Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60077344%3A_____%2F23%3A00569715" target="_blank" >RIV/60077344:_____/23:00569715 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11310/23:10458024

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0021925822011693?via%3Dihub" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0021925822011693?via%3Dihub</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.jbc.2022.102726" target="_blank" >10.1016/j.jbc.2022.102726</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared

Popis výsledku v původním jazyce

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two stra-tegies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity bio-tinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the popula-tion of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclu-sively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reli-ance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

Název v anglickém jazyce

Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared

Popis výsledku anglicky

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two stra-tegies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity bio-tinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the popula-tion of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclu-sively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reli-ance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

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 periodika

Journal of Biological Chemistry

ISSN

0021-9258

e-ISSN

1083-351X

Svazek periodika

299

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

15

Strana od-do

102726

Kód UT WoS článku

000923287700005

EID výsledku v databázi Scopus

2-s2.0-85146228423