Mass Spectrometric Investigations of Protein Charge State Distributions Using Alternating Current Electrospray Ionization Processes

• Main Authors: CHANG, YU-TZU, 張育慈
• Other Authors: WANG, SHAU-CHUN
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/84158439100648947424

碩士 === 國立中正大學 === 化學暨生物化學研究所 === 104 === In the early 80’s, John Fenn proposed and described how to use direct current ( dc ) electrospray ionization ( ESI ) mass spectrometry for identification and analysis of biological macromolecules, and confirmed that biological molecules such as proteins formed multiple charged gaseous ions. Researchers have investigated direct current electrospray ionization thoroughly for decades. In comparison, the research of the alternating current ( ac ) electrospray ionization, especially at high frequency range ( ≧ 100 kHz ) that can enhance signal intensity, has not been investigated until recent years. High mobility protons migrated back and forth between the Taylor cone tip and the bulk, whereas the protonated samples with low mobility remained inside the Taylor cone before aerosol ejection for ionization during each ac cycle. This study uses two large molecular weight proteins ( myoglobin and cytochrome c ) to explore the novel mechanism of alternating current electrospray ionization. Myoglobin dissolved in ammonium acetate solution adjusted various pH conditions by adding ammonia, have been studied at various frequencies ( 50 kHz，150 kHz，and 250 kHz ) of applied ac voltage. Under high frequency ( 250 kHz ) the charge state distributions do not change at different pH conditions. These observations are considerably different from the mass spectra obtained using dc voltage and are due to high temperature in ac ESI droplets. The high temperature effects denature myoglobin regardless pH conditions. We also examined the ESI spectrum using supercharging reagents of different concentrations including m-NBA，sulfolane，and glycerol of the same pH, and observed that charge state increase at dc ESI but unchanged states at ac ESI. We also investigated cytochrome c and found that charge state distributions were unchanged at ac ESI in low pH range.