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PFAIMS: An optimized workflow for quantitative multiplexed phosphorylation analysis using high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS)

ABSTRACT: Protein phosphorylation is vital for the regulation of cellular signaling. Isobaric tag-based proteomic techniques, such as tandem mass tags (TMT), can measure the relative phosphorylation states of peptides in a multiplexed format. However, the overall low stoichiometry of protein phosphorylation constrains the analytical depth of phosphopeptide analysis by mass spectrometry, thereby requiring robust and sensitive workflows. Here we evaluate and optimize high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid mass spectrometers for the analysis of TMT10plex-labeled phosphopeptides. We determined that using FAIMS-SPS-MS3 with three compensation voltages (CV) in a single method minimizes inter-CV overlap and maximizes peptide coverage (e.g., CV=-40V/-60V/-80V) and that consecutive analyses using CID-MSA and HCD fragmentation at the MS2 stage increases the depth of phosphorylation analysis.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Joao Paulo

LAB HEAD: Joao A. Paulo

PROVIDER: PXD014593 | Pride | 2020-01-10

REPOSITORIES: Pride

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Paradoxical mitotic exit induced by a small molecule inhibitor of APC/C Cdc20

Project description:The Anaphase Promoting Complex/Cyclosome (APC/C) is a mega-dalton ubiquitin ligase that initiates mitotic exit by targeting substrates for degradation. The APC/C is activated by Cdc20, which acts as a substrate receptor, and is inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated. We previously identified apcin, a small molecule ligand of Cdc20, as an inhibitor of APC/CCdc20. Surprisingly, we found that apcin paradoxically accelerates substrate degradation and promotes mitotic exit in cells with high SAC activity. Biochemical studies indicate that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin’s behavior as an antagonist of Cdc20 can thus result in either net inhibition of APC/C, delaying mitotic exit when SAC activity is slow, or net activation of APC/C, promoting mitotic exit when SAC activity is high. Genetic experiments suggest that the dual behaviors of apcin arise from targeting a common binding site in Cdc20 that is required for both substrate ubiquitination as well as efficient APC/C inhibition by MCC. We therefore establish a new mechanism through which a small molecule, by targeting a single site on a dynamic protein interface, can lead to opposing biological effects depending on the regulatory context.

2020-01-07 | PXD013786 | Pride

Multiplexed Proteome Profiling of Carbon Source Perturbations in Two Yeast Species with SL-SP3-TMT

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2020-09-02 | PXD021034 | Pride

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2022-09-02 | PXD016164 | Pride

ACP Acylation Is an Acetyl-CoADependent Modification Required for Electron Transport Chain Assembly

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Proteomic Landscape of Endoplasmic Reticulum Remodeling During Neurogenesis via Combinatorial Selective ER-phagy

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Investigating the effect of Aurora Kinase B inhibition in WT and CDH1 mutant primary neuron cultures

Project description:Primary cultures of mouse cerebellar granule neurons at DIV2 that were either WT or Cdh1 mutant were treated with and without the Aurora B inhibitor Barasertib and assessed by phosphoproteomic analysis

2023-10-12 | PXD045116 | Pride

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