Identification of Protein Interaction Partners in Bacteria Using Affinity Purification and SILAC Quantitative Proteomics

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60162694%3AG44__%2F24%3A00558811" target="_blank" >RIV/60162694:G44__/24:00558811 - isvavai.cz</a>

Výsledek na webu

<a href="https://link.springer.com/book/10.1007/978-1-0716-2863-8" target="_blank" >https://link.springer.com/book/10.1007/978-1-0716-2863-8</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/978-1-0716-2863-8_3" target="_blank" >10.1007/978-1-0716-2863-8_3</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Identification of Protein Interaction Partners in Bacteria Using Affinity Purification and SILAC Quantitative Proteomics

Popis výsledku v původním jazyce

Affinity purification, combined with mass spectrometry (AP-MS) is considered a pivotal technique in protein–protein interaction studies enabling systematic detection at near physiological conditions. The addition of a quantitative proteomic method, like SILAC metabolic labeling, allows the elimination of non-specifically bound contaminants which greatly increases the confidence of the identified interaction partners. Compared to eukaryotic cells, the SILAC labeling of bacteria has specificities that must be considered. The protocol presented here describes the labeling of bacterial cultures with stable isotope-labeled amino acids, purification of an affinity-tagged protein, and sample preparation for MS measurement. Finally, we discuss the analysis and interpretation of MS data to identify and select the specific partners interacting with the protein of interest. As an example, this workflow is applied to the discovery of potential interaction partners of glyceraldehyde-3-phosphate dehydrogenase in the gram-negative bacterium Francisella tularensis.

Název v anglickém jazyce

Identification of Protein Interaction Partners in Bacteria Using Affinity Purification and SILAC Quantitative Proteomics

Popis výsledku anglicky

Affinity purification, combined with mass spectrometry (AP-MS) is considered a pivotal technique in protein–protein interaction studies enabling systematic detection at near physiological conditions. The addition of a quantitative proteomic method, like SILAC metabolic labeling, allows the elimination of non-specifically bound contaminants which greatly increases the confidence of the identified interaction partners. Compared to eukaryotic cells, the SILAC labeling of bacteria has specificities that must be considered. The protocol presented here describes the labeling of bacterial cultures with stable isotope-labeled amino acids, purification of an affinity-tagged protein, and sample preparation for MS measurement. Finally, we discuss the analysis and interpretation of MS data to identify and select the specific partners interacting with the protein of interest. As an example, this workflow is applied to the discovery of potential interaction partners of glyceraldehyde-3-phosphate dehydrogenase in the gram-negative bacterium Francisella tularensis.

Druh

C - Kapitola v odborné knize

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název knihy nebo sborníku

SILAC. Methods and Protocols, vol. 2603

ISBN

978-1-0716-2862-1

Počet stran výsledku

12

Strana od-do

31-42

Počet stran knihy

286

Název nakladatele

Humana

Místo vydání

New York

Kód UT WoS kapitoly

—