新規プロテオーム解析法の開発とそのバイオマーカー探索への応用

This is the author's version of a work that was accepted for publication in Proteomics-Clinical Applications. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms, may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Proteomics-Clinical Applications, 4: 925-935 (2008), http://doi.org/10.1002/prca.200780111This is the author's version of a work that was accepted for publication in Rapid Communications in Mass Spectrometry. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms, may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Rapid Communications in Mass Spectrometry. 20: 31.38 (2006), http://doi.org/10.1002/rcm.2262This is the author's version of a work that was accepted for publication in Proteomics. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms, may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Proteomics, 6:2042-2049 (2006), http://doi.org/10.1002/pmic.200500575This is the author's version of a work that was accepted for publication in Rapid Communications in Mass Spectrometry. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms, may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Rapid Communications in Mass Spectrometry, 17: 1642.1650 (2003), http://doi.org/10.1002/rcm.110

Saliva from nymph and adult females of Haemaphysalis longicornis: a proteomic study

BACKGROUND: Haemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods. METHODS: H. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences. RESULTS: A total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products. CONCLUSIONS: This study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-015-0918-y) contains supplementary material, which is available to authorized users

Myeloperoxidase in vascular disease and autoimmunity

This dissertation defines mechanisms whereby myeloperoxidase (MPO) can mediate vascular damage when released into the vessel, and explores the pathogenesis of an autoimmune disease targeting MPO. The most abundant neutrophil granule protein, MPO generates powerful oxidants that contribute to innate host defense. However, these same oxidants cause host injury; the release of MPO into the vessel correlates with the impairment of vasoregulatory processes and cellular injury. Herein, we report that cytokeratin 1, an endothelial protein, mediates MPO binding and internalization. Cytokeratin 1 also functions as a scaffolding protein for the vasoregulatory plasma kallikrein-kinin system. This system produces bradykinin, a potent inducer of endothelial nitric oxide synthesis. Our investigations revealed that MPO bound and co-localized with high molecular weight kininogen on endothelial cells, and this interaction interfered with bradykinin cleavage by plasma kallikrein. Further, MPO oxidized and inactivated both kininogen and kallikrein, thus preventing bradykinin release. This work identified cytokeratin 1 as a facilitator of MPO-mediated responses, and provided a new paradigm by which MPO affects vasoregulatory processes during inflammation. One disease characterized by excess intravascular neutrophil degranulation is antineutrophil cytoplasmic autoantibody (ANCA)-mediated vasculitis. One major ANCA specificity is for MPO (MPO-ANCA). The origin of these pathogenic autoantibodies is unknown, though our group previously published studies implicating proteins complementary in sequence to autoantigens as the inciting elements of autoimmune disease in patients with proteinase 3-specific ANCA. In chapter 2, we demonstrated the presence of anti-complementary MPO antibodies in patients; this implied that the development of MPO-specific antibodies was a result of an anti-idiotypic response against the anti-complementary protein antibody, and in this way normal tolerogenic mechanisms were bypassed. For this dissertation, we tested the hypothesis that complementary proteins could cause disease in an MPO-ANCA mouse model. While results were not as we predicted, the work revealed the importance of identifying a pathogenic epitope. An epitope mapping study was carried out using a mass spectrometry-based technique, a tool that may generate powerful data for us in the near future. While the human data suggests a role for complementary proteins in MPO-ANCA disease, proving causation in an animal model remains an elusive goal

Microfluidic devices interfaced to matrix-assisted laser desorption/ionization mass spectrometry for proteomics

Microfluidic interfaces were developed for off-line matrix-assisted laser desorption/ionization mass spectrometry (MALDI). Microfluidic interfaces allow samples to be manipulated on-chip and deposited onto a MALDI target plate for analysis. For this research, microfluidic culturing devices and automated digestion and deposition microfluidic chip platforms were developed for the identification of proteins. The microfluidic chip components were fabricated on a poly(methyl methacrylate), PMMA, wafer using the hot embossing method and a molding tool with structures prepared via micromilling. One of the most important components of the chip system was a trypsin microreactor. An open channel microreactor was constructed in a 100 µm wide and 100 µm deep channel with a 4 cm effective channel length. This device integrated frequently repeated steps for MALDI-based proteomics such as digestion, mixing with a matrix solution, and depositing onto a MALDI target. The microreactor provided efficient digestion of proteins at a flow rate of 1 µL/min with a residence time of approximately 24 s in the reaction channel. An electrokinetically driven microreactor was also developed using a micropost structured chip for digestion. The micropost chip had a higher digestion efficiency due to the higher surface area-to-volume ratio in the channel. Also, the electrokinetic flow eliminated the need for an external pumping system and gave a flat flow profile in the microchannel. The post microreactor consisted of a 4 cm × 200 µm × 50 µm microfluidic channel with trypsin immobilized on an array of 50 µm in diameter micropost support structures with a 50 µm edge-to-edge inter-post spacing. This micropost reactor was also used for fingerprint analysis of whole bacterial cells. The entire tryptic digestion and deposition procedure for intact bacteria took about 1 min. A contact deposition solid-phase bioreactor coupled with MALDI-TOF MS allowed for low-volume fraction deposition with a smaller spot size and a higher local concentration of the analyte. A bacterial cell-culturing chip was constructed for growing cells on-chip followed by off-line MALDI analysis. Coupling MALDI-TOF MS whole cell analysis with microfluidic culturing resulted in more consistent spectra as well as reduction of the total processing time. The microfluidic cell culturing was performed in a PMMA chip with a polydimethylsiloxane (PDMS) cover to allow gas permeation into the culture channel, which contained a 2.1 μL volume active culture chamber. After incubation of E. coli in a microfluidic culture device at 37 ℃ for 24 h, the cultured cells were analyzed with MALDI MS. Also, a microfluidic cell culture device containing continuous perfusion of culture medium was developed using a polycarbonate membrane. This microfluidic culturing format was improved with a fluidic manifold and thermostatted microheaters. Fingerprint mass spectra distinguishing E. coli strains tested were obtained after a 6 h incubation time, which was shorter compared to the 24 h incubation time using conventional culturing techniques. In addition, an enhanced identification procedure for bacteria was achieved by integrating on-chip digestion of cultured bacteria

