Diese Arbeit wurde teilweise unterstützt durch den Natural Science Foundation of China (Zuschuss Zahlen 31471703, A0201300537 und 31671854, j.v. und l.l., Anzahl 31470435 gewähren, G.Y.), und die 100 ausländische Talente Plan (Grant-Nummer JSB2014012, j.v.).

1. die Probenahme
<ol><li>Verschiedene Sorten von frischem Rettich (Raphanus Sativus L.) zu erwerben.</li>
</ol>2. Isolierung von Proteinen aus Rettich
<ol><li>Ca. 100 g frische Radieschen mit einem Küche Mixer für 10 min zu homogenisieren.</li>
	<li>Übertragen Sie die Gülle 50 mL Zentrifugenröhrchen, Zentrifuge bei 14.000 × g bei 4 ° C für 20 min die unlösliche Material zu entfernen.</li>
	<li>Übertragen des Überstands vorsichtig in eine neue 50 mL Zentrifugenröhrchen und fügen Sie ein gleiches Volumen 2 M Trichloressigsäure Säure (TCA) Lösung. Hinweis: Hinzufügen von TCA wird löslich (Glyco) Proteine ausgefällt.</li>
	<li>Bei 14.000 × g bei 4 ° C für 30 min zentrifugieren und den überstand von gebeizte (Glyco) Proteine zu entfernen.</li>
	<li>Waschen Sie die Pellets mit 20 mL entionisiertem Wasser und Zentrifuge bei 14.000 × g bei 4 ° C für 5 min, lösliche Oligosaccharide und Polysaccharide zu entfernen.</li>
	<li>Wiederholen Sie Schritt 2.5. vier Mal.</li>
	<li>Wieder aussetzen Sie, die Pellets in 1 mL entionisiertem Wasser und Übertragung auf eine frische 1,5 mL Zentrifugenröhrchen.</li>
</ol>3. Vorbereitung der N- Glykane
<ol><li>Mix 50 µL Proteinlösung aus Schritt 2.7. (Dies entspricht 5 g frische Radieschen) mit 0,2 mU von rekombinanten PNGase H+ in 10 mM-Essigsäure.</li>
	<li>Inkubieren Sie die Reaktionsmischung bei 37 ° C für 12 h. Zentrifugieren Sie nach der Inkubation bei 14.000 × g für 5 min um die extra-Protein und Enzym zu entfernen.</li>
	<li>Übertragen Sie den überstand auf eine frische 1,5 mL Zentrifugenröhrchen.</li>
</ol>4. Reinigung des N- Glykane
<ol><li>Vorbereiten der Festphasen-Extraktion (SPE) Spalte zu bereichern die N- Glykane und Salze und andere Verunreinigungen zu entfernen, aus dem Reaktionsgemisch, Erhöhung der Selektivität des Fluorogenic 2-aminobenzamid (2-AB) Kennzeichnung Agenten für N- glycan Derivatisierung.<ol>
<li>Fügen Sie 3 mL deionisiertes Wasser, waschen Sie die Spalte hinzu.</li>
		<li>Fügen Sie 3 mL 80 % Acetonitril Lösung mit Trifluoroacetic Säure (TFA, 0,1 % V/V), um aktiv die SPE-Spalte hinzu.</li>
		<li>Hinzugeben Sie 3 mL deionisiertes Wasser für den SPE-Säulen equilibrate.</li>
	</ol>
</li>
	<li>Die Probe von Schritt 3.3 auf die Säule übertragen und die Durchströmung zu verwerfen.</li>
	<li>Hinzugeben Sie 1,5 mL deionisiertes Wasser, waschen Sie die Spalte und entsorgen Sie den Durchfluss.</li>
	<li>Eluieren freigegeben N- Glykane aus der Spalte mit 1,5 mL 20 % Acetonitril Wasserlösung mit 0,1 % TFA (V/V) in ein 2 mL Zentrifugenröhrchen.</li>
	<li>Entfernen Sie das Lösungsmittel durch zentrifugale verdunsten bei Raumtemperatur. Verdampfen Sie, bis die Probe vollständig trocken ist.</li>
</ol>(5) Fluoreszenz Derivatisierung von N- Glykanen
<ol><li>Bereiten Sie 1 mL 2-AB-Lösung, bestehend aus 35 mM 2-AB und 0,1 M-Natrium-Cyanoborohydride in Dimethyl Sulfoxid/Essigsäure Säurelösung (7:3, V/V).</li>
	<li>Fügen Sie sechs verschiedene Radieschen 5 µL 2-AB-Lösung für die getrockneten Proben (Schritt 4.5.) abgeleitet. Vortex: jede Probe, bis Sie vollständig aufgelöst.</li>
	<li>Inkubieren Sie die Mischung für 2 h bei 65 ° c</li>
	<li>Lassen Sie die Probe auf Raumtemperatur für 5 min abkühlen, und fügen Sie 5 µL deionisiertes Wasser und 40 µL Acetonitril. Zentrifugieren Sie die Röhrchen bei 14.000 x g für 3 min.</li>
