Gas-Phase Separations of Protein and Peptide Ion Fragments Generated by Collision-Induced Dissociation in an Ion Trap

Abstract

Ion mobility/time-of-flight mass spectrometry techniques have been used to examine distributions of fragment ions generated by collision-induced dissociation (CID) in a quadrupole ion trap. The mobility-based separation step prior to mass-to-charge (m/z) analysis reduces spectral congestion and provides information that complements m/z-based assignments of peaks. The approach is demonstrated by examining fragmentation patterns of insulin chain B (a 30-residue peptide), and ubiquitin (a protein containing 76 amino acids). Some fragments of ubiquitin show evidence for multiple stable conformations. Mass spectrometry (MS) techniques and tandem (MS/MS or MS n ) 1,2 strategies can provide detailed structural information about many different types of ions, including those generated from peptides, 3-5 proteins, 6-11 and carbohydrates. [12][13][14] Recently, a number of groups have investigated the ability to determine information about sequences from dissociating whole proteins, an approach that is referred to as top-down sequencing. 9 The ability to generate and assign fragments directly from protein parent ions offers several potential advantages, as compared with proteolytic approaches. For example, none of the solution chemistry associated with digestion and cleanup is required. Moreover, the entire protein sequence is potentially available from MS/MS analysis of the intact protein, whereas regions of sequence are often lost upon digestion and sample clean up. With this in mind, a variety of activation methods and instrumental configurations [6][7][8][9][10][11][15][16][17][18][19][20] have been used to produce and analyze fragment ions from whole proteins. After activation and dissociation are achieved, a significant issue in these systems is the complex pattern of peaks that arises from dissociation of multiply charged ions. Fragment ions may exist over a range of charge states and sizes, and peak assignments are often complicated by the inability to resolve isotopic structure in high-charge state fragments, as well as overlapping peaks associated with different fragments. Two strategies appear particularly well-suited for reducing spectral congestion in these systems. High-resolution techniques, such as Fourier transform (FT) MS often allow isotopic peaks (as well as overlapping isotope distributions from multiple ions) to be resolved, even for high-charge state fragments. 9,10 Additionally the high-mass accuracy associated with FTMS measurements is valuable for assigning peaks. Another approach that has emerged as a means of reducing spectral congestion involves exposing highly charged fragments to oppositely charged ions (ion/ion reactions). 20 As protons are removed from high-charge state fragment ions, peaks are shifted to higher m/z values, reducing spectral congestion and removing ambiguities associated with assigning charge states. 8,11,20 The kinetics of charge reduction is such that high-charge-state ions react faster than low-chargestate ions; it is possible to rapidly reduce a distribution of charge states and fragments to primarily singly and doubly charged ions. In this paper, we present an alternative strategy for reducing spectral congestion upon dissociation of multiply charged proteins, a combined ion mobility/time-of-flight (TOF) mass spectrometry approach to analysis of fragments that are produced from collisioninduced dissociation in an ion trap. The mobility-based separation of fragment ions that can be carried out prior to MS analysis appears to be an advantage that is unique to the injected-ion drift tube configuration. The separation reduces spectral congestion † Current address