New ultrasonic-based methods in proteomics

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia.A presente dissertação foi preparada no âmbito do convénio bilateral existente entre a Universidade Nova de Lisboa e a Universidade de Vigo.The present dissertation is devoted, essentially, to the development of new ultrasonic-based sample preparation methods for mass-spectrometry-based analysis of proteins. This dissertation is introduced by a brief overview about general aspects in proteomics such as protein identification, quantification and finally, tissue analysis by mass spectrometry. Special attention is given to fast methods for protein identification, as for example those using tools such as microwave energy and ultrasonic energy to speed-up enzymatic digestion. In addition, a general overview of label-based quantification approaches, such as SILAC, iTRAQ, ICAT, or 18O and, label-free methods are presented. Furthermore, stable 18O-isotopic labeling is described in detail. The last topic covered in the first chapter is the analysis of tissue samples by mass spectrometry, a short overview on this subject is done to introduce the reader on the importance of tissue analysis by mass spectrometry. As far as experimental concerns, different types of ultrasonic devices, such as ultrasonic bath, sonoreactor, ultrasonic probe and multiprobe were assessed in different steps of currents protocols for protein identification through mass spectrometry. Variables such as temperature, sonication time, amplitude and ultrasonic frequency were evaluated in order to identify the optimum conditions. On the first trial, ultrasonic energy was used to accelerate the in-solution enzymatic protein digestion. It was found that the application of the ultrasonic energy (ultrasonic probe and sonoreactor) to a liquid medium containing protein and trypsin speeds protein cleavage from overnight (12h) to 5min. To make the handling easier and faster, the ultrasonic energy was also successfully applied to the protein reduction and protein alkylation steps. Overall, the time was reduced from 3h to 10min, and from many single cleaning steps to just one for reduction and another for alkylation

Thiol-targeted microspray mass spectrometry of peptides and proteins through on-line EC-tagging

Modification strategies targeting specific amino acids in proteins are widespread in proteomic analysis. Cysteine residues have received deep consideration in view of their nucleophilic properties and their occurrence in the proteome. A recently developed micro-electrospray emitter for mass spectrometry was used to electrogenerate species reactive towards specific residues in biomolecules. When spraying L-cysteine in the presence of hydroquinone, the thiol cysteine moiety reacts via a 1,4-Michael addition with the benzoquinone electrochemically generated at the electrode. A series of electrogenerated selective electrophiles based on substituted benzoquinones was characterized as tags for L-cysteine. The rate constants pertaining to the addition of L-cysteine onto the benzoquinones were determined through electrochemical techniques. It was shown that the rate constants are primarily dependent on the electronic nature of the substituents. The apparent tagging extents observed for L-cysteine in microspray mass spectrometry experiments were shown to be highly dependent of the ionization efficiencies of the tag. The on-line mass spectrometric electrochemical tagging (EC-tagging) of cysteine residues was studied for peptides. The EC-tagging was tested with the different hydroquinones on an undecapeptide containing one cysteine residue. Methoxycarbonyl-1,4-hydroquinone was shown to be the most efficient probe and revealed to be suitable to count cysteine units in peptides containing up to three cysteines. The number of cysteines corresponds to the number of characteristic mass shifts observed from the unmodified peptide. The identification of bovine serum albumin and human a-lactalbumin digest samples in a peptide mapping strategy were greatly improved by the application of the EC-tagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provides powerful supplementary information to the masses. The tagging method was applied to the determination of four proteins in a model mixture. In parallel, the microspray emitter was characterized as an electrolysis flow cell for the EC-tagging of peptides. The Levich equation was validated as a first approximation for the calculation of the convection-diffusion limiting current in the device. A finite element simulation of the multi-tagging process of peptides was developed to yield the relative distribution and concentration of tags, untagged and tagged species in the microchannel. The main chemical parameters determining the kinetics of the labelling were assessed and discussed considering the microfluidic aspects of the process. The control of the tagging extent allows the simultaneous mass spectrometric analysis of both the unmodified and of the modified peptide(s). This theoretical work has established the range of optimum conditions for the determination of the number of cysteines in peptides containing up to five cysteine groups. The mass spectrometric EC-tagging of cysteine residues in proteins was studied to probe the cysteine environment. An analytical model was developed to calculate rapidly the tagging extent before the spray event. Experiments with unmodified proteins and their chemically reduced forms have highlighted the strong effect of the cysteine site reactivity on the tagging efficiencies. This study has shown relevant parameters for such on-line electrochemical derivatization / mass spectrometric detection strategies. The chemical derivatization of cysteines by benzoquinone reagents was also investigated. These alkylating reagents revealed efficient for diagonal liquid chromatography to isolate cysteinyl peptides by the retention time shifts due to the hydrophobicity of the tags. The work has demonstrated that the inherent electrochemistry of the electrospray can be employed as post column treatment to derivatize cysteinyl biomolecules. Analytical strategies have been developed to take advantage of this electrochemically-controlled modification