	<li>Übertragen Sie 48 µL aus der Überstand in ein 300 µL High-Erholung-HPLC-Fläschchen. Speichern Sie die derivatisierte N- Glycan Proben bei-20 ° C bis zu einem Monat.</li>
</ol>6. HILIC-UPLC Profilierung der N- Glykane
<ol><li>Analysieren Sie die Proben unter Verwendung eines Standardsystems UPLC bzw. an eine Online-Fluoreszenz-Detectorset bei der Anregung/Emission Wellenlängen von 330 nm/420 nm, angeschlossen.</li>
	<li>Verwenden Sie eine hydrophobe Wechselwirkung Flüssigkeitschromatographie (HILIC)-UPLC Glycan Spalte an eine Spalte Temperatur von 60 ° C für die Analyse.</li>
	<li>Bereiten Sie Lösungsmittel A durch Verdünnung 50 mL der Stammlösung (1 M Ammonium-Formiat Lösung, pH 4,5) mit 950 mL flüssige Gaschromatographie-Massenspektrometrie (LCMS)-Wasser zu benoten. Bereiten Sie die Stammlösung wie folgt:<ol>
<li>700 mL LCMS-Klasse H2O. 43 mL Ameisensäure hinzufügen</li>
		<li>Stellen Sie den pH-Wert auf 4,5 durch tropfenweise Zugabe von wässrigen Ammoniaklösung (25 % w/w).</li>
		<li>Das Lösungsmittel auf ein Messzylinder übertragen und LCMS-Klasse H2O. Store bei 4-6 ° C für bis zu 3 Monaten bis zu 1000 mL einfüllen.</li>
	</ol>
</li>
	<li>Verwenden Sie LCMS-Grade Acetonitril als Lösungsmittel B.</li>
	<li>N- Glykane mit folgenden Farbverlauf Elution zu trennen:<ol>
<li>Durch das Hinzufügen von 95 % der Lösungsmittel B in Lösungsmittel A. Set die Durchflussmenge bei 0,5 mL/min von 0 bis 44,5 min starten.</li>
		<li>Von 0 min bis 6 min verringern Sie allmählich den Anteil des Lösungsmittels B mit einem linearen Farbverlauf to78 %.</li>
		<li>Von 6 min bis 44,5 min verringern Sie allmählich den Anteil des Lösungsmittels B mit einem linearen Farbverlauf auf 55,9 %.</li>
		<li>Von 44,5 min bis 46,5 min schnell verringern Sie den Anteil des Lösungsmittels B mit einem linearen Farbverlauf von 55,9 % auf 0 % bei 0 % für 2 min halten Sie und initiieren Sie Waschen der Spalte zu. Reduzieren Sie die Durchflussmenge auf 0,25 mL/min von 44,5 auf 55 min.</li>
		<li>Waschen Sie die Spalte weiter durch Lösungsmittel B auf 95 % von 48,5 min bis 50,5 min erhöhen und bei 95 % für 4,5 min halten.</li>
		<li>Re equilibrate der Spalte um die Startbedingungen des 95 % Lösungsmittel B mit 0,5 mL/min von 50,5 auf 57 min.</li>
	</ol>
</li>
	<li>Injizieren Sie 45 µL der Probe in der UPLC-System.</li>
	<li>Eluieren Sie die N- Glykane von UPLC mit Retentionszeiten zwischen 15 und 40 min.</li>
	<li>Jeder beobachteten UPLC Peak Bruch mit 2 mL Zentrifuge Röhren sammeln und Trocknen der Proben durch zentrifugale verdampfen.</li>
	<li>Injizieren Sie ungefähr 1 Pmol 2-AB mit der Bezeichnung Dextran standard (2−20 Glucose-Einheiten), die Pflanze -N- Glycan Profilierung zu kalibrieren und ihre Elution Zeiten in standardisierten Glucose-Einheiten zuweisen.</li>
</ol>(7) MALDI-TOF-MS-Analyse
<ol><li>Auflösen der Proben aus Schritt 6,8 in 5 µL LCMS-Wasser zu benoten. Mix 1 µL der Probenlösung und 1 µL 6-Aza-2-Thiothymine (ATT) Lösung (0.3 % w/V in wässrigem Acetonitril 70 % V/V) und die Mischung auf die MALDI-TOF-Probenträger Pipette.</li>
	<li>Analysieren Sie die Proben mit einem MALDI-TOF Massenspektrometer Instrument im positiven Ionen-Modus durch Drücken der Schaltfläche " starten ".</li>
	<li>Analysieren Sie die Massenspektren mithilfe der begleitenden MALDI-TOF-MS-Analyse-Software.</li>
	<li>Interpretieren Sie die Spektren, die mit einer open-Source Glycan Auslegung Software19. Legen Sie die Suchparameter für 2-AB Kennzeichnung [M + Na]+, und legen Sie den Parameter Genauigkeit 2.0 da.</li>
</ol>

