Ion Trap Mass Analyzers

The design and operation of ion-trap mass analyzers is very different from that of triple quadrupoles. Whereas triple quads analyze and perform MS-MS on peptide ions "on the fly" as they pass through the analyzer, ion traps collect and store ions in order to perform MS-MS analyses on them. The analyzer is very simple in design. The ions from the source are directed into the ion trap, which consists of a top and bottom electrode (end caps) and a ring electrode around the middle (Fig. 8A). The trap itself is about the size of a grapefruit. Ions collected in the trap are maintained in orbits within the trap by a combination of DC and radiofrequency voltages. A small amount of helium is used as a "cooling gas" to help control the distribution of energies of the ions. In full-scan mode, the radiofrequency voltages on the electrodes are stepped or scanned to sequentially eject ions on the basis of their m/z values (Fig. 8B). This produces a spectrum representing all of the peptide ions in the trap at any given time. To monitor the ions coming from the source, the trap continuously

Quadrupole Ion Trap

Fig. 8. Schematic representation of an ion-trap MS instrument. (A) Trapping of ions within the analyzer; (B) the sequential "scanning out" of ions of differing m/z; (C) collision-induced dissociation (fragmentation) of a selected ion; (D) depicts sequential "scanning out" of product ions derived from fragmentation of the precursor ion in (C).

Fig. 8. Schematic representation of an ion-trap MS instrument. (A) Trapping of ions within the analyzer; (B) the sequential "scanning out" of ions of differing m/z; (C) collision-induced dissociation (fragmentation) of a selected ion; (D) depicts sequential "scanning out" of product ions derived from fragmentation of the precursor ion in (C).

repeats a cycle of: 1) filling the trap with ions, and 2) scanning the ions out according to m/z values. Thus, unlike the triple quadrupole, the ion trap produces a series of closely spaced analyses, rather than a continuous analysis. Like the triple quad, the ion trap detects multiply charged peptide ions formed by ESI, as long as their m/z values fall within the mass range limit of the analyzer.

To perform MS-MS analyses, the trap fills with ions from the source. Then a particular ion of interest is selected and the trap voltages are adjusted to eject ions of all other m/z values (Fig. 8B). The voltages on the trap then are quickly increased to increase the energies of the remaining ions, which results in energetic collisions of the peptide ions with the helium gas atoms in the trap and induces fragmentation of the ions (Fig. 8C). The fragments then are caught in the trap and scanned out in according to their m/z values (Fig. 8D).

A good analogy often used to describe MS analysis by ion traps is "rocks in a can." According to that analogy, we can summarize the ion trap MS-MS experiment: a handful of different-sized rocks are scooped up in a can. Then all but one are thrown out. The can is then rattled hard and the remaining rock fragments become pebbles, which then are let out one at a time and weighed.

A unique feature of traps is that fragment ions from an MS-MS experiment can themselves be retained in the trap and subjected to another round of fragmentation. Fragments from this secondary MS-MS analysis can likewise be retained and further fragmented. This type of analysis is referred to as MS" and can yield highly detailed fragmentation information is certain cases. However, MS" analyses are seldom used in proteomics, for two reasons. First, there is currently no way to anticipate what MS-MS-MS experiments need to be done while an analysis is underway. One does not necessarily know what ions will be formed in the MS-MS analysis of a peptide ion, so one cannot readily select a fragment for further fragmentation. Second, the total numbers of ions decrease with the number of MS cycles. After an MS-MS analysis, there frequently are not enough ions left in the trap to perform useful analyses.

There are a couple of other features that distinguish ion traps from triple quadrupoles for tandem MS analyses. The first is that fragmentation patterns generated by MS-MS of peptide ions in ion traps can differ somewhat from those produced by triple quadrupoles. Under the most commonly used operating conditions, traps tend to induce a much more complete fragmentation of the precursor ion than do quadrupoles. This means that more of the precursor ions are converted more efficiently to product ions (and thereby to sequence information) in ion traps. Indeed, the precursor ion signal usually is not seen in ion-trap MS-MS spectra, whereas it often is a prominent feature of triple quad MS-MS spectra. Although we shall consider the key features of peptide ion MS-MS spectra in Chapter 9, we can point out here that triple quads tend to induce a more diverse range of fragmentations in MS-MS than do ion traps. Most of the fragmentations produced by ion traps are those most directly useful in deducing sequence, whereas triple quad MS-MS spectra may yield additional features that can resolve ambiguities and provide additional detail. A final difference between ion traps and triple quadrupoles is the so-called "low m/z cutoff" for MS-MS in traps. Owing to the way the ion trap functions for MS-MS, it is not possible to record the masses of product ions whose m/z values are below about 25% of the m/z value of the precursor ion that was subjected to MS-MS. Thus, an ion at m/z 250 would be the lowest fragment ion that can be detected in MS-MS analysis of a m/z 1000 precursor ion. This is not usually a problem for peptide MS-MS analysis, because identities of low-mass peptide fragments can generally be deduced from the m/z values of corresponding larger fragments.

One last interesting feature to note about ion traps is that they actually are capable of very high mass resolution. However, the resolution of the trap decreases with the speed at which ions are scanned out and detected. At the scan rates typically used for full-scan and MS-MS analysis of peptides traps can adequately resolve ions that differ by at least 1 amu on the m/z scale. If the rate of scanning is slowed, traps can resolve species differing by as little as 0.05 units on the m/z scale. In automated operation, a combination of slow full scans over a limited mass range can be used to determine accurately the charge state of ions prior to MS-MS analysis. As we will see, information on the charge state of the precursor can be very helpful in determining peptide sequence from MS-MS data.

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