Development and Application of Theta Tips as a Novel NESI-MS Ion Source and Protein Identification Using Limited Trypsin Digestion and Mass Spectrometry

Feifei Zhao, Purdue University

Abstract

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 is one of the most important classes of biomolecules, and its structural changes including folding, unfolding, aggregation, degradation and post-translational modification all influence protein bioactivity. Therefore, characterization and identification of proteins are important to understand protein functional mechanism, and eventually may contribute to disease treatment development. Protein conformational 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 In this thesis project, theta tips were applied as a microreactor and nESI-MS emitter to perform fast protein manipulation immediately before MS analysis. Theta tips were operated in two modes. The first mode was for submillisecond time scale reactions. Proteins and reagents were loaded into different channels and sprayed out simultaneously. Proteins and reagents mixed and reacted in the Taylor cone and subsequent droplets for submillisecond time scale. Through this method, pH induced protein folding was investigated and protein folding intermediates were captured. The second mode was for milliseconds or longer reactions. Differential voltages were applied to each channel before ionization and spray. The electric field between the two channels induced in-tip electroosmosis, which led to an in-tip mixing and reaction. In this mode, the reaction time was not limited by the droplet lifetime as in the first mode, but was controlled by electroosmosis time. By changing the electroosmosis square wave frequency and cycles, the mixing time was elongated to milliseconds or longer, which was suitable for slower reaction study. Joule heating was discovered during theta tip electroosmosis when samples were dissolved in buffer. The Joule heating effect was high enough to heat up the aqueous solution to at least 75 °C based on Raman thermometry measurement, while the actual peak temperature could be higher. The Joule heating effect in theta tip electroosmosis was easily controlled by electroosmosis voltage, time, buffer concentration etc.. Proteins were thermally denatured by the Joule heating effect, and the denaturation extent was correlated with Joule heating parameters. With this results in hand, we developed a protein melting temperature measurement method using theta tip Joule heating effect and mass spectrometry. This new melting temperature measurement method measured 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 were highly avoided so a complete protein thermal unfolding process was monitored. Through theta tip electroosmosis denaturation of proteins and data processing using Gaussian fitting and sigmoidal fitting, protein melting voltages were identified, which could be converted to melting temperature with a calibrated temperature-voltage curve. The curve calibration still needs some further study to find a proper way to obtain standard protein melting temperature based on MS.

Degree

Ph.D.

Advisors

McLuckey, Purdue University.

Subject Area

Energy|Analytical chemistry|Atomic physics|Biophysics|Chemistry|Electromagnetics|Physics

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