UV radiation induces genome-mediated, site-specific cleavage in viral protein

Much research has been dedicated to understanding the molecular basis of UV damage to biomolecules, yet many questions remain regarding the specific pathways involved. Here we describe a genome-mediated mechanism that causes sitespecific virus protein cleavage upon UV irradiation. Bacteriophage MS2 was disinfected with 254 nm UV, and protein damage was characterized with ESI- and MALDI-based FT-ICR, Orbitrap, and TOF mass spectroscopy. Top-down mass spectrometry of the products identified the backbone cleavage site as Cys46–Ser47 in the virus capsid protein, a location of viral genome–protein interaction. The presence of viral RNA was essential to inducing backbone cleavage. The similar bacteriophage GA did not exhibit site-specific protein cleavage. Based on the major protein fragments identified by accurate mass analysis, a cleavage mechanism is proposed by radical formation. The mechanism involves initial oxidation of the Cys46 side chain followed by hydrogen atom abstraction from Ser47 Ca. Computational protein QM/MM studies confirmed the initial steps of the radical mechanism. Collectively, this study describes a rare incidence of genome-induced protein cleavage without the addition of sensitizers.

Analysis of expression of PDCP and MAL13P1.308 of Plasmodium falciparum employing a quantitative proteomics approach based on SILAC

Céline Freymond

Plasmodium falciparum is a deadly parasite that causes malaria in humans. This disease causes the death of one million people every year. To find new means to fight this parasite, it is important to learn more about its biology. As the pathways of protein expression are better understood, it becomes easier to find out how to block these mechanisms. The completion of the genome sequencing has opened new perspective of genome wide analysis. One of these studies was done by LaCount et al. who used the yeast1two1hybrid system to map the complete interaction network between proteins of P. falciparum. ¨ From this network of interaction an interesting protein was studied further by Daubenberger et al. which is PDCP. This protein is closely related to MSP11 in this protein network, a protein involved in erythrocyte invasion. PDCP is a CCCH1type zinc finger protein, a family of proteins that are involved in protein1protein interaction, nucleic acid binding and binding in small ligands. It was shown that its expression was dependant on the density of the parasites. In this study we used the SILAC technology to confirm these previous results. A culture is grown with isotopically heavy isoleucine in the medium as a control sample. After a few cell cycles, almost all the natural isoleucines are replaced with the labeled ones. Three cultures with parasitemia of 2, 5 and 10% are grown and mixed with an equal amount of the control culture. When the proteins are analyzed by mass spectrometry, the peptides that contained isoleucine will be detected as two separate peaks. After normalization of each peptide area with this internal control, the quantity of PDCP can be compared between cultures of different parasitemias. 11 peptides of PDCP have been detected by mass spectrometry, proving its existence for the first time. These peptides were labeled to more than 95%, allowing the comparison of PDCP expression between the cultures. But because of difficulty of reproducibility in in vitro cultures, the regulation of PDCP by the parasitemia has not been observed by SILAC. We wanted to study further the protein interaction network in which PDCP is involved. MAL13P1.308, a protein directly interacting with PDCP and MSP11 in the protein network map, was analyzed by IFA. It was found to be expressed during all stages of the asexual blood stage cycle and it was not imported in the host cell. The next step will be to see if it interacts with PDCP by co1localization studies

2008

Peptide Structure and Radical Chemistry in the Gas Phase Revealed by Product Ion Abundance Analysis in Electron Capture and Transfer Dissociation Mass Spectrometry

