이 작품은 중국의 자연 과학 재단에 의해 부분적으로 지원 (부여 번호 31471703, A0201300537 및 31671854 J.V. 하 L.L., 번호 31470435 G.Y.에 부여), 그리고 100 외국 재능 계획 (보조금 번호 JSB2014012 J.V.).

1. 샘플 컬렉션
<ol><li>신선한 무 (Raphanus sativus L.)의 다른 cultivars을 구입.</li>
</ol>2입니다. 무에서 단백질의 격리
<ol><li>10 분 동안 부엌 믹서와 신선한 무의 약 100 g을 균질.</li>
	<li>50 mL 원심 분리기 튜브 및 불용 성 물질을 제거 20 분 4 ° C에서 14000 × g 에서 원심 슬러리를 전송 합니다.</li>
	<li>새로운 50 mL 원심 분리기 튜브에는 상쾌한을 신중 하 게 전송 하 고 2m trichloroacetic 산 (TCA) 솔루션의 동일한 볼륨을 추가 합니다. 참고: TCA를 추가 수용 성 (glyco) 단백질 침전 됩니다.</li>
	<li>30 분 동안 4 ° C에서 14000 × g 에서 centrifuge 고 수송과 (glyco) 단백질에서는 상쾌한을 제거 합니다.</li>
	<li>녹는 oligosaccharides 및 다 당 류를 제거 하 5 분 동안 4 ° C에서 14000 × g 에서 이온된 수와 원심 분리기의 20 mL와 펠 릿을 세척.</li>
	<li>2.5 단계를 반복 합니다. 4 번입니다.</li>
	<li>다시 1 ml의 이온을 제거 된 물과 신선한 1.5 mL 원심 분리기 튜브에 펠 릿을 일시 중단 합니다.</li>
</ol>3. N-Glycans의 준비
<ol><li>2.7 단계에서 단백질 해결책의 믹스 50 µ L. (신선한 무의 5 g와 같음) 10 mM 아세트산에 재조합 PNGase H+ 의 0.2 뮤와 함께.</li>
	<li>12 h 37 ° C에서 반응 혼합물을 품 어. 부 화 후 5 분 여분의 단백질 및 효소 제거 14000 × g 에서 원심.</li>
	<li>신선한 1.5 mL 원심 분리기 튜브는 상쾌한 전송.</li>
</ol>4. NGlycans의 정화
<ol><li>Nglycans 풍부와 Nglycan에 대 한 fluorogenic 2 aminobenzamide (2-AB) 레이블 에이전트의 선택도 증가 하는 반응 혼합물에서 소금 및 기타 불순물을 제거 고체 상 추출 (SPE) 열을 준비 derivatization입니다.<ol>
<li>씻어 열 이온된 물 3 mL를 추가 합니다.</li>
		<li>SPE-열 활성 trifluoroacetic 산 (TFA, 0.1 %v / v)을 포함 하는 80% 이기 솔루션의 3 mL를 추가 합니다.</li>
		<li>SPE-열 equilibrate 하 이온된 물 3 mL를 추가 합니다.</li>
	</ol>
</li>
	<li>3.3 열에 단계에서 샘플을 전송 하 고 흐름을 통해 삭제.</li>
	<li>이온된 물 흐름을 통해 삭제를 열을 씻어 1.5 mL를 추가 합니다.</li>
	<li>0.1%를 포함 20% 이기 용액의 1.5 mL를 사용 하 여 열에서 출시 된 Nglycans elute TFA (v/v) 2 mL 원심 분리기 관으로.</li>
	<li>상 온에서 원심 증발에 의해 용 매를 제거 합니다. 이 샘플은 완전히 건조 될 때까지 증발.</li>
</ol>5. 형광 Derivatization NGlycans의
<ol><li>2 AB 솔루션, 디 메 틸 ⁚ / 아세트산 산 성 용액 (7:3, v/v)에 35 m m 2-AB와 0.1 M 나트륨 cyanoborohydride의 구성의 1 mL를 준비 합니다.</li>
	<li>6 다른 무에서 파생 된 말린 샘플 (4.5 단계.) 2-AB 솔루션의 5 µ L를 추가 합니다. 와 동 각 샘플까지 완전히 해산.</li>
	<li>2 h 65 ° c.에 대 한 혼합물을 품 어</li>
	<li>5 분 동안 실내 온도 아래로 샘플 식 고 이온된 수의 5 µ L 및 이기의 40 µ L를 추가 합니다. 3 분 14000 x g에서 튜브 원심</li>
