On-line analysis and tandem mass spectrometry on an ion trap storage/reflectron time-of-flight mass spectrometer.

Abstract

The development and performance of a quadrupole ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) for on-line analysis and tandem mass spectrometry (MS/MS) have been presented herein. This unique and versatile hybrid instrument serves as a rapid and sensitive detector for HPLC and capillary electrophoresis (CE) over a broad mass range with a high duty cycle. Microbore and packed capillary HPLC were coupled with the MS detector for model protein digest analysis and for structure and mutation verification of real recombinant proteins. A low cost data system has also been developed for on-line analysis of HPLC and capillary electrophoresis (CE) separations using the IT/reTOF-MS detector. The system is capable of conducting the data acquisition, display, and real-time storage at a rate exceeding 10 Hz. A data reduction scheme allows the storage of a full set of data in the computer RAM circumventing the slow disk storage process. In addition, a 2-dimensional color contour map describing a separation process can be rapidly generated for the unambiguous identification of analyses in a complex mixture. MS/MS on the IT/reTOF-MS was also demonstrated in this work. Ion isolation and activation are achieved resonantly using an analog circuit. Product ion spectra of small oligopeptides are obtained, which are comparable in fragmentation to those obtained on sector or hybrid mass spectrometers. Several important parameters governing the MS/MS process were optimized. Although the limited energy deposition in the trap does not allow efficient fragmentation of large singly-charged peptides with m/z higher than 1000, the problem may be partially solved by taking advantage of fragmenting the multiply-charged ions produced from the electrospray ionization source. The above idea is demonstrated for fragmentation of multiply-charged peptide ions with low picomole sensitivity and a resolution nearly 1000 for the fragment products. The influence of the primary structure of the peptides on the observed collision-induced fragmentation patterns is discussed.