High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

Abstract:

:AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

journal_name

Nat Protoc

journal_title

Nature protocols

authors

Yu X,LaBaer J

doi

10.1038/nprot.2015.044

subject

Has Abstract

pub_date

2015-05-01 00:00:00

pages

756-67

issue

5

eissn

1754-2189

issn

1750-2799

pii

nprot.2015.044

journal_volume

10

pub_type

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