Hisham Ben Hamidane

Peptide and protein ion abundances in mass spectrometry (MS) and tandem mass spectrometry (MS/MS) may provide orthogonal, or complementary, information to their mass measurements. Collision induced dissociation (CID), the most commonly used MS/MS technique, demonstrates high variability of fragmentation pathways and their relative product abundances as a function of experimental parameters. Moreover, a reliable prediction of relative abundances of CID products is possible only for relatively small systems, but not for larger biomolecules. Recently developed radical-induced fragmentation techniques, e.g., electron capture and transfer dissociation (ECD/ETD), appear to demonstrate more repeatable and reproducible fragmentation patterns. However, it has been reported that ECD/ETD product ion abundance distribution for peptides is rather flat and extracting additional peptide sequence information is yet to be done. In the present work, an attempt is made to understand the peptide's ECD/ETD product ion abundance formation fundamentals and to reveal the possible implications of this source of information for improving peptide and protein structural characterization. Specifically, we have shown that ECD/ETD of peptides is sensitive to the chemical environment in terms of peptide primary structure (amino acid nature) and possibly higher order structure (hydrogen bond connectivity). After demonstrating the analytical validity of the ECD/ETD product ion abundance methodology, its parameter dependence and its applicability to different experimental setups, results will be presented and rationalized along two main axes of comprehension: gas phase peptide radical chemistry and structure. Whereas fundamental aspects of ECD/ETD (mechanism, thermodynamics) can be addressed with the former, additional ECD/ETD yield modulations were observed and presumably relate to structural properties of ions in the gas phase. ECD specificity will be demonstrated on the class of amphipathic peptides, where periodicity is revealed in product ion abundance and correlated to peptide primary and secondary structure. To decipher between both contributions, sequence variations of amphipathic peptides were probed by ECD and correlated to gas phase structures suggested by ion mobility mass spectrometry and supported by molecular dynamics computations. After probing amino acid side chain influence, unraveling the chemical environment specificity of ECD/ETD led us to investigate peptide backbone modifications. Peptides containing beta amino acids revealed two main effects in ECD and ETD. We demonstrate that individual substitutions yield c and z type fragments only for amino acids that provide sufficient z• radical stabilization, whereas multiple substitutions shift the preferential fragmentation pathway to the otherwise minor a-type ion formation pathway indicating possible change in peptide protonation. Finally, understanding the partitioning of radical ions between N- and C-terminal fragments led us to investigate ion internal energy dependence. The extent of hydrogen atom transfer, the [c+z] complex lifetime and secondary fragmentation pathways (w-type ion formation) are directly modulated by ion internal energy as demonstrated by decoupling ETD from charge reduced CID. To summarize, ECD/ETD product ion abundance methodology developed in the thesis advances biomolecule radical chemistry understanding and provides a potentially useful methodology for peptide and protein structural characterization.

EPFL2011

Novel Analytical Methods for Allergy-Related Studies

Mikaël Frossard

Managing and treating diseases require a deep understanding of the pathologies coupled with accurate diagnostic procedures. The constant development and improvement of bioanalytical methods reflect the need of diagnosis ameliorations with respect to accuracy, sensitivity and efficiency. The field of allergy diagnosis is a good example. Despite affecting almost a third of the population in western countries, allergies still present complex challenges for the physicians in terms of diagnosis and allergen characterization. This thesis presents novel analytical methods developed for the fast and personalized allergy component-resolved diagnosis (CRD), the allergen identification and quantification in food matrices and the characterization of allergen modifications, notably nitration. Immunomagnetic separation (IMS) was used to perform the allergy diagnosis at the molecular level by extracting the IgE antibodies from allergic patient blood sera and probing them against individual allergens and natural allergenic extracts. The fast and highly selective IMS procedure was coupled to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS) to identify accurately and with high sensitivity the proteins recognized as sensitizers by the patient antibodies. The developed CRD methods were successful to diagnose cowâs milk, egg, peanut and tree nuts allergies, allowing the resolution of several complex clinical cases. Additionally, precious information was collected for the cross-reactivity and cross-sensitization in peanut and tree nuts allergies using blood sera from the PronNut clinical study. Allergen nitration is one of the potential causes for the increase in allergy prevalence observed recently and requires thorough investigation. Herein, various nitrating agents were tested and compared for in vitro allergen nitration. After modifying allergens using two of the most efficient nitrating procedures, the nitration sites were identified by bottom-up proteomics (BUP) and compared to the known epitopes, bringing useful information and novel insights to the problem of correlation between protein nitration and allergenicity. The presence of a minute amount of allergens in a food product is responsible for potential life-threatening reactions in highly allergic patients and sensitive analytical methods are therefore required in food quality assessments. A proteomic-based method was used for the quality control of several food products suspected of causing severe reactions for peanut and hazelnut-allergic patients. Peptide markers were identified in a first screening of the food extracts by BUP using a high-resolution MS instrument. A standard addition method was then used to quantify peanut and hazelnut proteins by BUP using an untargeted âshotgunâ approach.

EPFL2020