Fishing for newly synthesized proteins with phosphonate-handles.

Abstract:

:Bioorthogonal chemistry introduces affinity-labels into biomolecules with minimal disruption to the original system and is widely applicable in a range of contexts. In proteomics, immobilized metal affinity chromatography (IMAC) enables enrichment of phosphopeptides with extreme sensitivity and selectivity. Here, we adapt and combine these superb assets in a new enrichment strategy using phosphonate-handles, which we term PhosID. In this approach, click-able phosphonate-handles are introduced into proteins via 1,3-dipolar Huisgen-cycloaddition to azido-homo-alanine (AHA) and IMAC is then used to enrich exclusively for phosphonate-labeled peptides. In interferon-gamma (IFNγ) stimulated cells, PhosID enabled the identification of a large number of IFN responsive newly synthesized proteins (NSPs) whereby we monitored the differential synthesis of these proteins over time. Collectively, these data validate the excellent performance of PhosID with efficient analysis and quantification of hundreds of NSPs by single LC-MS/MS runs. We envision PhosID as an attractive and alternative tool for studying stimuli-sensitive proteome subsets.

journal_name

Nat Commun

journal_title

Nature communications

authors

Kleinpenning F,Steigenberger B,Wu W,Heck AJR

doi

10.1038/s41467-020-17010-0

subject

Has Abstract

pub_date

2020-06-26 00:00:00

pages

3244

issue

1

issn

2041-1723

pii

10.1038/s41467-020-17010-0

journal_volume

11

pub_type

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