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Site-specific characterization of endogenous SUMOylation across species and organs.

ABSTRACT: Small ubiquitin-like modifiers (SUMOs) are post-translational modifications that play crucial roles in most cellular processes. While methods exist to study exogenous SUMOylation, large-scale characterization of endogenous SUMO2/3 has remained technically daunting. Here, we describe a proteomics approach facilitating system-wide and in vivo identification of lysines modified by endogenous and native SUMO2. Using a peptide-level immunoprecipitation enrichment strategy, we identify 14,869 endogenous SUMO2/3 sites in human cells during heat stress and proteasomal inhibition, and quantitatively map 1963 SUMO sites across eight mouse tissues. Characterization of the SUMO equilibrium highlights striking differences in SUMO metabolism between cultured cancer cells and normal tissues. Targeting preferences of SUMO2/3 vary across different organ types, coinciding with markedly differential SUMOylation states of all enzymes involved in the SUMO conjugation cascade. Collectively, our systemic investigation details the SUMOylation architecture across species and organs and provides a resource of endogenous SUMOylation sites on factors important in organ-specific functions.

SUBMITTER: Hendriks IA

PROVIDER: S-EPMC6018634 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Site-specific characterization of endogenous SUMOylation across species and organs.

Hendriks Ivo A IA Lyon David D Su Dan D Skotte Niels H NH Daniel Jeremy A JA Jensen Lars J LJ Nielsen Michael L ML

Nature communications 20180625 1

Small ubiquitin-like modifiers (SUMOs) are post-translational modifications that play crucial roles in most cellular processes. While methods exist to study exogenous SUMOylation, large-scale characterization of endogenous SUMO2/3 has remained technically daunting. Here, we describe a proteomics approach facilitating system-wide and in vivo identification of lysines modified by endogenous and native SUMO2. Using a peptide-level immunoprecipitation enrichment strategy, we identify 14,869 endogeno ...[more]

PMID: 29942033

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Project description:Cysteine S-nitrosylation is a post-translational modification regulating protein function and nitric oxide signaling. Herein the selectivity, reproducibility, and sensitivity of a mass spectrometry-based proteomic method for the identification of endogenous S-nitrosylated proteins are outlined. The method enriches for either S-nitrosylated proteins or peptides through covalent binding of the cysteine sulfur with phenylmercury at pH=6.0. Phenylmercury reacts selectively and efficiently with S-nitrosocysteine since no reactivity can be documented for disulfides, sulfinic or sulfonic acids, S-glutathionylated, S-alkylated or S-sulfhydrylated cysteine residues. A specificity of 97±1% for the identification of S-nitrosocysteine peptides in mouse liver tissue is achieved by the inclusion of negative controls. The method enables the detection of 36 S-nitrosocysteine peptides starting with 5pmolS-nitrosocysteine/mg of total tissue protein. Both the percentage of protein molecules modified as well as the occupancy by S-nitrosylation can be determined. Overall, selective, sensitive and reproducible enrichment of S-nitrosylated proteins and peptides is achieved by the use of phenylmercury. The inclusion of appropriate negative controls secures the precise identification of endogenous S-nitrosylated sites and proteins in biological samples.Biological significanceThe current study describes a selective, sensitive and reproducible method for the acquisition of endogenously S-nitrosylated proteins and peptides. The acquisition of endogenous S-nitrosoproteomes provides robust data that is necessary for investigating the mechanism(s) of S-nitrosylation in vivo, the factors that govern its selectivity, the dependency of the modification on different isoforms of nitric oxide synthases (NOS), as well as the physiological functions of this protein modification. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Dataset Information

Site-specific characterization of endogenous SUMOylation across species and organs.

Publications

Site-specific characterization of endogenous SUMOylation across species and organs.

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