يقر MH بدعم من &quot;جمعية الألمانية للبحوث&quot; (DFG). الكاتب المساهمات: تصميم MH البحوث؛ تنفيذ KT، JS وMT البحوث وتحليل البيانات؛ كتب KT وMH الورقة.

الكتاب تعلن أي مصلحة مالية المتنافسة.

وقد نشرت دراسات البروتين الكمي المختلفة باستخدام الوسم النظائر مستقرة في السنوات الأخيرة. في هذه التجارب عادة ما تتم مقارنة اثنين من عينات مختلفة، منها هو المسمى عينة واحدة مع النظائر مستقرة. بعد ذلك يتم الجمع بين البروتينات أو الببتيدات من العينتين في نسبة متساوية ...

ويمكن لعمليات التمثيل الضوئي في الأغشية الثايلاكويد من النباتات والطحالب تعمل في وضع الخطية ودوري. خلال الخطية تدفق الإلكترونات (LEF) الضوئي الأول (PSI)، الضوئي الثاني (PSII) والسيتوكروم ب 6 / و في نهاية المطاف نقل الالكترونات من الماء إلى NADP + 1، مما يؤدي إلى توليد NADPH و ATP 2. في المقابل، دوري تدفق الإلكترونات (CEF)، والذي يعرف ليكون ذلك حافزا تحت ظروف بيئية متنوعة مثل الدولة 2 3 4 شروط واللاهوائية، والنتائج في إعادة الحد من PSI أكسدة عن طريق حقن الإلكترونات مرة أخرى إلى سلسلة نقل الإلكترون. هذه العملية يمكن أن تتم إما في الجانب اللحمية من السيتوكروم ب 6/1 و المعقدة أو في بركة كينون صانعي 5 ويولد ATP، ولكن لا NADPH 2. الهدف من البروتوكول هو عرض ليبرهن على وجود الطيف الكتلي (MS) على أساس مethod للمقارنة، والتحليل الكمي من المجمعات multiprotein في الأغشية الثايلاكويد من كلاميدوموناس reinhardtii إلى التبصر في تكوين هذه المجمعات في ظل ظروف مختلفة (يتضح من المقارنة بين سلالات مختلفة وراثيا). تم تطبيق هذا النهج في منشور من قبل Terashima وآخرون في عام 2012 مما يدل على كا 2 + التي تعتمد على التنظيم من مركز دراسات الحدود في C. reinhardtii بوساطة مجمع multiprotein بما في ذلك البروتينات CAS، ANR1، وPGRL1 6. وسيتم شرح الإجراء من خلال تحليل مقارن لتكوين CEF-supercomplex في اثنين من سلالات مختلفة وراثيا، وبالتالي الاستفادة من وضع العلامات واحدة من سلالتين مع النيتروجين الثقيلة (15 N). لفترة وجيزة، ويتضمن بروتوكول إعداد الأغشية الثايلاكويد، تليها الانحلالية المنظفات وتجزئة المجمعات الضوئي في التدرج كثافة السكروز. بعد تجزئة التدرج، اختار فاركستعقد من سلالتين مختلطة على أساس حجم مساو، مفصولة SDS-PAGE تليها الهضم في جل واللاحقة تحليل MS الكمي. كما ذكر أعلاه، هو فعل CEF تحت ظروف بيئية مختلفة ونشر اعتبارا من عام 2010 يدل على عزلة وظيفية CEF-supercomplex من الدولة 2 الخلايا المؤمنة من C. reinhardtii 7، التي كان يؤديها عن طريق فصل الأغشية الثايلاكويد solubilized على التدرج كثافة السكروز خلال تنبيذ فائق. مختلفة من إيواي وآخرون. 7، يصف بروتوكول المعروضة عزلة CEF-supercomplex من اللاهوائية نمت C. الثقافات reinhardtii باتباع إجراء بديل. هذا وتضم التغييرات في بروتوكول العزلة الثايلاكويد فضلا عن الاختلافات بشأن الخطوة الانحلالية وفصل المجمعات البروتين بواسطة تنبيذ فائق. في هذا البروتوكول، والأغشية الثايلاكويديتم عزل بتطبيق الإجراء الذي نشرته تشوا وBennoun 8، في حين أن مخازن تستخدم لإعداد الثايلاكويد بواسطة إيواي وآخرون. تضم 25 ملي مس، 0.33 M السكروز، 5 ملي MgCl 2، 1.5 ملي مول كلوريد الصوديوم (الرقم الهيدروجيني 6.5) كما هو موضح 9. تم إجراء الانحلالية مع المنظفات 0.7-0.8٪ (ن tridecyl-β-D-مالتوزيد) لمدة 30 دقيقة على الجليد في حالة إيواي وزملاء العمل، في حين أن طريقة الإذابة وصفها هنا تعتمد