Electrospray ionization mass spectrometry and on-line capillary zone electrophoresis - mass spectrometry for the characterization of citrate synthase

Abstract

The enzyme citrate synthase from E. coli is a protein with a molecular weight (Mr) of 47 885 Da (wild type). This enzyme has been studied extensively, and its amino acid sequence has been characterized. This model protein has been used in this work for development and validation of methods involving capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). The Mr determinations were conducted using sample infusion ESI-MS, and the tryptic digestion products of wild-type citrate synthase were characterized by on-line CE-ESI-MS coupled with a sheathless interface. On-line experiments were conducted on two different mass spectrometers, a Quattro-LC triple quadrupole instrument equipped with a Z-SprayTM source (Micromass), and a reflecting time-of-flight (TOF) mass spectrometer built in-house in the Time-of-Flight Laboratory, Department of Physics, University of Manitoba. This is the first article to be written on the interfacing of a Z-SprayTM source with CE. Unmodified fused silica capillaries gold-coated sheathless interfaces were used. The on-line CE separations yielded theoretical plate numbers greater than 104 on average. Selected ion electrophorograms (SIE) of the tryptic peptides recorded on the Quattro-LC displayed S/N ratios ranging from ca. 14 to 120 on raw data. These SIE enabled identification of each peptide. The use of reflecting time-of-flight mass spectrometry (TOFMS) afforded mass resolution values of ca. 6000 (m/deltamFWHM), which enabled isotopic resolution of the peptide components. CE-ESI-MS and CE-ESI-TOFMS experiments enabled the generation of a complete tryptic map of citrate synthase.Key words: capillary electrophoresis, electrospray ionization, mass spectrometry, citrate synthase, tryptic digestion, triple quadrupole analyzer, time-of-flight analyzer.