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Formation and Characterization of Protein Post-Translational Modifications and Assessment of Cellular Responses by Application of Metals in Biological Systems

Periodic Reporting for period 4 - METALS (Formation and Characterization of Protein Post-Translational Modifications and Assessment of Cellular Responses by Application of Metals in Biological Systems)

Reporting period: 2020-03-01 to 2021-02-28

The chemistry of metals is rich and viewed in a biological context its diversity is crucial for a multitude of molecular functions in the living cell. In this proposal, I plan to develop novel applications of metal compounds to solve immediate challenges in mass spectrometry-based proteome research, and also assess the potential risks of using nano-sized metals in our society. Presently, C-terminal peptide amidation poses a challenge in pharmaceutical production due to limitations of the two enzymes used for this purpose. The suggested approach in METALS will examine if C-terminal amidated peptides can be produced by specific binding of uranyl to phosphorylated peptides with subsequent UV irradiation of the complex. Attempt will be made to minimize the bias inherent in current phosphopeptide analysis. Application of a recently developed digallium complex with high affinity to protein phosphorylations can potentially advance this line of research. Finally, humans are now exposed to increasing amounts of artificially nano-metals applied via consumer products, food packages, and cosmetics. I will investigate nano-bio interactions using advanced mass spectrometry, confocal microscopy, and biochemical assays to assess the responses in human neural cells to nano-metal particles.

The Main conclusions that can be drawn from the METALS program are:
1) The invented digallium complex is a valuable chemical for phosphoproteomics studies as it can limit the extent of miscleavages by trypsin during digestion of phosphoproteins, 2) formation of C-terminally amidated peptides is possible by application of photo-induced dissociation of uranyl-bound phosphopeptides, but a limitation exists in the lack of specificity of bond breakage. 3) Proteomics studies of cells and animals exposed to various nanoparticles reveal unprecedented details of cellular mechanistic for toxicity that can assist future legislation, but also propel new strategies for cancer treatment.
The work in METALS have been focused within these areas:
Topic I. Limitations in Characterization of Phosphoproteins
A) Stabilization of the phosphorylation motif in tandem mass spectrometry using vibrational excitation.
ActionA1: We have written the first beta version of software to interpret the fragment spectra produced when the gallium complex is attached to phosphorylated peptides
Action A2: We have found that the DIMPES approach for phosphate group protection also has application for the analysis of phospholipids.
B) Enrichment of phosphorylated peptides using the gallium complex.
Action B1: To use for enrichment of phosphopeptides using the gallium tag I have collaborated with Börje Sellergren at Malmø University and Carsten Skøjt at the University hospital Odense.
C) Co-fragmentation of peptides from complex samples.
Action C1: to address the great complexity of biological proteomics samples we have developed software (SuperQuant) that can efficiently deconvolute spectra containing fragments from multiple peptide ions.
Action C2: Another benefit of the SuperQuant software is that it can also assist in peptide identification when peptide sequences are not the databases. This is termed de novo sequencing.
Topic II. Formation of native peptides (C-terminally amidated) from recombinant gene products
D) Comprehensive mass spectrometric analysis of the cleavage mechanism and yield from uranyl photo cleavage
Action D1: A key to this technology is to understand the mechanism of the process. We have now published a paper revealing this mechanism for the first time.
Topic III. Cellular responses to nano-sized metals.
F) Deep proteomics of in vitro nano-bio experiments
Action F1: We have succeeded in establishing a blood-brain-barrier (BBB) in vitro using a transwell membrane setup.
Action F2: Using the transwell membrane we are now succeeding with have the first proteomics data of the impact of silver nanoparticles towards astrocytes and endothelia cells.
Action F3: We have characterized using advanced mass spectrometry the protein corona of silver nanoparticles using different condition.
Action F4: An important area in nanoparticle exposure is the possible synergetic effects of other contaminants like heavy metal and other types of nanoparticles. Therefore, we have studied the synergetic effect of cadmium together with silver nanoparticles.

Below is listed and commented on the results and disseminations of the three main work packages.
Topic I. Limitations in Characterization of Phosphoproteins
Three publications document a data processing approach to interrogate tandem mass spectra to separate and quantify cofragmented peptide ions. For a HeLa cell lysate SuperQuant increased the number of protein quantifications with 10%.
We have also proposed a method termed PhosphoShield, to mitigate salt bridge formation by adding a digallium complex that exhibits high binding affinity to the phosphate group. PhosphoShield significantly enhances the cleavage frequency of at least 17% of the tryptic phosphopeptides that have cleavage sites close to the phosphate group in human liver cancer cells.
Topic II. Formation of native peptides (C-terminally amidated) from recombinant gene products
We applied high mass accuracy mass spectrometry to demonstrated that peptides formed by uranyl photocleavage are C-terminally amidated. Our data led to the hypothesize that the photocleavage reaction preferentially follows an α-amidation-like pathway.
Topic III. Cellular responses to nano-sized metals.
Protein corona composition and structural properties are crucial for the interaction of nanoparticles with living cells. Here we report on a comprehensive quantitative characterization (>350 proteins) of the corona formed on 60 nm silver NPs interacted with human blood plasma under various pH and temperature conditions. Our findings show major overlap between quantified proteins. The study of 544 physicochemical properties for protein suggests that hydrophobicity and favorable partition energy (solvent exclusion) of proteins favor binding to silver NP.

We succeeded with achieving large-scale and fast analysis of samples useful for nano-bio interaction studies. This has allowed us to analyze more than 170 biological samples from mouse bronchoalveolar lavage fluid in just 2.7 days. Among many enriched biological pathways, the highest expression increase was found for neutrophil extracellular trap (NET) formation which is a strong marker for lung inflammation.

Synergy: We found significant effects in cells exposed to more than one nanoparticle in a co-exposure study. Co-exposure to two types of nanoparticles synergistically inhibited proliferation of both cell types, to a greater extent for endothelial cells. In addition, nanoparticle-induced toxicity depended on the cell type; astrocytes were more tolerant to nanoparticles.
In terms of studying nano-bio interactions the use of proteomics has proved very efficient and I think that we have demonstrated unprecedented details of cellular mechanistic not previously obtained for such studies. Especially, in our study using a fast chromatographic system, we show that we can analyze hundreds of biological samples in few days and quantify significant biological markers useful in risk assessment of nanoparticles. We hope more such studies will be done in this type of biological studies making it a gold standard.
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