على استخدام المنظفات بنسبة 0.9٪ (ن دوديسيل-β-D-مالتوزيد (β-DM)) ويتم تنفيذ سوى 20 دقيقة على الجليد. تستخدم كلتا المجموعتين 0.8 ملغ من الكلوروفيل لكل مل لإذابة المنظفات مع كل منها. لفصل المجمعات الضوئي من الأغشية الثايلاكويد solubilized إيواي وآخرون. تطبيق تركيزات السكروز بين 0،1-1،3 م، في حين أن واضعي هذا البروتوكول يستخدم بتركيزات تتراوح 0،4-1،3 م الفرق الأخير هو سرعة الطرد المركزي، والذي هو أقل مقارنة إلى عصامنشر rlier. تم تطبيقه بالفعل الانحلالية الأغشية الثايلاكويد مع المنظفات غير أيوني تليها كثافة السكروز التدرج التجزئة في العديد من الدراسات تتراوح من 1980s وحتى اليوم 7، 9-14، وكذلك تطبيق وضع العلامات الأيضية للبروتينات هو الأسلوب على نطاق واسع في مجال تحليل البروتينات. النهج وصف ينطبق على وضع العلامات 15 N الأيض واحد من اثنين من سلالات مقارنة عن طريق زرع في وجود النيتروجين الثقيل كمصدر للنتروجين وحيد في شكل 15 N NH 4 CL، التي يتم تضمينها في جميع الأحماض الأمينية التي تؤدي الى كتلة التحول تبعا لتسلسل الأحماض الأمينية من الببتيد. عند تحليل خليط من 14 N و 15 N ضمن واحدة MS تشغيل، ويمكن استخدام هذا التحول الشامل إلى تحديد منشأ العينة لكل الببتيد ببتيد والنسبية يمكن حساب الكميات المتوفرة تمثل فرة نسبية لتتوافقالبروتين 15 جي. العديد من الدراسات الكمية على تحليل البروتينات C. هي reinhardtii المتاحة، التي تقارن كمية محددة من البروتين لتحليل التغيرات في بروتيوم بين الظروف التجريبية (مثل التغيرات في بروتيوم بسبب المغذيات 16-19 أو ضوء التوتر 20،21). مقارنة مع تلك الدراسات، في نهج مطروحة حاليا يتم الجمع بين أحجام متساوية من العينات وتحليلها. هذا الإعداد يسمح لدراسة سلوك الهجرة من البروتينات داخل التدرج وعلاوة على ذلك لتحليل تكوين مجمعات مختلفة فيما يتعلق سلالات التحقيق. وسيتم شرح هذه الطريقة من خلال التركيز بشكل رئيسي على ثلاثة بروتينات: المرشح الأول هو البروتين استشعار الكالسيوم CAS-المترجمة بلاستيدات الخضراء، والتي تبين أن تشارك في الصور التأقلم في C. reinhardtii 22 يعتبر الكالسيوم لتكون إشارة مهمةايون جي للمسارات التي يتم تفعيلها نظرا لمختلف الضغوط الحيوية وغير الحيوية الرائدة في النهاية إلى تغيرات في التعبير الجيني والخلايا الفسيولوجيا 23 واقترح أن البلاستيدات الخضراء يمكن أن تسهم في الخلوية كا 2 + إشارات عبر البروتينات CAS 22،24،25. البروتين الثاني هو ANR1 (استجابة اللاهوائية 1 6)، وهو البروتين الذي تبين أن يسببها نقص الأكسجين تحت ظروف النمو في C. reinhardtii 26. ولا سيما، CAS وكذلك ANR1 تم تحديدها باعتبارها مفارز من CEF-supercomplex وعلاوة على ذلك، باستخدام النهج الوراثية العكسية، وقد تبين أن كلا من البروتينات تسهم ظيفيا لمركز دراسات الحدود في الجسم الحي 6، ودعم دورها في مفارز وظيفية من هذا المجمع البروتين. البروتين الثالث هو الثايلاكويد البروتين PGR5 على غرار 1 (PGRL1)، والذي يظهر أن تشارك في CEF في كلاميدوموناس 4،27 وكذلك في نبات الأرابيدوبسيس 5،28 وكان أيضا معرفentified في عمل إيواي وآخرون. 7 وسيقدم هذا النهج من خلال إظهار نتائج تجربتين مختلفتين: wildtype (WT) مقابل (مقابل) سلالة ΔPSI 29، واظهار حذف الجين psab، والترميز لالضوئي ضروري أنا الوحيدات، والتي هي أيضا جزء من CEF-supercomplex وWT مقابل لpgrl1 خروج المغلوب سلالة 4. لكل من هذه التجارب تكوين كمية من CEF-supercomplex بين 15 و N وقد تم مقارنة سلالة N 14 المسمى.