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Das Protokoll, die, das wir hier vorgestellt haben, erlaubt den Vergleich der N- Glycan Profile von verschiedenen Sorten von Rettich. Ein wesentlicher Vorteil dieser Methode im Vergleich zu bestehenden Protokolle ist, dass keine Puffer Änderungen zwischen die enzymatische Freisetzung von N- Glykanen und der Derivatisierung Reaktion mit 2-AB erforderlich sind. Der wichtigste Schritt dieses Verfahrens ist die Reinigung von N- Glykanen mit der Spalte "SPE" als Verstoß gegen Salze zu entfernen od...

N- Glykane in Pflanzen haben erhöhte Aufmerksamkeit in den letzten Jahren gezogen, wie die bisherige Forschung N- Glykane als eine potenzielle Quelle für immunologische Kreuzreaktionen hervorhob, die allergische Reaktionen1,2 provozieren kann . Es nachweislich bereits, dass N- Glykane auf Pflanze Glykoproteine katalytische Aktivität3,4, Thermostabilität und klappbare5,6 oder subzelluläre Lokalisierung beeinflussen kann und Sekretion7. Um die Glycan-Strukturen mit ihren jeweiligen Funktionen zu korrelieren, muss N- Glykane zuerst aus Glykoproteinen entweder chemisch oder enzymatisch entlassen werden. Die klassische chemische Methode für die Freigabe von N- und O- Glykanen ist β-Eliminierung, in denen alkalische Probenbehandlung durch Reduktion mit Natriumborhydrid ein Alditol8weichen begleitet wird. Jedoch dieses Verfahren schließt mit einem Fluorophore beschriften und verursacht erhebliche Verfall der Monosaccharid-Einheiten aus der reduzierenden Ende der Glycan Struktur. Chemische Deglycosylation basierend auf Ammonium Hydroxid/Carbonat Behandlung ist auch eine häufig verwendete alternative Methode9. Keines dieser Verfahren chemische Freisetzung verschlechtert intakte Proteine, die massenspektrometrische Analyse der unbeschrifteten Glycan Pools ohne die Einmischung von Peptid-Fragmente in den gleichen Massenbereich ermöglicht. Ein Nachteil dieser Methoden ist jedoch die erhöhte Abbaurate von α1, 3-Fucosylated N- Glykane, eine gemeinsame Struktur der Kohlenhydrate in Pflanzen10gefunden. Alternativ, enzymatischen Freisetzung von Methoden mit Peptid:N- Glycanases (PNGases, EC 3.5.1.52) werden auch eingesetzt. Rekombinante PNGase F (von Flavobacterium Meningosepticum) ist die häufigste Wahl und erlaubt die Freigabe aller Arten von N- Glykanen, außer die Strukturen mit einem Kern α1, 3 Fucose11,12. PNGase A (isoliert aus Mandel Samen) ist daher in der Regel für die Analyse der Anlage N- Glykane13verwendet. Jedoch dieses Enzym Deglycosylates nur proteolytisch abgeleitete Glykopeptide, und ist nicht in der Lage, Deglycosylate native Glykoproteine14. Daher ist eine mehrstufige Probe Aufarbeitung vor weiteren Analyse erforderlich, die umfangreichen Verlust von Glykoproteinen, insbesondere der niedrige Fülle15führt. Das übergeordnete Ziel der Methode ist, einen optimierten Arbeitsfluss für N- Glycan Release und Fluoreszenz-Beschriftung in eine einfache und robuste Art und Weise zu präsentieren. Die zugrunde liegende Logik ist, dass PNGase H+, das war vor kurzem in Terriglobus Roseus entdeckt und in E. Colirekombinant ausgedrückt werden kann, können N- Glykane direkt aus dem Protein-Gerüst in sauren hydrolyseneigung Bedingungen-16. Ein entscheidender Vorteil der Verwendung von PNGase H+ gegenüber alternativen Methoden ist, dass fluoreszierende Kennzeichnung Reaktionen in der selben Reaktionsgefäß durchgeführt werden können, ohne die Reaktion Puffer17,18. Die einfache Zubereitung Bedingungen und hohe Rückgewinnung von niedrig-Fülle Oligosaccharide machen diese Methode ein wertvolles Werkzeug in der Analyse von N- Glykanen. Dieses Protokoll eignet sich für die Analyse der N- Glykane von verschiedenen Pflanzenarten.

