No description available.

Analysis of low-abundance components in small samples remains a challenge within bioanalytical chemistry, and new techniques for sample pretreatments followed by sensitive and informative detection are required. In this thesis, procedures for preconcentration and separation of proteins and peptides in open microchannels fabricated on silicon microchips are presented. Analyte electromigration was induced by applying a voltage along the channel length, and detection was performed either by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) within the open channel, or by sampling a nL fraction containing the preconcentrated analytes from the channel for subsequent nano-electrospray ionization- (nESI-) or MALDI-MS. Utilizing solvent evaporation from the open system during sample supply, sample volumes exceeding the 25-75 nL channel volume could be analyzed. For preconcentration/separation of components in the discrete channel volume a lid of inert fluorocarbon liquid was used for evaporation control. In Papers I and II, aqueous, carrier-free solutions of proteins and peptides were analyzed, and the method was successfully applied for fast and simple preconcentration of amyloid beta (Aβ) peptides, related to Alzheimer’s disease. The impact of possible impurities in the analysis of carrier-free solutions was investigated in Paper III with the 1D simulation software GENTRANS, and a method for open-channel isoelectric focusing in a tailor-made pH gradient was developed. The latter approach was used in Paper IV for preconcentration and purification of Aβ peptides after immunoprecipitation from cerebrospinal fluid and blood plasma, followed by MALDI-MS from a micropillar chip. Paper V includes simulations of an isotachophoretic strategy for selective enrichment of Aβ peptides. GENTRANS simulations were used to select the electrolyte composition, and 2D simulations in a geometry suitable for on-chip implementation were performed using COMSOL Multiphysics. / <p>QC 20160930</p>

<div><div><div><div>Mass spectrometry is a widely used tool for efficient chemical characterization and identification. The development of electrospray ionization as a soft ionization method enables mass spectrometry for large biomolecule investigation. Protein as one of the most important classes of biomolecules, its structural changes including folding, unfolding, aggregation, degradation and post-translational modification all influence protein bioactivity. Protein characterization and identification are important for protein behavior mechanism understanding, which may further contribute to disease treatment development. Protein conformation changes are normally very fast, and the initial stages, which significantly influence the conformation changing pathway, normally occur in milliseconds or shorter time scale. Such a fast structural change is hard to be monitored using traditional bulk solution manipulations, and fast sample preparation methods are required. </div><div><br></div><div>In this thesis project, theta tips are applied as a microreactor and nESI-MS emitter to perform fast protein manipulation immediately before MS analysis. Theta tips can be operated in two modes. The first mode is for submillisecond time scale reactions. Proteins and reagents are loaded into different channels and directly sprayed out simultaneously. Proteins and reagents mix and react in the Taylor cone and subsequent droplets for submillisecond time scale. Through this method, pH induced protein folding is investigated and protein folding intermediates were captured. The second mode is for milliseconds or longer reactions. Differential voltages can be applied to each channel before ionization and spray. The electric field between the two channels induces in-tip electroosmosis, which lead to an in-tip mixing and reaction. In this mode, the reaction time is not limited by the droplet lifetime as in the first mode, but is controlled by electroosmosis time. By changing the electroosmosis square wave frequency and cycles, the mixing time can be elongated to milliseconds or longer, which is suitable for slower reaction study. </div><div>Joule heating is discovered during theta tip electroosmosis when samples are dissolved in buffer. The Joule heating effect is high enough to heat up the aqueous solution to at least 75 oC based on Raman thermometry measurement, while the actual peak temperature could be higher. The Joule heating effect in theta tip electroosmosis can be easily controlled by electroosmosis voltage, time, buffer concentration etc.. Proteins are thermally denatured by the Joule heating effect, and the denaturation extent correlates with Joule heating parameters. </div><div>With this results in hand, we are developing a protein melting temperature measurement method using theta tip Joule heating effect and mass spectrometry. This new melting temperature measurement method measures changes in protein mass and charge state distributions. Therefore, it could sensitively detect ligand loss and protein tertiary structural changes, which is an important compensation to current protein melting temperature measurement techniques like CD or DSC. Since the heating time is short and protein concentration for MS is low, protein aggregation and thermal fragmentation are highly avoided so a complete protein thermal unfolding process is monitored. Theta tip electroosmosis combining MS characterized protein thermal denaturation behavior from a new aspect.</div><div><br></div><div>Besides single protein folding and unfolding, protein identification and post-translational modification are important for proteomics study. The traditional bottom-up, top-down and middle-down methods are not able to both preserve intact protein mass and efficiently generate enough fragment peaks easily without performing gas phase dissociation. In this thesis, we also developed a new way to identify proteins combining limited trypsin digestion and mass spectrometry. Intact protein mass was preserved for protein size and PTM identification. Enough tryptic peptides were also generated for protein identification through database search.</div></div></div></div><div><br></div>

No description available.