Name: protease- and acid-catalyzed labeling workflows employing 18o-enriched water
Uploaded: 2013-02-20T12:00:00
Description: Watch this Scientific Journal Video about Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water at JoVE.com

Эта работа была поддержана NIH / NIDCR Грант 1R01DE019796.

Представленные Лео-процессов позволяют стабильный изотоп маркировки белков дайджесты и синтетических пептидов. Эти временные Конечно экспериментов (рис. 1) применяются к сравнительной протеомики и количественные исследования, а также протеазы исследований. Каждый рабочий п...

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Объединив стабильного изотопа и маркировки высокого разрешения масс-спектрометрии в времяразрешенных образом, Палео-TimeCourse метод позволяет динамический анализ поколение пептидов продукции. Анализ может быть использован для создания стабильной меченных изотопами пептидов для колич?...

<table border="1"><tbody><tr><td> Название материала </td><td> Компания </td><td> Номер в каталоге </td></tr><tr><td> PepClean C-18 Спин Столбцы </td><td> Thermo </td><td> 89870 </td></tr><tr><td> Opti-TOF 384 MALDI мишень </td><td> AB SCIEX </td><td> 1016629 </td></tr><tr><td> 4800 MALDI TOF / TOF </td><td> AB SCIEX </td><td></td></tr></tbody></table> Таблица 1. Материалов <table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td></tr><tr><td> Альфа-циано-4-гидроксикоричной кислоты </td><td> Sigma Aldrich </td><td> 70990-1G-F </td></tr><tr><td> Бычий сывороточный альбумин (BSA) </td><td> Sigma Aldrich </td><td> A3294-10G </td></tr><tr><td> Dithiothreitol (DTT) </td><td&gt; Acros </td><td> 16568-0050 </td></tr><tr><td> Иодацетамида (IAM) </td><td> Sigma Aldrich </td><td> 1149-5G </td></tr><tr><td> Эндотелина-превращающего фермента-1 (ECE-1) </td><td> R &amp; D Systems </td><td> 1784-ZN </td></tr><tr><td> Трипсин золото </td><td> Promega </td><td> V5280 </td></tr><tr><td> Вода-18 O, 97% атома 18 O </td><td> Sigma Aldrich </td><td> 329878-1G </td></tr><tr><td> Трифторуксусной кислоты (ТФК) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> Массовый набор стандартов для калибровки AB SCIEX TOF / TOF инструменты </td><td> AB SCIEX </td><td> 4333604 </td></tr></tbody></table> Таблица 2. Реагенты

protease- and acid-catalyzed labeling workflows employing 18o-enriched water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Мы использовали Палео-TimeCourse рабочего процесса для динамического наблюдения за включение 18 O-стабильных изотопов в продуктах расщепления пептида порожденных протеолитических ферментов. Изложенный подход является универсальным инструментом для изучения сравнительно протеолит...

Watch this Scientific Journal Video about Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water at JoVE.com

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Стабильный изотоп маркировки рабочих процессов использования<sup&gt; 18</sup&gt; O обогащенной воды (Leo-процессы) являются универсальными инструментами для количественного и качественного исследования протеомики. В протеазы помощь (палео) рабочих процессов,<sup&gt; 18</sup&gt; O-атомы вводятся путем протеолитического расщепления и карбоксильных реакций обмена кислорода опосредовано протеаз. В кислотно-катализируемых (Aleo) рабочий процесс,<sup&gt; 18</sup&gt; O-атомы вводятся карбоксильной обмен кислорода при низких рН.

Протеазы и кислотно-катализируемой маркировки рабочих процессов Применение<sup&gt; 18</sup&gt; O обогащенной воды

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

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Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

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