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="880">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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+

In den letzten Jahren haben die Kohlenhydrat-Moieties Pflanzen beträchtliche Aufmerksamkeit empfangen, da sie eine potentielle Quelle von kreuzreaktiven, Allergie provozieren Immunantworten sind. Darüber hinaus auch spielen Kohlenhydrat-Strukturen eine entscheidende Rolle im Stoffwechsel der Pflanze. Hier präsentieren wir Ihnen eine einfache und schnelle Methode für die Vorbereitung und Analyse von N- Glykanen aus verschiedenen Sorten von Rettich (Raphanus Sativus) mit einem N- Glycanase speziell für die Freisetzung von pflanzliche Kohlenhydrat-Strukturen. Um dies zu erreichen, wurden grobe Trichloressigsäure Säure Ausscheidungen von Rettich Homogenates mit PNGase H+behandelt und mit 2-aminobenzamid als fluoreszierende Tag gekennzeichnet. Die beschrifteten N- Glycan Proben wurden anschließend durch ultra-Performance liquid Chromatography (UPLC) Trennung und Matrix-assisted Laser Desorption ionisation-Zeit der Massenspektrometrie Flug (MALDI-TOF) für eine detaillierte Struktur-analysiert. Bewertung und die relative Abundancies der Rettich abgeleitet N- Glycan Strukturen zu quantifizieren. Dieses Protokoll kann auch für die Analyse von N- Glykanen aus verschiedenen anderen Pflanzenarten verwendet werden und kann für weitere Untersuchungen der Funktion und Wirkung von N- Glykanen auf die menschliche Gesundheit nützlich sein.

Abbildung 1 zeigt einen schematischen Überblick über die beschriebene Protokoll, einschließlich der Isolierung von Proteinen (Glyco-) aus Rettich, der Vorbereitung von N- Glykanen, UPLC Analyse und die MALDI-TOF-MS-Analyse dieser Komponenten. Abbildung 2 zeigt repräsentative UPLC Chromatogramme von derivatisierte N- Glykane der analysierten Rettich Sorten. Abbildung 3 zeigt die ...

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+

Analyse der N- Glykane von Raphanus Sativus Sorten verwenden PNGase H+

Wir beschreiben eine einfache und schnelle Methode für die Aufbereitung und Analyse von N- Glykanen aus verschiedenen Sorten von Rettich (Raphanus Sativus).

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

Research

JoVE Journal

Biochemistry

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

6.5K Aufrufe.  Nanjing Agricultural University. Wir beschreiben eine einfache und schnelle Methode für die Aufbereitung und Analyse von N- Glykanen aus verschiedenen Sorten von Rettich (Raphanus Sativus).

Serie: 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.

Experimentelles Design für Laser Mikrodissektion RNA-Seq: Lehren aus einer Analyse von Maisblattentwicklung

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

Forschung

Biochemie

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.

Hochauflösende Einzelteilchenanalyse aus Elektronen-Kryomikroskopie-Bildern mit SPHIRE

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.

Isolierung von Zwischenfilamentproteinen aus mehreren Mausgeweben zur Untersuchung von altern-assoziierten posttranslationalen Modifikationen

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.

Dissektion der menschlichen Netzhaut und RPE-Aderhaut für Proteomic Analysen

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.

Messung der mitochondrialen Sauerstoffverbrauch in Permeabilized Fasern von Drosophila mit minimalen Mengen von Gewebe

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.

Dynamische Proteomic und MiRNA Analyse des Polysomes von isolierten Maus Herz nach Langendorff Perfusion

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.

Analyse der Fucosylated menschlichen Milch Trisaccharides in biotechnologischen Zusammenhang mit genetisch codierte Biosensoren

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.

Studie über den Stoffwechsel von sechs systemischen Insektiziden in einer neu etablierten Zellsuspensionskultur abgeleitet aus Tee (Camellia Sinensis L.) Blätter

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.

Isolierung von Histon von Sorghum Blattgewebe für Top Down Mass Spektrometrie Profilierung von potenziellen epigenetischen Markern

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.

Herstellung von Probenträgerfilmen in der Transmissionselektronenmikroskopie mit einem Stütz-Floatationsblock

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