No abstract

A new technique for studying the time dependence of conformational changes of gas-phase protein ions is described. In this approach, a short pulse of electrosprayed protein ions is introduced into an ion trap and stored. After a defined time period, the distribution of ions is ejected from the trap into an ion mobility/time-of-flight mass spectrometer. Combined measurements of mobilities and flight times in the mass spectrometer provide information about the abundances of different conformer types and charge-state distributions. By varying the storage time in the trap, it is possible to monitor changes in ion conformation that occur over extended time periods (approximately 10-200 ms). The method is demonstrated by examining changes in cytochrome c ion conformations for the +7 to +10 charge states.

An ion mobility/mass spectrometry technique has been developed to record collision-induced dissociation patterns for multiple ions in a parallel fashion. In this approach, a mixture of ions is separated in a drift tube on the basis of differences in mobilities through a buffer gas. As the ions exit the drift tube, they are accelerated into a collision cell and the ensuing fragment ions are dispersed by differences in mass-to-charge (m/z) ratios in a time-of-flight mass spectrometer. Fragment ions that are formed in the collision cell have drift times that are coincident with their antecedent parent ions, allowing the origin of all fragments formed from the mixture of ions to be determined. The approach is demonstrated by examining fragmentation patterns of the [M + H]+ parent and a series of a-, b-, and y-type fragments of [D-Ala2,3]methionine enkephalin.

Cross sections for mixtures of tryptic digest peptide ions formed by electrospray ionization have been measured by a new ion mobility/time-of-flight mass spectrometry technique. Analysis of a series of 113 peptides containing 5−10 residues and having a single lysine group located at the C-terminal end show that cross sections are largely dependent upon the amino acid composition of each peptide. Reduced cross sections (which take into account differences in mass) are found to correlate with the fractions of nonpolar or polar aliphatic residues. Average intrinsic contributions to size for individual amino acid residues (referred to as intrinsic size parameters) have been obtained by solving a system of equations that relates the 113 reduced cross sections to the occurrence frequency of each residue within the different sequences. These parameters fall into families according to the physical sizes and chemical properties of the amino acids; contributions to cross section from nonpolar residues are significantly larger than those from polar groups. Calculated cross sections that are obtained by combining intrinsic size factors with peptide sequences are remarkably accurate:  >90% of calculated values are within 2% of experimental measurements.

An ion trap/ion mobility/quadrupole/collision cell/time-of-flight mass spectrometer that incorporates a differentially pumped orifice-skimmer cone region at the back of the drift tube has been developed for the analysis of peptide mixtures. The combined approach allows a variety of strategies to be employed for collisionally activating ions, and fragments can be monitored by subsequent stages of mass spectrometry in a parallel fashion, as described previously (Anal. Chem. 2000, 72, 2737). Here, we describe the overall experimental approach in detail. Applications involving different aspects of the initial mobility separation and various collisional activation and parallel sequencing strategies are illustrated by examining several simple peptide mixtures and a mixture of tryptic peptides from beta-casein. Detection limits associated with various experimental configurations and the utility for analysis of complex systems are discussed.