Collision‐Induced Dissociation (CID) of Peptides and Proteins
Introduction
Evidence for the gas‐phase CID of molecular ions is apparent in the first mass spectra recorded by Sir J. J. Thomson with his parabola mass spectrograph, and the phenomenon was a subject of study throughout the development of MS during the first half of the twentieth century. A summary of the early work on CID has been published (Cooks, 1995). The modern application of CID to the detection, identification, and structural analysis of organic molecules, to complex mixture analysis, and to biopolymer sequencing can be traced to multiples works (Beynon 1973, Futrell 1972, Jennings 1968, McLafferty 1973, Wachs 1972) on CID itself and on the closely allied topic of metastable ion dissociation (Cooks et al., 1973). Detailed study of CID during the latter half of the twentieth century has resulted in an understanding of the energy transfer mechanisms and dissociation chemistry of small ions (<500 Da) (Cooks 1978, McLafferty 1983, McLuckey 1992, Shukla 2000, Turecek 1996). Tandem mass spectrometers, which use gas‐phase collision regions to produce product ions from precursor ions, have proven extremely useful for the identification and characterization of ions and for complex mixture analysis. This usefulness is due to several factors, including the ease of implementation of CID (relative to alternative methods such as photo‐ or surface‐induced dissociation [SID]), the fact that CID is universal (i.e., all ions have a collision cross section), and the fact that CID cross sections are typically high.
The last 20 years has seen a revolution in the application of MS and tandem MS to biological problems, due to the advent of ionization methods such as matrix‐assisted laser desorption ionization (MALDI) (Hillenkamp 2000, Karas 1985) and electrospray ionization (ESI) (Cole 1997, Whitehouse 1985), capable of producing ions from biologically derived samples such as peptides, proteins, and nucleic acids. Given the popularity and wide application of CID for use in tandem MS of small ions, it is not surprising that CID has been extended to the tandem MS of these larger biological ions (Biemann 1994, Hunt 1986). Indeed, tandem MS with CID has become an important tool in the growing field of proteomics, the effort to elucidate the protein complement for a given species as a function of physiological conditions (Aebersold 2001, Larsen 2000, Yates 1998). As is often the case in science, the application of the technique has outpaced the understanding of the underlying phenomena that dictate the appearance of the resulting data. The energy‐transfer mechanisms that operate during collisions of large (>1000 Da) possibly multiply charged ions with small target gas atoms or molecules are not as well understood as they are for smaller ions. The dissociation chemistry of large multiply charged biological ions upon activation by collisions is also the subject of continuing study. The intent of this chapter is to describe the important experimental variables that affect the CID of biologically derived macro‐ions and to describe what is known about how such ions behave as a function of those variables, with reference to examples from the literature. The discussion is largely focused on the behavior of peptide and protein ions, as these species constitute by far the most widely studied biological ions by CID. The ranges of the relevant variables that can be accessed with available instrumentation is discussed. Note that in terms of instrumentation, the discussion is kept narrowly focused on CID behavior; other characteristics of tandem mass spectrometers, such as precursor and product mass resolution, efficiency of ion transfer between mass analysis stages, sensitivity, and others, are not discussed. The reader should be aware that although a given instrument may access a range of CID variables, which yields useful tandem mass spectra in terms of the dissociation that occurs, other instrument criteria must also be considered when evaluating the applicability of a tandem mass spectrometer to a particular problem.
Section snippets
Experimental Variables that Influence the CID Behavior of Biological Ions
A wide variety of conditions has been used to effect CID of biological ions as new types of tandem mass spectrometers have been developed or as existing types have been fitted with ion sources capable of producing biological ions. Some activation methods have been developed specifically to improve the quality of tandem MS data for biological ions. A set of figures of merit for CID are outlined here to aid in the discussion of how peptides and proteins dissociate as a function of the CID
Summary of Commonly Used CID Conditions
The values for any individual figure of merit discussed earlier may vary widely with the type of instrument used and the design of the experiment, and the various figures are not independent but influence one another. In this section, three commonly used regimens for effecting CID of gas‐phase biological ions are described in terms of the figures of merit outlined earlier. Table I summarizes the information given in this section with typical ranges of values used for the operating parameters
Summary and Conclusions
This chapter has focused exclusively on the effects of experimental variables, described in terms of the set of figures of merit, on CID. At least an equal amount could be said about the effects of ion structure on CID behavior, even if the discussion were limited to what is known about peptides and proteins. An exhaustive summary of what is known about biological ion structural effects is beyond the scope of this chapter. The interested reader is referred to the literature on the effects of
Acknowledgments
S. A. M. acknowledges support of his research program by the U.S. Department of Energy, Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Sciences, U.S. Department of Energy, under Award No. DE‐FG02–00ER15105 and the National Institutes of Health, under grant GM45372.
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