Dataset Information

Identification of myristoylated proteins in Human cell lines (HEK293, HeLa, MCF7)

ABSTRACT: Multi-functional capture reagents reported here enable robust identification of metabolically-tagged myristoylated proteomes with unprecedented confidence resulting from the combination of chemical probe-based enrichment, and release and direct detection of lipid-modified peptides by MS. Whilst capture reagents containing enzymatically cleavable linkers have been previously reported they typically require an extra proteolytic step and their capacity to enable detection of lipidated peptides has not been demonstrated. Herein, we report the largest database (85 counts) of experimentally validated human proteins that are myristoylated at an endogenous level in living cells. Furthermore, we demonstrate the first profile of myristoylation in a living multicellular organism and the confident identification of over 50 novel targets. Importantly, this is also the first example of analysis of any protein lipidation event during development. Our methodology is novel in analytical/chemical approach, and provides quantitative and dynamic information. This submission concerns myristoylated proteomes of human origin.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Remigiusz Serwa

LAB HEAD: Edward W. Tate

PROVIDER: PXD001863 | Pride | 2015-03-30

REPOSITORIES: Pride

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Publications

Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic profiling of protein lipidation during vertebrate development.

Broncel Malgorzata M Serwa Remigiusz A RA Ciepla Paulina P Krause Eberhard E Dallman Margaret J MJ Magee Anthony I AI Tate Edward W EW

Angewandte Chemie (International ed. in English) 20150325 20

Novel multifunctional reagents were applied in combination with a lipid probe for affinity enrichment of myristoylated proteins and direct detection of lipid-modified tryptic peptides by mass spectrometry. This method enables high-confidence identification of the myristoylated proteome on an unprecedented scale in cell culture, and allowed the first quantitative analysis of dynamic changes in protein lipidation during vertebrate embryonic development. ...[more]

PMID: 25807930

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Project description:Rhinovirus causes the common cold and drives exacerbation in asthma and COPD, leading to substantial morbidity and healthcare cost. The single positive strand RNA genome of this non-enveloped virus is translated into a single polyprotein that is processed by virus 3C protease to form three structural capsid proteins VP0, VP1 and VP3, and other proteins required for viral life cycle completion. Capsid proteins assemble into the 5S capsid protomer, five of which form a 14S pentamer, and 12 pentamers then assemble to form the 150S icosahedral provirion into which the viral genome is incorporated. During the final step of viral maturation, VP0 is cleaved into VP2 and VP4. VP4 is encoded at the N-terminus of the viral polyprotein, and in many Picornaviruses is N-myristoylated by host cell N-myristoyltransferase (NMT). We have explored the impact of IMP-1088 (the first sub-nanomolar IC50 dual inhibitor of human N-myristoyl transferases HsNMT1 and HsNMT2) on HRV capsid myristoylation in cells. Global identification of proteins for which myristoylation is selectively inhibited by IMP-1088 was performed by quantitative chemical proteomic analysis of YnMyr-tagged proteins in HeLa cells infected with RV16 treated with 50 nM IMP-1088 or vehicle (DMSO). Proteins were subjected to ligation to AzRB, an azide capture reagent bearing a trypsin-cleavable linker and biotin dual label followed by affinity enrichment on Neutravidin beads, on-bead digestion with trypsin, nanoLC-MS/MS analysis of tryptic peptides, and data processing by label-free protein quantification (LFQ) in MaxQuant. 60 N-myristoylated proteins were identified, including the RV16 polyprotein which responded to IMP-1088 inhibition, with no significant change in protein abundances across the whole proteome in the presence of inhibitor. The majority of viral capsid peptides identified in the pull-down mapped to the VP0 domain of the polyprotein, and the N-terminal YnMyr-tagged peptide was readily identified by MS/MS. These data provide the first direct evidence for HRV capsid myristoylation in human cells, and demonstrate that this lipid modification is highly susceptible to inhibition by a selective NMT inhibitor.

Dataset Information

Identification of myristoylated proteins in Human cell lines (HEK293, HeLa, MCF7)

Publications

Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic profiling of protein lipidation during vertebrate development.

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