	<li>300 µ L 높은 복구 HPLC 유리병에는 상쾌한에서 48 µ L를 전송. 최대 한 달 동안-20 ° C에서 derivatized N-glycan 샘플을 저장 합니다.</li>
</ol>6. HILIC UPLC Nglycans의 프로 파일링
<ol><li>각각 330 nm/420 nm의 여기/방출 파장에서 형광 온라인 detectorset에 연결 된 표준 UPLC 시스템을 사용 하 여 샘플을 분석 합니다.</li>
	<li>소수 성 상호 작용 액체 크로마토그래피 (HILIC)를 사용 하 여-UPLC glycan 열 분석을 위한 60 ° C의 열 온도에.</li>
	<li>950 mL의 액체 크로마토그래피-질량 분석 (LCMS)와 재고 솔루션 (1m 암모늄 편대 솔루션, pH 4.5) 50 mL를 희석 하 여 준비 하는 용 매 A-등급 물. 준비 재고 솔루션을 다음과 같습니다.<ol>
<li>LCMS 급 H2o.의 700 mL에 포 름 산의 43 개 mL를 추가</li>
		<li>4.5 pH 수성 암모니아 솔루션 (25 %w / w)의 dropwise 추가 조정.</li>
		<li>측정 실린더 용 매를 전송 하 고 LCMS 급 H2o. 저장소 4-6 ° C에서 최대 3 개월까지 최대 1000 mL 채우기.</li>
	</ol>
</li>
	<li>솔벤트 B. LCMS 급 이기 사용</li>
	<li>N-glycans 다음 그라데이션 차입 구분:<ol>
<li>44.5 분을 0에서 세트로 용 A. 0.5 mL/min 유량 용 매 B의 95%를 추가 하 여 시작 합니다.</li>
		<li>0 분-6 분에서 점차적으로 용 매 B 선형 그라데이션 to78%의 비율을 감소.</li>
		<li>44.5 분 6 분에서 점차적으로 55.9%를 선형 그라데이션으로 용 매 B의 비율을 감소.</li>
		<li>46.5 분 44.5 분에서 급속 하 게 용 매 B 0 %55.9 %에서 선형 그라데이션으로 비율을 감소 하 고 2 분 동안 0%에서 누른 열 세척을 시작. 55 분 44.5에서 0.25 mL/분 유량을 줄일 수 있습니다.</li>
		<li>50.5 분 48.5 분에서 용 매 B 95% 증가 하 여 추가 열을 세척 하 고 4.5 분 95%에서 개최.</li>
		<li>다시-57 분 50.5에서 95% 용 매 B 0.5 mL/min의 시작 조건에 열을 equilibrate.</li>
	</ol>
</li>
	<li>UPLC 시스템으로 샘플의 45 µ L를 주사.</li>
	<li>N-glycans UPLC 여 15 그리고 40 분 사이 보존 시간 elute</li>
	<li>2 mL 원심 분리기 튜브를 사용 하 여 각 관찰된 UPLC 피크 분수를 수집 하 고 샘플 원심 증발에 의해 건조.</li>
	<li>2 AB dextran 표준 (2−20 포도 당 단위) 식물-N-glycan 파일링, 보정 하 고 표준화 된 포도 당 단위로 그들의 차입 시간을 할당 하 라는의 약 1 pmol 주사.</li>
</ol>7. MALDI TOF MS 분석
<ol><li>LCMS의 단계 5에서 6.8 µ L에서 샘플을 분해-등급 물. 샘플 솔루션 및 6-아 자-2-thiothymine (ATT) 솔루션 (0.3 w/v %70 %v / v 수성 이기에), 그리고 피 펫 MALDI TOF 샘플 캐리어에 혼합물의 1 µ L의 믹스 1 µ L.</li>
	<li>긍정적인 이온 모드 시작 버튼을 누르면 MALDI TOF 질량 분석기 악기를 사용 하 여 샘플을 분석 합니다.</li>
	<li>동반 MALDI TOF MS 분석 소프트웨어를 사용 하 여 질량 스펙트럼을 분석 합니다.</li>
	<li>오픈 소스 glycan 해석 소프트웨어19를 사용 하 여 스펙트럼을 해석 합니다. 2-AB, [M + 나]+에 대 한 검색 매개 변수를 설정 하 고 2.0 다를 정확도 매개 변수를 설정 합니다.</li>
</ol>

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저자는 공개 없다.

