><li> PNGase F - 소화 샘플로 시작, 다음과 같이 각각의 하나를 ziptip. </li><li> 10 μl 100 % acetonitrile, 10 μl 80 % acetonitrile, 0.1 % 개미 산성 다음과 젖은 ziptip. </li><li> 20 μl 0.1 %의 개미의 소산과 ziptip을 평형. </li><li> ziptip 매트릭스를 통해 10 번 그것을 pipetting 아래로 ziptip에 샘플을로드합니다. </li><li> 10 μl 0.1 %의 개미의 산, 5 번 pipetting하여 ziptip 씻으십시오. </li><li> 10 μl 80 % acetonitrile, 0.1 % 개미 산성과 깨끗한 eppendorf 튜브에 샘플을 Elute. </li><li> 용리를 반복하고 같은 튜브에 두 번째 10 μl를 추가합니다. </li><li> &lt;2 μl의 볼륨에 샘플을 Speed​​vac. </li><li> 20 μl 0.1 %의 개미의 산이 Resuspend 샘플. 샘플 지금 LC-MS/MS로 분석 준비가되어 있습니다. </li></ol> 9) 대표 검색 결과 : 20 MG / ML - POROS의 활용은 일반적으로 2의 범위에서 - 비드 렉틴 농도를 산출. 이것은 친화도 크로마 토그래피 최적입니다. 트립신 - 소화 MARS - 고갈 인간 플라즈마의 렉틴 크로마 토그래피는 일반적으로 바운드 분수에 glycopeptides을 풍요롭게. glycopeptides가 PNGase 이전 LC-MS/MS에 F - 대우, 그들은 N - 연결 합의 순서와 펩티드로 식별되며, 아스파라긴가로 변환되어있는 NXS / T (여기서 X는 어떤 잔류물하지만 프롤린됩니다) 이러한 특성을 가진 PNGase F.의 펩티드의 작업을 통해 아스파르트산는 deglycosylated 펩티드 (또는 &quot;deglycopeptides&quot;)로 간주됩니다. 바운드 분수에서 가져온 펩티드의 30-50%가 deglycopeptides 반면 평균 흐름을 통해 AAL 또는 SNA 크로마 토그래피에서 (FT) 분수, 2-4 %의 deglycopeptides가 포함되어 있습니다. 일반적으로 QStar 엘리트를 사용하여, 1000-1300 펩티드는 FT 분율에 표시됩니다, 그리고 200-400 펩티드는 바운드 분수에 표시됩니다. 두 개 이상의 서로 다른 Invertase의 deglycopeptides는 FT 분율과 바운드 분율 (표 1)에 두 개 이상의 별개의 락토페린의 deglycopeptides에서 관찰해야합니다. <img src="/files/ftp_upload/1398/1398table.jpg" alt="표 1" /> 표 1.

<ol>
	<li>Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=multiplexed+assays+for+low+abundance+proteins+in+plasma+by+targeted+mass+spectrometry+and+stable+isotope+dilution.">multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.</a> Mol Cell Proteomics. 6, 2212-2229 (2007).</li></ol>

이 프로토콜은 glycan 특정 방식으로 glycopeptides를 분리하고 확인하는 빠른 방법을 제공합니다. 글리코 실화 특히 개발과 pathogenesis 동안 생체 내에 광범위하게 다릅니다,이 기술은 질문의 다양한 해결하기 위해 적용할 수 있습니다. 특히, 우리는 선택 biomarker 발견을 향한 첫 단계로서 암 관련 epitopes로 변경되었습니다 glycopeptides을 풍요롭게하는 방법을 사용하고 있습니다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Human plasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>We make this in house</td>
</tr>
<tr>
<td>Human lactoferrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L0520</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Yeast invertase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I0408</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Oass HLB SPE cartridges</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT094225</td>
<td>1 cc volume</td>
</tr>
<tr>
<td>ZipTip pipet tips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>ZTC1 8M0 96</td>
<td>Millipore, C18 for 10 ml pipette</td>
</tr>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP154-1</td>
<td>99%</td>
</tr>
<tr>
<td>Ammonium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A6141</td>
<td>99%</td>
</tr>
<tr>
<td>Calcium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4901</td>
<td>96%</td>
</tr>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>98%</td>
</tr>
<tr>
<td>Acetic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>242853</td>
<td>99.7%</td>
</tr>
<tr>
<td>pH indicator paper</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>M95903</td>
<td>Range 0-14</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A998</td>
<td>99.9%</td>
</tr>
<tr>
<td>Formic acid</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28905</td>
<td>99%</td>
</tr>
<tr>
<td>Phosphate-buffered saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-136</td>
<td>Without calcium and magnesium</td>
</tr>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>71289</td>
<td>99.5%</td>
</tr>
<tr>
<td>PNGase F</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>P0705L</td>
<td>Glycerol-free</td>
</tr>
<tr>
<td>Vacuum-filter flasks</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SCGVU05RE</td>
<td>0.2 mm pores</td>
</tr>
<tr>
<td>POROS-AL beads</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>1-6028-02</td>
<td>&nbsp;</td>
<tr>
<td>Aleuria aurantia lectin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1390</td>
<td>&nbsp;</td>
<tr>
<td>Sambucus nigra agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1300</td>
<td>&nbsp;</td>
<tr>
<td>Sodium cyanoborohydride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>296945</td>
<td>5.0 M solution</td>
</tr>		</tbody>
		</table>
		</div>

a lectin hplc method to enrich selectively-glycosylated peptides from complex biological samples

