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

Lei Wei; Jesse G. Meyer; Birgit Schilling

doi:10.3791/57209

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Cuantificación de proteína específica lisina acetilación y Succinylation estequiometría usando Spectrometry total de la adquisición de datos-independiente

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

JoVE Revista
Biología

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JoVE Revista Biología

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

Cuantificación de proteína específica lisina acetilación y Succinylation estequiometría usando Spectrometry total de la adquisición de datos-independiente

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11,653 Views

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12:49 min

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April 04, 2018

DOI:

10.3791/57209-v

DOI:

10.3791/57209-v

•

12:49 min

April 04, 2018

•

11636 Views

¹Buck Institute for Research on Aging

Transcripción

The overall goal of this novel workflow is to determine the occupancy or stoichiometry of protein post-translational modifications at specific acetylation sites.This novel mass spectrometric workflow provides a higher level of precision of quantification.This method can help answer key questions about reversible protein regulation by post translational modification such as lysine acetylation or succinylation sites are most highly modified and may alter protein function.The main advantage of this technique is that site level stoichiometry of lysine acetylation and succinylation and succinylation are accurately determined and the method can determine stoichiometry from peptides with multiple lysine residues.This technique is highly relevant because it reveals acetylation and succinylation stoichiometry or proportion modified versus unmodified instead of measuring a dimensionless relative abundance change.I first had the idea for this method when I realized that using data independent acquisitions and MS2 based quantification for stoichiometry analysis would greatly improve measurement accuracy.Prepare three replicates of 100

Summary

Aquí, presentamos imparcial cuantificación de proteína específica acetilación o succinylation ocupación (estequiometría) de un proteoma completo mediante un análisis radiométrica de modificaciones endógenas a modificaciones introducidas después de cuantitativo química acilación con anhídridos marcado con el isótopo estables. En combinación con la espectrometría de masas adquisición independiente de los datos sensibles, se obtienen mediciones de ocupación del sitio exacto.