우리가 여기 제시 하는 프로토콜 무의 다양 한 재배의 N-glycan 프로 파일의 비교를 허용 한다. 기존 프로토콜에 비해이 방법의 중요 한 장점은 Nglycans의 효소 출시와 2 AB와 derivatization 반응 사이의 버퍼 변경할 필요가 없습니다입니다. 이 절차의 가장 중요 한 단계는 N-glycans SPE 열을 사용 하 여 염 분을 제거 하는 실패의 정화 또는 반응 혼합물에 다른 불순물 형광 derivatization 효율성 저하 될 수 있습니다. 여기 다른 N-glycans;의 상대적인 나타났는데 평가 허가 제시 방법 절대 정량화 단계 2.1에서에서 (예: maltopentose)는 내부 표준의 추가 해야 합니다.
또는, derivatization 에이전트 여기 사용 (2-AB) 2-aminopyridine (2-AP) 또는 다른 derivatization 에이전트를 사용 하 여 쉽게 수정할 수 있습니다. 5.3 단계에서 얻은 N-glycan 파생 상품 또한 크기 배제 크로마토그래피 (SEC), 강한에 의해 분리 될 수 있다 또는 약한 음이온 교환 크로마토그래피 (색소폰 또는 왁 스), 또는 역 상 HPLC 방법 (비록 가능성이 열 등 한 해상도). 추가로 N-glycans의 구조를 확인 순차적 exoglycosidase 치료 사용할 수 있습니다 단계 5.3 후 자세한 내용 Nglycans의 구조 특성. 마지막으로, 단계 6.8에서에서 샘플 분석 될 수 있었다 MALDI TOF MSn을 사용 하 여 추가-조각화 메서드를 기반으로. 우리의 프로토콜의 가능한 미래 애플리케이션으로 N-glycans 무 또는 다른 식물 종에서의 기능과 건강 효과 조사 하 고 있다.

