64 research outputs found
Microfluidic front-end technologies for protein electrospray mass spectrometry
In the present work, a polymer microfabricated microsprayer is characterised and tested for the mass spectrometric analysis of biomolecules, in particular in the context of protein mass spectrometry and proteomics. As the core of this work deals with analytical sciences and device development, Chapter 1 puts this work in perspective with the emergence of proteomics and more generally systems biology. In particular, major epistemological concepts that allowed the emergence of these new disciplines among life science research are reviewed. It is shown that the new paradigm of integrative biology did not emerge ex nihilo, but was preceded by major epistemological shifts in medicine, physics, and engineering. As such, the rise of systems biology appears as a new episode in the balance between reductionist and holistic approaches in the history of biology and medicine, and its biomedical promises are discussed. Chapter 2 reviews the state of the art in the hyphenation of microfluidic devices with electrospray mass spectrometry, both from a technical and applicative standpoint. Chapter 3 and 4 present the characterisation of the new polymer microspray for the analysis of peptides, proteins and glycoconjugates when coupled with various electrospray ion sources and instruments. In particular, a detailed comparison with classical pulled nanospray capillaries is performed, that established the performances in terms of sensitivity, stability and applicable ranges of flow rates and solvents. Chapter 5 introduces a functionalised polymer microspray for online sample clean-up. Basically, a hydrophobic polyvinylidene fluoride membrane is interfaced at the inlet of the polymer microspray to serve as a capture solid-phase of hydrophobic compounds; once captured, they can be cleaned up, and further eluted by the spray solution containing organic solvents. The process is further investigated in the presence of compounds usually used in protein sample preparation, such as chaotropes, reducing agents or detergents. Further microchip developments include the introduction of a dual channel microsprayer in Chapter 6, that allows to spray two solutions at the same time; given the microchip design, the two solutions are mixed only in the Taylor cone, where the electrospray is generated, and their respective flow rates can be controlled independently. This unique feature allows to analyse pure aqueous samples with integration of minimum amounts of organic sheath flow to promote desolvation and ionisation. Moreover, when proteins are analysed, it allows to measure them in their folded state, as demonstrated by their charge state distribution in the mass spectrum. Further tuning of the organic sheath flow allows to denature the protein within the Taylor cone, which can be monitored by the evolution of the protein charge state distribution in the mass spectrum. Potential of this technology for protein thermodynamic stability measurement is discussed. Lastly, Chapter 7 presents in silico evaluation of two technologies developed in the laboratory, namely Off-Gel electrophoresis for isoelectric fractionation of proteins and peptides mixtures, and online counting of cysteine residues within peptides during their analysis by electrospray mass spectrometry, to provide useful information to speed-up proteome profiling: if one wants to seek within a whole digested proteomes for peptides that are unique, measurement of their mass alone is hardly sufficient to perform useful protein identification, even with very high resolution, high mass accuracy instruments. The purpose of these simulations is to estimate how much information peptide isoelectric point and number of cysteines within each peptide provide in terms of number of unique peptides (and hence unambiguous peptide identification without MS/MS). Applicability of this strategy for high-throughput proteome profiling and its limitations are further discussed
Dual-Channel Electrospray Microchip
A dual-channel electrospray microchip has been developed for electrospray ionization mass spectrometry (ESI-MS) where aqueous samples are mixed at the Taylor cone with an organic buffer. Due to the fast and effective mixing in the Taylor cone, the aqueous sample can be well ionized with a high ion intensity. The influence of geometric parameters such as the distance between the two microchannels at their junction at the tip of the emitter has been investigated together with chemical parameters such as the organic buffer compositio
About the Electrospray Ionization Source in Mass Spectrometry: Electrochemistry and On-chip Reactions
The present work shows that the electrochemical properties of electrospray ionization (ESI) can be used to add functions to the process. As example, we show how the choice of the electrode material can be used to study interactions between metal ions and biomolecules in mass spectrometry
(MS). In positive ionization MS, an electrospray device acts as anode, which implies oxidation reactions. Sacrificial electrodes (made of copper or zinc) are used to supply the electrospray current and to produce cations that are able to react on-line with compounds of interest. Thus, the
interactions between copper ions and ligands or peptides were investigated by using a copper electrode. Another example is the in situ electrogeneration of a dinuclear zinc(II) complex for the mass tagging of phosphopeptides when working with a zinc electrode. In order to perform these
reactions on the same microchip, a dual-channel microsprayer was used, where one channel was dedicated to the tag electrogeneration and the other to the infusion of a phosphopeptides solution. Finally, this dual-channel microsprayer was used to study complexation at liquid-liquid interfaces
in biphasic ESI-MS, such as thioether crowns and lead ions or peptides and phospholipids complexes. These examples illustrate the use of electrochemistry and on-chip reactions in ESI-MS analysis
Capillary Electrophoresis as a Second Dimension to Isoelectric Focusing for Peptide Separation
Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes
The storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transporter, an anchor site for the cytoskeleton and other membrane proteins as well as a binding site for glycolytic enzymes and bears blood group antigens. In the present study, band 3 complexes were isolated from RBCs stored for 7 and 42 days in average (n = 3), as well as from microvesicles (n = 3). After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE and analyzed by bottom-up proteomics. It revealed that three proteins were present or absent in band 3 complexes stemming from long-stored or short-stored ECs, respectively, whereas the membrane protein contents remained equivalent. These potential markers for storage-induced RBC aging are adenylosuccinate lyase (ADSL), alpha-adducin and flotillin-2, and were further analyzed using western blots. ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas alpha-adducin mainly tended to stay onto the membrane extract. Interestingly, flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage (1.6-fold-change, p = 0.0024). Moreover, flotillin-2 was enriched (almost threefold) in RBCs compared to microvesicles (MVs) (p < 0.001) and the amount found in MVs was associated to band 3 complexes. Different types of band 3 complexes are known to exist in RBCs and further studies will be required to better understand involvement of this protein in microvesiculation during the storage of RBC
α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer
Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes
Biomarker Analysis of Stored Blood Products: Emphasis on Pre-Analytical Issues
Millions of blood products are transfused every year; many lives are thus directly concerned by transfusion. The three main labile blood products used in transfusion are erythrocyte concentrates, platelet concentrates and fresh frozen plasma. Each of these products has to be stored according to its particular components. However, during storage, modifications or degradation of those components may occur, and are known as storage lesions. Thus, biomarker discovery of in vivo blood aging as well as in vitro labile blood products storage lesions is of high interest for the transfusion medicine community. Pre-analytical issues are of major importance in analyzing the various blood products during storage conditions as well as according to various protocols that are currently used in blood banks for their preparations. This paper will review key elements that have to be taken into account in the context of proteomic-based biomarker discovery applied to blood banking
Thin chip microsprayer system coupled to quadrupole time-of-flight mass spectrometer for glycoconjugate analysis
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