Glycans 게시할 - translational 수정의 중요한 클래스입니다. 일반적으로 분비 및 세포 분자에있는, glycan 구조는 세포의 내부 상태를 신호. 종양 세포에 Glycans 풍부한 sialic 산성 및 fucose의 moieties이 필요한 경우가 많다. 우리는이 암 관련 glycan 변종이 초기 단계 질병을 진단하기위한 biomarker 개발에 이용할 것을 제안합니다. 따라서, 우리는 대량 lectins, 특정 glycan 구조를 바인딩 단백질의 형성 친화 매트릭스에서 크로마 토그래피를 통합 분광 기반의 워크플로우를 개발했습니다. 각각 sialic 산성 및 fucose를 묶어 lectins Sambucus 니그라 (SNA)과 Aleuria aurantia (AAL)는, covalently POROS 비즈 (응용 Biosystems)로 결합하고 고압 액체 크로마 토그래피 (HPLC)에 대한 피크 컬럼으로 포장했다. 간단히, 플라즈마는 여러 친화 제거 시스템 (; 애질런트 MARS - 14)를 사용하여 열네 가장 풍부한 단백질의 고갈되었다. 고갈 플라즈마 트립신 - 소화와 흐름을 통하여 SNA 또는 AAL HPLC에 의해 구속 분수로 분리되었다. 분수는 N - glycans 링크를 제거하는 PNGaseF 취급하고 QStar 엘리트에 LC-MS/MS로 분석되었다. 데이터 마스콧 소프트웨어를 사용하여 분석했다. 실험 설계는 제어 - fucosylated 및 sialylated 긍정을 포함하는 인간 락토페린의 glycopeptides 및 Saccharomyces에서 부정적인 제어 높은 mannose glycopeptides cerevisiae가 - 렉틴 캡처의 특이성을 모니터링하는 데 사용되었습니다. 이 워크플로의 주요 기능은 당단백질 표준을 사용하여 HPLC 형식들은 deamidated Asn-Xxx-Ser/Thr 주제에서 캡처 PNGaseF - 처리 glycopeptides의 긍정적인 식별 및 품질 평가에서 파생 재현성을 포함합니다. 프로토콜 최적화는 또한, 열 용량 자료를 시작 가장 효율적인 캡처 및 용출 버퍼를 파악하고, 전체 deglycosylation을 보장하기 위해 PNGaseF - 치료를 모니터링의 적절한 비율을 결정 포함되어 있습니다. 미래의 방향 유방암 환자 및 제어 개인의 플라즈마 질량 분석법에 기반 검색 실험을 수행하기 위해이 워크플로우를 사용하여 포함됩니다.

Watch this Scientific Journal Video about A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples at JoVE.com

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

렉틴 - 복합 POROS 비즈는 HPLC를 위해 고용되었다. 당펩타이드 기준은 긍정적이고 부정적인 컨트롤을 역임 하였다. MARS - 14 고갈, 트립신 - 소화 인간의 플라즈마는 chromatographed와 흐름 -을 통해 (FT)와 바운드 분수는 ESI-LC-MS/MS 분석을 위해 수집되었다. FT에 비해 Glycopeptides는 바운드 분수에 풍부했다.

복잡한 생물 학적 샘플에서 선택적으로 - glycosylated 펩티드을 풍요롭게하는 렉틴 HPLC 방법

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

a-lectin-hplc-method-to-enrich-selectively-glycosylated-peptides-from

Research

JoVE Journal

Biology

15.9K 조회수.  University of California, San Francisco - UCSF. 렉틴 - 복합 POROS 비즈는 HPLC를 위해 고용되었다. 당펩타이드 기준은 긍정적이고 부정적인 컨트롤을 역임 하였다. MARS - 14 고갈, 트립신 - 소화 인간의 플라즈마는 chromatographed와 흐름 -을 통해 (FT)와 바운드 분수는 ESI-LC-MS/MS 분석을 위해 수집되었다. FT에 비해 Glycopeptides는 바운드 분수에 풍부했다.

비디오: A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T - 파 이온 모빌리티 분석법 - 질량 : 단백질 복잡한 분석을위한 기본 실험 절차

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

연구

생물학

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

파지 디스플레이 도서관에서 펩티드는 Mesenchymal 세포의 유전자 발현을 조절하고 Unicortical 뼈 결함에 Osteogenesis을 힘을 주다

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

의 야생 - 타입과 장식 품종을 구별하기 PCR 기반 Genotyping 방법 Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

을 통해 생산 단지 항생제 에리스로 마이신을 자랑하는 논리, 실험 단계, 그리고 Heterologous 자연 제품 생합성의 잠재적<em&gt; E. 대장균</em

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

의 미세 주입을위한 방법<em&gt; Patiria의 minata</em&gt; 접합자

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

불순한 정화 : 연속 Metagenomes 및 Metatranscriptomes을 복잡한 동물 관련 샘플에서

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

가금류 생산 샘플에서 세균 공동체의 정성 및 정량 평가를위한 하이브리드 DNA 추출 방법

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

고대 생물 커뮤니티 숨겨 내부를 조사하기 위해 냉동 코어의 바깥 부분에서 외인성 물질의 제거

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

주사 투과 전자 단층 촬영에 의해 준비 및 두꺼운 생물 샘플의 관측

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.