Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - METALS (Formation and Characterization of Protein Post-Translational Modifications and Assessment of Cellular Responses by Application of Metals in Biological Systems)
Teaser
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. Many of these reactions are very attractive to both academia and industry. In this proposal, I plan to develop novel...
Summary
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. Many of these reactions are very attractive to both academia and industry. In this proposal, I plan to develop novel applications of metal compounds to solve immediate challenges in mass spectrometry-based proteome research, but also will assess the potential risks of using nano-sized metals in our society. First, it is important to develop an efficient enzyme-independent method to synthesize large amounts of biologically relevant C-terminal amidated peptides. 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 the specific binding of uranyl to artificially phosphorylated recombinant peptides. Data reveal that subsequent UV irradiation produces C-terminal amidated peptides. I will attempt to minimize the bias inherent in current phosphopeptide analysis, which comes from inefficient inhibition of phosphatases during cell lysis. Application of a recently developed gallium complex during cell lysis should limit the extent of this bias by binding phosphorylated proteins. The neutral conditions involved with the gallium complex reaction should also facilitate the possibility of enrichment of acid labile phospho-histidine peptides of which only a handful have been characterized. Finally, humans are now exposed to increasing amounts of artificially nano-metals applied via consumer products, food packages, and cosmetics. I will investigate this problem using advanced mass spectrometry, confocal microscopy, and biochemical assays of the response of human neural cells to nano-metal particles. The particular focus area will be to elucidate whether the action of nanoparticles in human neural cells may shed new light on understanding of diseases like Parkinson´s disease.
To the society an unbiased mass spectrometry characterization of phosphopeptides will lift a long-standing hurdle to proteomics studies. It is of considerable importance in clinical and functional proteomics and will boost the research areas of cell biology, molecular medicine, and systems biology. Revealing the dynamics of the phosphorylation patterns can lead to a more comprehensive understanding of protein functions, cell-signaling, and basic understanding of many diseases. Production of C-terminally amidated pharmaceutical peptides/proteins via a chemical approach is of considerable importance in the pharmaceutical industry and in clinical treatment of many diseases. Many potential peptide pharmaceuticals cannot be efficiently synthesized due to the present limitations of the enzymatic approach. The suggested approach utilizes the specific binding of uranyl to artificially phosphorylated recombinant peptides. Subsequent UV irradiation produces the amidated peptide. The success of this program will remove many of the existing limitations, which will be beneficial for established and new pharmaceutical companies in Europe. Assessing the potential human health risk of metal nanoparticles will be of much importance to toxicology research, industry, public health, law makers, and society. Furthermore, the action of nanoparticles in human neural cells may also shed new light on diseases like Parkinson´s disease that despite decades of research is far from fully understood.
Work performed
Topic I. Limitations in Characterization of Phosphoproteins
This topic encompasses a number of research challenges:
A) Stabilization of the phosphorylation motif in tandem mass spectrometry using vibrational excitation.
ActionA1: Here 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. In this regard I have started collaboration with prof. Markus Wenk at the University of Singapore. The potential outcome could significantly assist in important analysis of spares levels 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 to synthesis molecular imprints and raising anti-bodies, respectively. The molecular imprint did not work as intended, however, we have recently measured the dissociation constant for the antibody and its results are very promising.
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. Publication made.
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. A publication is made
Topic II. Formation of native peptides (C-terminally amidated) from recombinant gene products
This topic encompasses a number of research challenges:
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. The results have highlighted the next targets to investigate. Including the binding affinity as well as the primary sequence effects. We are investigating peptide libraries for this purpose.
E) Click-chemistry to recombinant peptides with unnatural alkyne amino acids
Action E1: appropriate click molecules have been selected and peptide for their coupling is planned. Also the process of initiating collaboration to achieve incorporation of these unnatural amino acids into peptides using recombinant technology has begun.
Topic III. Cellular responses to nano-sized metals.
This topic encompasses a number of research challenges:
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. This was not the original goal, which was to establish the BBB using a dynamic flow cell. However, this has turned out to be quit complicated due to the poor attachment of cells in the flow cell. We therefore now work in parallel using both the tranwell membrane and still attempting to succeed with the dynamic flow-cell.
Action F2: Using the transwell membrane we are now succeed 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 and mercury together with silver nanoparticles. Publications are underway.
Action F5: A series of experiments of the uptake mechanism of silver nanoparticles is planned and will be performed
Final results
We will advance both analysis of nanoparticle induced toxicity in cells using sophisticated proteomics tools. The DIMPES approach appears to hold larger potential than first expected. Now including all phosphorylated biomolecules.