식물에서 N-glycans 그린 관심 증가 최근 몇 년 동안, 이전 연구로 알레르기 성 응답1,2 유도할 수 있는 면역 cross-reactions의 N-glycans을 강조 표시 . 그것은 입증 되었습니다 이전 촉매 활동3,4, 내열성 및 접는5,6 또는 subcellular 지 방화 N-glycans 공장 glycoproteins에 영향을 미칠 수 있는 고 분 비7. Glycan 구조 그들의 각각 기능 연관, 위하여 N-glycans 먼저 해제 되어야 합니다 glycoproteins에서 화학적 또는 효소. N-및 O-glycans 방출에 대 한 고전적인 화학 방법은 β-제거, 알칼리 샘플 치료 borohydride alditol8를 가진 감소에 의해 동반입니다. 그러나,이 절차는 fluorophore로 하는 걸로 하 고 glycan 구조의 줄이는 끝에서 단 당 류 단위의 상당한 감퇴의 원인. 수산화 암모늄/탄산 치료에 따라 화학 deglycosylation는 또한 일반적으로 사용 되는 대체 방법9. 이러한 화학 릴리스 방법의 그대로 단백질 같은 대량 범위에 레이블이 없는 glycan 풀 펩 티 드 파편의 간섭 없이 대량 spectrometric 분석을 수 있는 성능이 저하 됩니다. 그러나, 이러한 방법의 한 가지 단점이 일반적인 탄수화물 구조 발견 식물10에서 α1, glycans-3-fucosylated N의 증가 저하 속도입니다. 또는, 펩 티 드를 사용 하 여 효소 분리 방법:N-glycanases (PNGases, EC 3.5.1.52) 또한 광범위 하 게 적용 됩니다. 재조합 PNGase F ( Flavobacterium meningosepticum)에서 가장 일반적인 선택 하 고 N-glycans, 코어 α1, fucose 311,12베어링 구조를 제외 하 고 모든 종류의 릴리스 합니다. 따라서, PNGase A (아몬드 씨에서 분리)으로 식물 N-glycans13의 분석을 위해 사용 됩니다. 그러나,이 효소 deglycosylates만 proteolytically-파생 된 glycopeptides, deglycosylate 기본 glycoproteins14수입니다. 따라서, 다단계 샘플 검사 결과 추가 분석, glycans, 특히 낮은 풍부15의 그의 광범위 한 손실이 발생 하기 전에 필요 합니다. N-glycan 릴리스 및 형광 간단 하 고 강력한 방식으로 라벨에 대 한 최적화 된 워크플로우를 제시 하는 방법의 전반적인 목표가입니다. 기본 근거 PNGase H+는 Terriglobus roseus 에서 최근에 발견 되었다 recombinantly 대장균에 표현 될 수 있다, N-glycans 산 성에서 단백질 비 계에서 직접은 수은 조건16. PNGase H+ 를 사용 하 여 대체 방법의 주요 장점은입니다 형광 라벨 반응 반응 버퍼17,18를 변경 하지 않고 동일한 반응 관에서 수행할 수 있습니다. 간단한 준비 조건 및 낮은 풍부 oligosaccharides의 높은 복구 확인이 방법은 유용한 도구 Nglycans의 분석에. 이 프로토콜은 다양 한 식물 종에서 N-glycans의 분석에 적합 합니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="880">
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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chemicals:</td>
			<td style="width:123px;"></td>
			<td style="width:207px;"></td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloroacetic acid&#160;</td>
			<td>SCR, Shanghai</td>
			<td>80132618</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid glacial</td>
			<td>Huada, Guangzhou</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>General-reagent</td>
			<td>G80988C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid</td>
			<td>Energy chemical</td>
			<td>W810031</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-aminobenzamide</td>
			<td>Heowns, Tianjin</td>
			<td>A41900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium cyanoborohydride</td>
			<td>J&#38;K Scientific Ltd</td>
			<td>314162</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide</td>
			<td>Huada, Guangzhou</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2AB-labeled dextran ladder, 200 pmol</td>
			<td>Agilent Technologies</td>
			<td>AT-5190-6998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Aza-2-thiothymine&#160;</td>
			<td>Sigma</td>
			<td>275514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tools/Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kitchen blender</td>
			<td>Bear, Guangzhou</td>
			<td>LLJ-A10T1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Techcomp</td>
			<td>CT15RT</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifugal Evaporator</td>
			<td>Hualida, Taicang</td>
			<td>LNG-T120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SPE column</td>
			<td>Supelco</td>
			<td>Supelclean ENVI Carb SPE column</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>Autoflex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC Analysis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-recovery HPLC vial</td>
			<td>Agilent Technologies</td>
			<td>&#160;# 5188-2788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC System</td>
			<td>Shimadzu</td>
			<td>Nexera</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Detector for HPLC</td>
			<td>Shimadzu</td>
			<td>RF-20Axs&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Column oven</td>
			<td>Hengxin</td>
			<td>CO-2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Column</td>
			<td>Waters</td>
			<td>Acquity UPLC BEH glycan column</td>
			<td>2.1 &#215; 150 mm, 1.7 &#956;m particle size</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Water</td>
			<td>Merck Millipore</td>
			<td>#WX00011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Acetonitrile</td>
			<td>Merck Millipore</td>
			<td># 100029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Aladdin</td>
			<td>F112034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonia solution</td>
			<td>Aladdin</td>
			<td>A112080</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of n-glycans from raphanus sativus cultivars using pngase h+

최근 몇 년 동안, 식물의 탄수화물 moieties 받은 관심, 그들은 cross-reactive, 알레르기 자극 면역 반응의. 또한, 탄수화물 구조 또한 식물 물질 대사에 중요 한 역할을 담당할. 여기, 우리가 준비 하 고 사용 하는 N-glycanase 특정 식물에서 파생 된 탄수화물 구조의 릴리스에 대 한 무 (Raphanus sativus)의 다른 재배 품 정에서 N-glycans를 분석 하는 간단 하 고 빠른 방법을 제시. 이 위해 무 homogenates의 원유 trichloroacetic 산 침전 PNGase H+로 치료 되었고 형광 태그 2-aminobenzamide를 사용 하 여 표시. 레이블이 지정 된 N-glycan 샘플 이후 울트라 성능 액체 크로마토그래피 (UPLC) 분리 및 세부 구조에 대 한 비행 (TOF MALDI) 질량 분석의 매트릭스 보조 레이저 탈 착 이온화 시간으로 분석 되었다 평가 및 무 파생 N-glycan 구조 상대 abundancies 척도를. 이 프로토콜 사용할 수 있습니다 또한 다양 한 다른 식물 종에서 N-glycans의 분석 그리고 함수 추가 조사 및 인간의 건강에 N-glycans의 효과 대 한 유용할 수 있습니다.

