Analyzing peptides and proteins by mass spectrometry: principles and applications in proteomics

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32166The study of proteins has been a key element in biomedicine and biotechnology because of their important role in cell functions or enzymatic activity. Cells are the basic unit of living organisms, which are governed by a vast range of chemical reactions. These chemical reactions must be highly regulated in order to achieve homeostasis. Proteins are polymeric molecules that have taken on the evolutionary process the role, along with other factors, of control these chemical reactions. Learning how proteins interact and control their up and down regulations can teach us how living cells regulate their functions, as well as the cause of certain anomalies that occur in different diseases where proteins are involved. Mass spectrometry (MS) is an analytical widely used technique to study the protein content inside the cells as a biomarker point, which describes dysfunctions in diseases and increases knowledge of how proteins are working. All the methodologies involved in these descriptions are integrated in the field called Proteomics

Measurements and predictions of a liquid spray from an air-assist nozzle

Droplet size and gas velocity were measured in a water spray using a two-component Phase/Doppler Particle Analyzer. A complete set of measurements was obtained at axial locations from 5 to 50 cm downstream of the nozzle. The nozzle used was a simple axisymmetric air-assist nozzle. The sprays produced, using the atomizer, were extremely fine. Sauter mean diameters were less than 20 microns at all locations. Measurements were obtained for droplets ranging from 1 to 50 microns. The gas phase was seeded with micron sized droplets, and droplets having diameters of 1.4 microns and less were used to represent gas-phase properties. Measurements were compared with predictions from a multi-phase computer model. Initial conditions for the model were taken from measurements at 5 cm downstream. Predictions for both the gas phase and the droplets showed relatively good agreement with the measurements

End-group functionalization of poly(2-oxazoline)s using methyl bromoacetate as initiator followed by direct amidation

Poly(2-alkyl/aryl-2-oxazoline)s (PAOx) are an alluring class of polymers for many applications due to the broad chemical diversity that is accessible for these polymers by simply changing the initiator, terminating agent and the monomer(s) used in their synthesis. Additional functionalities (that are not compatible with the cationic ring-opening polymerization) can be introduced to the polymers via orthogonal post-polymerization modifications. In this work, we expand this chemical diversity and demonstrate an easy and straightforward way to introduce a wide variety of functional end-groups to the PAOx, by making use of methyl bromoacetate (MeBrAc) as a functional initiator. A kinetic study for the polymerization of 2-ethyl-2-oxazoline (EtOx) in acetonitrile (CH3CN) at 140 degrees C revealed relatively slow initiation and slower polymerization than the commonly used initiator, methyl tosylate (MeOTs). Nonetheless, well-defined polymers could be obtained with MeBrAc as initiator, yielding polymers with near-quantitative methyl ester end-group functionality. Next, the post-polymerization modification of the methyl ester end-group with different amines was explored by introducing a range of functionalities, i.e. hydroxyl, amino, allyl and propargyl end-groups. The lower critical solution temperature (LCST) behavior of the resulting poly(2-ethyl-2-oxazoline)s was found to vary substantially in function of the end-group introduced, whereby the hydroxyl group resulted in a large reduction of the cloud point transition temperature of poly(2-ethyl-2-oxazoline), ascribed to hydrogen bonding with the polymer amide groups. In conclusion, this paper describes an easy and fast modular approach for the preparation of end-group functionalized PAOx

Particle-laden weakly swirling free jets: Measurements and predictions

A theoretical and experimental investigation of particle-laden, weakly swirling, turbulent free jets was conducted. Glass particles, having a Sauter mean diameter of 39 microns with a standard deviation of 15 microns, were used. A single loading ratio of 0.2 was used in the experiments. Measurements are reported for three swirl numbers, ranging from 0.0 to 0.3. The measurements included mean and fluctuating velocities of both phases, and particle mass flux distributions. Measurements were compared with predictions from three types of multiphase flow analysis: locally homogeneous flow (LHF); deterministic separated flow (DSF); and stochastic separated flow (SSF). For the particle-laden jets, the LHF and DSF models did not provide very satisfactory predictions. The LHF model generally overestimated the rate of decay of particle mean axial and angular velocities with streamwise distance, due to the neglect of particle inertia. The LHF model predictions of particle mass flux also showed poor agreement with measurements due to the assumption of no-slip between phases. The DSF model also performed quite poorly for predictions of particle mass flux, because turbulent dispersion of the particles was neglected. The SSF model, which accounts for both particle inertia and turbulent dispersion of the particles, yielded reasonably good predictions throughout the flow field for the particle-laden jets

Total body calcium analysis

A technique to quantitate total body calcium in humans is developed. Total body neutron irradiation is utilized to produce argon 37. The radio argon, which diffuses into the blood stream and is excreted through the lungs, is recovered from the exhaled breath and counted inside a proportional detector. Emphasis is placed on: (1) measurement of the rate of excretion of radio argon following total body neutron irradiation; (2) the development of the radio argon collection, purification, and counting systems; and (3) development of a patient irradiation facility using a 14 MeV neutron generator. Results and applications are discussed in detail