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			<td height="21" style="height:21px;width:392px;">Chemicals</td>
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			<td style="width:119px;"></td>
			<td style="width:199px;"></td>
		</tr>
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			<td height="42" style="height:42px;width:392px;">Acetic acid</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0662</td>
			<td></td>
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			<td height="42" style="height:42px;width:392px;">Acetone</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2300</td>
			<td></td>
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			<td height="63" style="height:63px;">Acetonitrile Optigrade f&#252;r LC-MS</td>
			<td style="width:427px;">Diagonal 
			website: https://www.diagonal.de/</td>
			<td style="width:119px;">9340</td>
			<td style="width:199px;">harmful, work with gloves 
			see protocol text for further precautions</td>
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			<td height="45" style="height:45px;">Ammonium chloride 15N</td>
			<td style="width:427px;">Cambridge Isotope Laboratories 
			website: http://www.isotope.com/cil/index.cfm</td>
			<td style="width:119px;">39466-62-1</td>
			<td></td>
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			<td height="42" style="height:42px;">Ammonium chloride 14N</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0988</td>
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			<td height="42" style="height:42px;">Ammonium hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3583</td>
			<td></td>
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			<td height="42" style="height:42px;">Ammonium sulfate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3598</td>
			<td></td>
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			<td height="42" style="height:42px;">Coomassie brilliant blue R-250</td>
			<td style="width:427px;">Fisher Scientific 
			website: http://www.de.fishersci.com/index.php/deindex</td>
			<td>10041653</td>
			<td></td>
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			<td height="42" style="height:42px;">n-Dodecyl-&#946;-D-maltoside</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0819</td>
			<td></td>
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			<td height="42" style="height:42px;">EDTA</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2937</td>
			<td></td>
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			<td height="42" style="height:42px;">Formic acid&#160;&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3858</td>
			<td></td>
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			<td height="42" style="height:42px;">Hepes</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3724</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">Magnesium chloride</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td>A4425</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2954</td>
			<td></td>
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			<td height="42" style="height:42px;">Phosphorous acid</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0989</td>
			<td></td>
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			<td height="42" style="height:42px;">Di-Potassium hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1042</td>
			<td></td>
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			<td height="42" style="height:42px;">Potassium di-hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1043</td>
			<td></td>
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			<td height="42" style="height:42px;">Sodium hydroxide</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1551</td>
			<td></td>
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			<td height="42" style="height:42px;">Sucrose</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1125</td>
			<td></td>
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			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3954</td>
			<td></td>
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			<td height="42" style="height:42px;width:392px;">Tris</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2264</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">Trypsin (sequencing grade modified) and Trypsin buffer</td>
			<td style="width:427px;">Promega 
			website: http://www.promega.de/</td>
			<td style="width:119px;">V5111</td>
			<td></td>
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			<td style="width:427px;"></td>
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			<td height="21" style="height:21px;width:392px;">Equipment&#160;</td>
			<td style="width:427px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="41" style="height:41px;">Neboulizer (BioNeb cell disruptor)</td>
			<td style="width:427px;">Glas-Col 
			website: http://www.glascol.com/product/subproduct/id/75</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Centrifuge tubes (14 x 89 mm)&#160; 
			for preparation of takahashi style gradients</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;">331372</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Centrifuge tubes 25 x 89 mm 
			for preparation of thylakoid isolation gradients&#160;</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;">344058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Coulter Avanti Centrifuge J-20 XP</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">Fuchs-Rosenthal cell couting chamber</td>
			<td style="width:427px;">Diagonal 
			website: https://www.diagonal.de/</td>
			<td style="width:119px;">449/72</td>
			<td></td>
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			<td height="63" style="height:63px;">Homogenizer (Potter) 50 ml&#160;</td>
			<td style="width:427px;">Fisherbrand 
			website: http://www.de.fishersci.com/index.php/defisherbrand</td>
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			<td></td>
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			<td style="width:427px;">Fisherbrand 
			website: http://www.de.fishersci.com/index.php/defisherbrand</td>
			<td style="width:119px;">105252220</td>
			<td></td>
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			<td height="42" style="height:42px;width:392px;">Ultracentrifuge (Optima XPN-80 Ultracentrifuge)</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:392px;">Other</td>
			<td style="width:427px;"></td>
			<td style="width:119px;"></td>
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			<td style="width:427px;">Agrisera 
			website: http://www.agrisera.com/en/index.html</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a new approach for the comparative analysis of multiprotein complexes based on 15n metabolic labeling and quantitative mass spectrometry

