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Quantitative nascent proteome profiling by pulse labeling of O-propargyl-puromycin and stable isotope labeled amino acids

ABSTRACT: Recently, a clickable puromycin analog, O-propargyl-puromycin (OPP), was developed and applied to label C-termini of nascent polypeptide chains (NPCs), followed by a proteomic analysis of NPCs that can be affinity purified via click reaction. Despite its advantage, the affinity purification of NPCs using magnetic beads or resins inherently suffers from dozens of non-specific protein binding, which results in hindering accurate quantification of the nascent proteins. To address this issue, we performed dual pulse labeling of NPCs with both OPP and stable isotope labeled amino acids that allows us to distinguish bona fide NPCs from non-specific proteins, thereby enabling accurate quantitative profiling of NPCs.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD019459 | JPOST Repository | Thu Sep 17 00:00:00 BST 2020

REPOSITORIES: jPOST

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Quantitative nascent proteome profiling by dual-pulse labelling with O-propargyl-puromycin and stable isotope-labelled amino acids.

Uchiyama Junki J Ishihama Yasushi Y Imami Koshi K

Journal of biochemistry 20210301 2

Monitoring translational regulation in response to environmental signals is crucial for understanding cellular proteostasis. However, only limited approaches are currently available for quantifying acute changes in protein synthesis induced by stimuli. Recently, a clickable puromycin analogue, O-propargyl-puromycin (OPP), was developed and applied to label the C-termini of nascent polypeptide chains (NPCs). Following affinity purification via a click reaction, OPP allows for a proteomic analysis ...[more]

PMID: 32926143

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