그림 1 (glyco-) 무, N-glycans의 준비, UPLC 분석 및 이러한 구성 요소의 MALDI TOF MS 분석에서 단백질의 격리를 포함 하 여 설명된 프로토콜의 구조 개요를 보여 줍니다. 그림 2 분석된 무 재배의 derivatized Nglycans의 대표 UPLC chromatograms를 보여 줍니다. 그림 3 2AB derivatized N-glycan 구조 MALDI TOF 질량 분석을 사용 하 여 얻은 결과 보여 줍니다. 그림 4 는 각 무 품종에서 N-glycans의 양적 구성.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57979/57979fig1.jpg"> 그림 1 : N-glycans 무 glycoproteins에서 발표에 대 한 기술된 분석 방법의 개요 개요. 메서드 (glyco-) 단백질 및 N-glycans UPLC 프로 파일링, MALDI TOF MS 분석의 준비를 포함 한다. <a href="https://www.jove.com/files/ftp_upload/57979/57979fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57979/57979fig2.jpg"> 그림 2 : N 2 ab 표시-glycan 프로필 N -glycans. Nglycans 빨간 무 그린 무, gegeol 무, 무, 앵 무, 수 박 무에서 격리 됩니다. <a href="https://www.jove.com/files/ftp_upload/57979/57979fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a> 
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57979/57979fig3.jpg"> 그림 3 : 대량 spectrometric 분석 N -glycan 샘플 HILIC UPLC에 의해 분리. N-glycans 각 스펙트로그램에 같은 피크에 의해 수집 됩니다. <a href="https://www.jove.com/files/ftp_upload/57979/57979fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a> 
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57979/57979fig4.jpg"> 그림 4 : 양적 구성 N -각 무 품종에서 glycans. 크기의 원형 및 조각 무 glycoproteins에서 2AB 표시 된 N-glycans의 관계 되는 풍부를 나타냅니다. <a href="https://www.jove.com/files/ftp_upload/57979/57979fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>

Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

N-glycans Raphanus sativus Cultivars 사용 PNGase H+ 에서 분석

우리는 준비 및 N-glycans 무 (Raphanus sativus)의 다른 재배에서의 분석을 위한 간단 하 고 빠른 방법을 설명합니다.

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

analysis-of-n-glycans-from-raphanus-sativus-cultivars-using-pngase-h

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

6.5K 조회수.  Nanjing Agricultural University. 우리는 준비 및 N-glycans 무 (Raphanus sativus)의 다른 재배에서의 분석을 위한 간단 하 고 빠른 방법을 설명합니다.

비디오: Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

레이저 이외에 Microdissection RNA-서열에 대한 실험 디자인 : 옥수수 잎 개발의 분석 교훈

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

연구

생화학

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE를 사용한 전자 저온 현미경 이미지의 고해상도 단일 입자 분석

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

노화 관련 post-translational 수정을 공부하기 위해 여러 마우스 조직에서 중간 필라멘트 단백질의 분리

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

Proteomic 분석에 대 한 인간의 망막과 맥락 막의 RPE의 해 부

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

조직의 최소 금액을 사용 하 여 초파리의 Permeabilized 섬유에 미토 콘 드리 아 산소 소비량의 측정

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

동적 Proteomic 그리고 미르 Langendorff 관류 후 절연 마우스 마음에서에서 Polysomes의 분석

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

Fucosylated 인간 우유 Trisaccharides 유전자를 사용 하 여 바이오 컨텍스트에서 분석 인코딩된 바이오 센서

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

차에서 파생된 새로 설립된 세포 현탁액 배양에서 6개의 전신 살충제의 물질대사에 관한 연구(동백나무시넨시스 L.) 잎

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

잠재적 인 후성 유전학 마커의 하향 식 질량 분광프로파일링을위한 수줌 잎 조직에서 히스톤의 격리

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

지원 부동 블록을 사용하여 전송 전자 현미경 검사법에서 샘플 지원 필름의 준비

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...

N-glycans Raphanus sativus Cultivars 사용 PNGase H⁺ 에서 분석

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