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Capillary Gradient Chromatofocusing-Mass Spectrometry: A Sensitive Approach for Protein Analysis

Hribar, James Anthony
http://rave.ohiolink.edu/etdc/view?acc_num=csu1306436593

Abstract Details

2011, Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, College of Sciences and Health Professions.
Gradient chromatofocusing-mass spectrometry is a new technique for protein analysis recently introduced by our research group. Capable of separating and identifying proteins according to pI values and molecular weight, gradient chromatofocusing-mass spectrometry has been achieved by integrating a new ion-exchange chromatography technique called gradient chromatofocusing with a newly discovered buffer system that promotes mass spectrometry detection. Differing from traditional ion-exchange chromatography techniques, gradient chromatofocusing employs specific low molecular weight, volatile buffer components that are introduced onto an ion-exchange HPLC column by programming a binary gradient pumping system to deliver the correct proportions of acidic mobile phase to overcome buffering of the column’s stationary phase initially equilibrated with a basic mobile phase thus creating a linear pH gradient through the column. Offering greater control of the slope of the pH gradient and improving separation capabilities through usage of buffers at higher concentrations, gradient chromatofocusing buffer systems offer compatibility with mass spectrometry detection that is not possible using polyampholyte buffers commonly used with traditional ion-exchange chromatography techniques. This compatibility led to the first reporting of ion-exchange chromatography being interfaced with mass spectrometry by a previous group member who used a 2.1 mm i.d DEAE weak anion-exchange column and a 25 mM buffer system consisting of ammonium bicarbonate, pyridine, lactic acid and acetic acid. Furthermore, the focus of this dissertation will be to develop an optimized capillary gradient chromatofocusing-mass spectrometry system (Chapter 4) capable of detecting at the low-levels associated with proteomics by miniaturizing the HPLC system (Chapter 2) and effectively operating with the lowest buffer concentrations possible to generate linear pH gradients to promote compatibility with the mass spectrometer (Chapter 3). Similar to capillary gradient chromatofocusing, other commonly used protein characterization techniques separate proteins according to charge before determining the molecular mass by introducing analytes into a mass spectrometer preferably using capillary chromatography. Advantages and considerations for using capillary columns will be discussed in Chapter 1. In Chapter 2, sensitivity gains and detection limits were compared for various DEAE weak anion-exchange columns with inner diameters ranging from 255 µm to 508 µm using UV detection. Comparison of sensitivity gains using 255 and 508 µm i.d. columns gave results as theoretically expected. Novel to the completion of the work in this study is the packing of PEEK columns in-house using a pressurized column packing technique and development of an on-line pH measurement system for measuring pH gradients prior to analysis of proteins. In Chapter 3, various trials of generating linear pH gradients with various buffer systems are displayed with goals of selecting a buffer system with the lowest concentrations of buffer components possible to promote mass spectrometry compatibility. Linear pH gradients were achieved by reducing concentrations of the initial buffer system composed of ammonium bicarbonate, pyridine, lactic acid and acetic acid from 25 mM to 10 mM by trial and error programming of the gradient system. Efforts were also directed towards including additional buffer components such as collidine to minimize pH drops in unbuffered regions of the gradient resulting from reduced buffering capacity when reducing buffer concentrations. Capable of providing the buffering capacity needed to generate linear pH gradients, the buffer system containing collidine was not used based upon inaccurate pK values determined for proteins, undesirable UV absorbance and shortened lifetime of columns due to incompatibilities with this buffer component. In Chapter 4, the optimized capillary gradient chromatofocusing system using a 255 µm i.d. DEAE weak anion-exchange column with a 10 mM ammonium bicarbonate, pyridine, lactic acid and acetic acid buffer system was defined and interfaced with an electrospray ionization triple quadrupole mass spectrometer for detection. Significant improvements are emphasized using the optimized capillary gradient chromatofocusing-mass spectrometry system opposed to the gradient chromatofocusing-mass spectrometry system initially introduced. Detection limits have been reduced significantly and pK values were determined using the optimized capillary GCF-MS system thus showing the utility of this qualitative and quantitative tool in the field of proteomics.
David Anderson (Advisor)
Baochuan Guo (Committee Member)
Xiang Zhou (Committee Member)
Stan Duraj (Committee Member)
Mike Kinter (Committee Member)
141 p.
Analytical Chemistry
HPLC; LC-MS; protein analysis

Recommended Citations

Citations

  • Hribar, J. A. (2011). Capillary Gradient Chromatofocusing-Mass Spectrometry: A Sensitive Approach for Protein Analysis [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1306436593

    APA Style (7th edition)

  • Hribar, James. Capillary Gradient Chromatofocusing-Mass Spectrometry: A Sensitive Approach for Protein Analysis. 2011. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1306436593.

    MLA Style (8th edition)

  • Hribar, James. "Capillary Gradient Chromatofocusing-Mass Spectrometry: A Sensitive Approach for Protein Analysis." Doctoral dissertation, Cleveland State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=csu1306436593

    Chicago Manual of Style (17th edition)

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© 2011, all rights reserved.
This open access ETD is published by Cleveland State University and OhioLINK.