Chemical sensor system

A chemical sensing apparatus and method for the detection of sub parts-per-trillion concentrations of molecules in a sample by optimizing electron utilization in the formation of negative ions is provided. A variety of media may be sampled including air, seawater, dry sediment, or undersea sediment. An electrostatic mirror is used to reduce the kinetic energy of an electron beam to zero or near-zero kinetic energy

Instrumentation and development of a mass spectrometry system for the study of gas-phase biomolecular ion reactions

Gas-phase reactions of biomolecular ions are highly relevant to the understanding of structures and functions of the biomolecules. Mass spectrometry is a powerful tool in investigating gas-phase ion chemistry. Various mass spectrometers have been developed to explore ion/molecule reactions, ion/ion reactions, ion/photon reactions, ion/radical reactions etc., both at atmospheric pressure and in vacuum. In-vacuum reactions have an advantage of involving pre-selecting the ions for the reactions using a mass analyzer. Over the decades, a variety of mass analyzers have been employed in the research of ion chemistry. Hybrid configurations, such as quadrupole ion trap with a time-of-flight and or a quadrupole ion trap tandem with an Orbitrap, have been utilized to improve the performances for both the reaction (in trapping mode) and the mass analysis (accurate mass measurements). Complicated instrument structures, including ion optics, multiple mass analyzers and differential pumping for high vacuum, are typically required for the mass spectrometers for gas phase ion chemistry study. An alternative approach is to simplify the instrumentation by using pulsed discontinuous atmospheric pressure interfaces for introducing ionic or neutral reactants and a single ion trap as both the reactor and the mass analyzer. Such a simple mass spectrometry system was set up and demonstrated using two discontinuous atmospheric pressure interfaces in the study for this thesis. It was capable of carrying out ion/molecule and ion/ion reactions at an elevated pressure without the needs of ion optics or differential pumping system. Together with a pyrolysis radical source, in-vacuum ion/radical reactions were performed and their associated chemistry was studied. Radicals are important intermediates related to biochemical processes and biological functions. There are very limited techniques to monitor the reactive intermediates in-situ during a multi-step reaction in aqueous phase. On the other hand, these intermediates can be cooled down and preserved into a single-step procedure in gas-phase reactions since they only occur via collisions. Therefore, the fundamental study of gas-phase radical ion chemistry will provide evidences of the reactivity, energetics, and structural information of biological radicals, which has the potential to solve puzzles of aging, disease biomarker identification, and enzymatic activities. Using the system described above, a new reaction between protonated alkyl amines and pyrolysis formed cyclopropenylidene carbene was discovered, as the first experimental evidence of the reactivity of cyclopropenylidene. Given the important role of cyclopropenylidene in the combustion chemistry, organic synthesis, and interstellar chemistry, it is highly desirable to establish a fundamental understanding of their physical and chemical properties. The amine/cyclopropenylidene reactions were systematically studied using both theoretical calculation and experimental evidences. A proton-bound dimer reaction mechanism was proposed, with the amine and the carbene sharing a proton to form a complex as the first step, which was closely related to the high gas-phase basicity of cyclopropenylidene. Subsequent unimolecular dissociation of the complex yielded three possible reaction pathways, including proton-transfer to the carbene, covalent product formation, and direct separation. These reactions were studied with a variety of alkyl amines of different gas-phase basicities. For the covalent complex formation, partial protonation on cyclopropenylidene within the dimer facilitates subsequent nucleophilic attack to the carbene carbon by the amine nitrogen and leads to a C-N bond formation. The highest yield of covalent complex was achieved with the gas-phase basicity of the amine slightly lower but comparable to cyclopropenylidene. The results demonstrated a new reaction pathway of cyclopropenylidene besides the formation of cyclopropenium, which has long been considered as a dead end in interstellar carbon chemistry. Further reactivity study of cyclopropenylidene towards biomolecular ions was also carried out for nucleobases, nucleosides, amino acids, peptides, proteins, and lipids. The reaction to form proton-bound dimer for protonated biomolecular ions remained as the dominant reaction pathway. Interestingly, other possible reaction pathways, such as modifications of thiyl group or disulfide bonds, double bond addition, and single bond insertion, were inhibited in gas-phase ion/carbene reactions. Such results inferred that the reactivity of neutral species was not directly applicable to ion reactions, with the proton involved in the gas-phase biomolecular ion reactions

Zero-gravity cloud physics laboratory: Experiment program definition and preliminary laboratory concept studies

The experiment program definition and preliminary laboratory concept studies on the zero G cloud physics laboratory are reported. This program involves the definition and development of an atmospheric cloud physics laboratory and the selection and delineations of a set of candidate experiments that must utilize the unique environment of zero gravity or near zero gravity