Dataset Information

Isobaric Labeling Proteomics Allows a High-Throughput Investigation of Protein Corona Orientation.

ABSTRACT: The formation of the biomolecular corona represents a crucial factor in controlling the biological interactions and trafficking of nanomaterials. In this context, the availability of key epitopes exposed on the surface of the corona, and able to engage the biological machinery, is important to define the biological fate of the material. While the full biomolecular corona composition can be investigated by conventional bottom-up proteomics, the assessment of the spatial orientation of proteins in the corona in a high-throughput fashion is still challenging. In this work, we show that labeling corona proteins with isobaric tags in their native conditions and analyzing the MS/MS spectra of tryptic peptides allow an easy and high-throughput assessment of the inner/outer orientation of the corresponding proteins in the original corona. We put our results in the context of what is currently known of the protein corona of graphene-based nanomaterials. Our conclusions are in line with previous data and were confirmed by in silico calculations.

SUBMITTER: Liessi N

PROVIDER: S-EPMC7818227 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Isobaric Labeling Proteomics Allows a High-Throughput Investigation of Protein Corona Orientation.

Liessi Nara N Maragliano Luca L Castagnola Valentina V Bramini Mattia M Benfenati Fabio F Armirotti Andrea A

Analytical chemistry 20201207 2

The formation of the biomolecular corona represents a crucial factor in controlling the biological interactions and trafficking of nanomaterials. In this context, the availability of key epitopes exposed on the surface of the corona, and able to engage the biological machinery, is important to define the biological fate of the material. While the full biomolecular corona composition can be investigated by conventional bottom-up proteomics, the assessment of the spatial orientation of proteins in ...[more]

PMID: 33285070

Similar Datasets

Project description:BackgroundMiddle-down mass spectrometry (MS), i.e., analysis of long (~50-60 aa) polypeptides, has become the method with the highest throughput and accuracy for the characterization of combinatorial histone posttranslational modifications (PTMs). The discovery of histone readers with multiple domains, and overall the cross talk of PTMs that decorate histone proteins, has revealed that histone marks have synergistic roles in modulating enzyme recruitment and subsequent chromatin activities. Here, we demonstrate that the middle-down MS strategy can be combined with metabolic labeling for enhanced quantification of histone proteins and their combinatorial PTMs in a dynamic manner.MethodsWe used a nanoHPLC-MS/MS system consisting of hybrid weak cation exchange-hydrophilic interaction chromatography combined with high resolution MS and MS/MS with ETD fragmentation. After spectra identification, we filtered confident hits and quantified polypeptides using our in-house software isoScale.ResultsWe first verified that middle-down MS can discriminate and differentially quantify unlabeled from heavy labeled histone N-terminal tails (heavy lysine and arginine residues). Results revealed no bias toward identifying and quantifying unlabeled versus heavy labeled tails, even if the heavy labeled peptides presented the typical skewed isotopic pattern typical of long protein sequences that hardly get 100% labeling. Next, we plated epithelial cells into a media with heavy methionine-(methyl-13CD3), the precursor of the methyl donor S-adenosylmethionine and stimulated epithelial to mesenchymal transition (EMT). We assessed that results were reproducible across biological replicates and with data obtained using the more widely adopted bottom-up MS strategy, i.e., analysis of short tryptic peptides. We found remarkable differences in the incorporation rate of methylations in non-confluent cells versus confluent cells. Moreover, we showed that H3K27me3 was a critical player during the EMT process, as a consistent portion of histones modified as H3K27me2K36me2 in epithelial cells were converted into H3K27me3K36me2 in mesenchymal cells.ConclusionsWe demonstrate that middle-down MS, despite being a more scarcely exploited MS technique than bottom-up, is a robust quantitative method for histone PTM characterization. In particular, middle-down MS combined with metabolic labeling is currently the only methodology available for investigating turnover of combinatorial histone PTMs in dynamic systems.

Dataset Information

Isobaric Labeling Proteomics Allows a High-Throughput Investigation of Protein Corona Orientation.

Publications

Isobaric Labeling Proteomics Allows a High-Throughput Investigation of Protein Corona Orientation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets