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Miniaturised interaction proteomics on a microfluidic platform with ultra-low input requirements.

ABSTRACT: Essentially all cellular processes are orchestrated by protein-protein interactions (PPIs). In recent years, affinity purification coupled to mass spectrometry (AP-MS) has been the preferred method to identify cellular PPIs. Here we present a microfluidic-based AP-MS workflow, called on-chip AP-MS, to identify PPIs using minute amounts of input material. By using this automated platform we purify the human Cohesin, CCC and Mediator complexes from as little as 4 micrograms of input lysate, representing a 50?100-fold downscaling compared to regular microcentrifuge tube-based protocols. We show that our platform can be used to affinity purify tagged baits as well as native cellular proteins and their interaction partners. As such, our method holds great promise for future biological and clinical AP-MS applications in which sample amounts are limited.

SUBMITTER: Furlan C

PROVIDER: S-EPMC6449397 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Miniaturised interaction proteomics on a microfluidic platform with ultra-low input requirements.

Furlan Cristina C Dirks René A M RAM Thomas Peter C PC Jones Robert C RC Wang Jing J Lynch Mark M Marks Hendrik H Vermeulen Michiel M

Nature communications 20190404 1

Essentially all cellular processes are orchestrated by protein-protein interactions (PPIs). In recent years, affinity purification coupled to mass spectrometry (AP-MS) has been the preferred method to identify cellular PPIs. Here we present a microfluidic-based AP-MS workflow, called on-chip AP-MS, to identify PPIs using minute amounts of input material. By using this automated platform we purify the human Cohesin, CCC and Mediator complexes from as little as 4 micrograms of input lysate, repres ...[more]

PMID: 30948724

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