يوفر بروتوكول أدخلت أداة لتحليل المجمعات multiprotein في الغشاء الثايلاكويد، من خلال الكشف عن نظرة ثاقبة تكوين معقدة في ظل ظروف مختلفة. في هذا البروتوكول ويتجلى هذا النهج من خلال مقارنة تكوين البروتين المسؤول معقدة للدوري تدفق الإلكترونات (CEF) في كلاميدوموناس reinhardtii، معزولة عن سلالات مختلفة وراثيا. ويتألف الإجراء عزل الأغشية الثايلاكويد، تليها انفصالهما في المجمعات multiprotein بواسطة السكروز الطرد المركزي التدرج الكثافة، SDS-PAGE، مناعي والمقارنة، قياس الطيف الكتلي الكمي (MS) على أساس وضع العلامات الأيضية التفاضلية (14 N / 15 N) من سلالات تحليلها. يتم تحميل الأغشية الثايلاكويد solubilized المنظفات على التدرجات كثافة السكروز في تركيز الكلوروفيل متساوية. بعد تنبيذ فائق، يتم فصل التدرجات إلى كسور، التي يتم تحليلها بواسطة الشامل spectrometراي يعتمد على حجم مساو. هذا النهج يتيح التحقيق في تكوين داخل الكسور التدرج وعلاوة على ذلك لتحليل سلوك الهجرة من البروتينات المختلفة، مع التركيز خصوصا على ANR1، CAS، وPGRL1. علاوة على ذلك، ويتجلى هذا الأسلوب من خلال التأكد من نتائج مع immunoblotting وبالإضافة إلى ذلك بدعم النتائج من الدراسات السابقة (تحديد والهجرة تعتمد على المبادرة من البروتينات التي تم وصفها سابقا لتكون جزءا من CEF-supercomplex مثل PGRL1، FNR، و CYT و). والجدير بالذكر أن هذا النهج ينطبق على معالجة مجموعة واسعة من الأسئلة التي هذا البروتوكول يمكن اعتمادها وعلى سبيل المثال تستخدم لتحليلات مقارنة للتكوين معقدة multiprotein معزولة عن الظروف البيئية المتميزة.

<html> ويهدف النهج الكمي البروتيوميات قدم لوصف تكوين المجمعات multiprotein في الأغشية الثايلاكويد يتبين من تحليل مقارن لمكونات CEF-supercomplex في مختلف وراثيا C. reinhardtii السلالات. وقد تم بنجاح تطبيق الطريقة الموصوفة من قبل Terashima وآخرون. 6 وتضم عزلة الأغشية الثايلاكويد ...

Watch this Scientific Journal Video about A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry at JoVE.com

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

ويهدف النهج وصفها النسبية، كمية البروتين في الحصول على نظرة ثاقبة لتكوين المجمعات multiprotein في ظل ظروف مختلفة، ويتجلى من خلال مقارنة سلالات مختلفة وراثيا. للتحليل الكمي يتم خلط كميات متساوية من كسور مختلفة من التدرج كثافة السكروز وتحليلها بواسطة مطياف الكتلة.

نهج جديد لتحليل مقارن لMultiprotein مجمعات بناء على<sup&gt; 15</sup&gt; N الأيض وصفها والكمية الطيف الكتلي

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

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A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

12.1K Views.  University of Münster.  ويهدف النهج وصفها النسبية، كمية البروتين في الحصول على نظرة ثاقبة لتكوين المجمعات multiprotein في ظل ظروف مختلفة، ويتجلى من خلال مقارنة سلالات مختلفة وراثيا. للتحليل الكمي يتم خلط كميات متساوية من كسور مختلفة من التدرج كثافة السكروز وتحليلها ب?...

Video: A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI تحضير العينة : الترا أسلوب طبقة رقيقة

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

والتقييم الكمي لاستخدام الطيف Lipidome الخميرة Electrospray قداس التأين

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

تحليل البروتين المجمعات الكبيرة التي الطيف الكتلي الإنشائية

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

زرقاء هلام بولي أكريلاميد الأصلية الكهربائي (BN - PAGE) للتحليل من المجمعات Multiprotein Lysates الخليوي

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

أميد الهيدروجينية / الديتريوم الطيف الكتلي تبادل و- TOF MALDI تحليل Pak2 التنشيط

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

بروتوكول لتحديد البروتين البروتين التفاعلات على أساس<sup&gt; 15</sup&gt; N صفها الأيض، مناعي، الكمي والتحوير الطيف الكتلي الانجذاب

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

الأيضية وصفها وغشاء التقطير للالمقارن تحليل البروتين من<em&gt; نبات الأرابيدوبسيس thaliana</em&gt; الثقافات تعليق خلية

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

النهج ChroP يجمع بين الشذرة والطيف الكتلي لتشريح المناظر الطبيعية البروتيومية الحالة رقم محدد من الكروماتين

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

تقنية جديدة لتحليل الكمي لتساقط الشعر في الفئران عن طريق تحليل درجات الرمادي

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

التحديد الكمي للبروتين خاصة بالموقع يسين Acetylation و Succinylation Stoichiometry الكتلي حيازة مستقلة عن البيانات باستخدام

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...