The Whys and Wherefores of Quadrupole Ion Trap Mass Spectrometry

Karen R. Jonscher and John R. Yates, III University of Washington at Seattle

A sensitive and versatile analytical system, capable of identifying both large and small molecules and determining their molecular structure, is required to address the complex mixtures of molecules found in many types of biological problems. Of fundamental importance to the biochemist and biologist is the existence of robust, easy-to-use, and inexpensive instrumentation for application to their studies.

Developments over the last 10 years have made the quadrupole ion trap mass spectrometer (ITMS) an excellent tool for biomolecular analysis. A quadrupole ion trap is an instrument roughly the size of a tennis ball whose size is inversely proportional to its versatility. Three hyperbolic electrodes, consisting of a ring and two endcaps, form the core of this instrument. In the early 1950s, Wolfgang Paul and co-workers invented two instruments that could be used to determine mass -to-charge (m/z) ratios of ions (1). The first was the quadrupole mass filter that rapidly was applied to a wide range of analytical problems (2). The second was the quadrupole ion trap. A diagram of the two mass spectrometers is presented in Figure 1. The difficulty of machining the hyperbolic electrodes, coupled with limited performance, restricted interest in this instrument primarily to the physics community. A user of note was Hans Dehmelt at the University of Washington who recently won the Nobel Prize for employing the ion trap to investigate the physical properties of isolated ions (3). The ion trap was operated at that time in the so-called "mass-selective stability" mode of operation. In this mode, analogous to the operation of a quadrupole mass filter, the amplitudes of rf and dc voltages applied to the ring electrode were ramped at a constant rf/dc ratio to allow stability, hence storage, of a single (increasing) m/z value in the ion trap.

The chemistry community's interest in the trap was confined to several research groups until 1983 when George Stafford and co-workers at Finnigan MAT made two major advances. First, they developed the mass-selective instability mode of operation (4). The fundamental difference between this mode of operation and previous methods is that all ions created over a given time period were trapped and then sequentially ejected from the ion trap into a conventional electron multiplier detector. Thus, all ions were stored while mass analysis was performed, unlike the mass-selective stability mode of operation where only one value of m/z at a time was stored. This new method for operating the ion trap simplified the use of the instrument. Stafford's group's second breakthrough was finding that a helium gas of about 1 mtorr within the trapping volume greatly improved the mass resolution of the instrument by contracting the ion trajectories to the center of the trap and reducing the kinetic energy of the ions (5). This allows ions of a given m/z to form a packet. The ion packet is ejected more quickly and efficiently than a diffuse cloud of ions may be ejected, thus improving resolution. Both these discoveries led to the successful development of a commercial ion trap mass spectrometer.

Figure 1: Comparative diagram, roughly to scale, of a quadrupole mass filter and a cutaway view of a quadrupole ion trap mass spectrometer. Both instruments may be interfaced to an ion source and a detector.

A glossary of ion trap terminology is included with this article. The first occurrence in the text of a term defined in the glossary is shown by italics.

Subsequent innovations have been rapid. The nominal mass range of the instrument has been extended from m/z 650 to m/z 70,000. This mass range is considerably higher than that obtained on a quadrupole instrument and lower than that achievable on a time-of-flight mass spectrometer (TOF-MS). Quadrupole instruments typically have unit mass resolution throughout the mass range. The mass resolution on highly modified ion traps allows the separation of ions of m/z 106 and m/z 106 + 1 to achieve a resolution of 1 million. Perhaps the biggest strength of the ion trap technique lies in the ability to perform multiple stages of mass spectrometry, unlike a triple quadrupole instrument or a TOF-MS. Up to 12 stages of tandem mass spectrometry (MS12) have been performed using an ion trap, greatly increasing the amount of structural information obtainable for a given molecule. Quadrupole ion trap mass spectrometers are also exquisitely sensitive. Molecular weight information has been recorded with as few as 1.5 million peptide molecules. These and other developments have improved the performance of the ion trap and created interest in its application to biological molecules. Graphical user interfaces on the newest commercial ion traps provide easy access to the different scan modes an ion trap uses for different experiments without the need for extensive manual setup and tuning. This obviates the need for an in-depth understanding of the theoretical aspects of ion trap use and brings the power of ITMS to biologists and biochemists in addition to analytical chemists.

General Overview

Ion Trapping Ions created by electron impact (EI), electrospray (ESI), or matrix-assisted laser desorption (MALDI) ionization are focused using an electrostatic lensing system into the ion trap. An electrostatic ion gate pulses open (-V) and closed (+V) to inject ions into the ion trap. The pulsing of the ion gate differentiates ion traps from "beam" instruments such as quadrupoles where ions continually enter the mass analyzer. The time during which ions are allowed into the trap, termed the "ionization period", is set to maximize signal while minimizing space-charge effects. Space-charge results from too many ions in the trap that cause a distortion of the electrical fields leading to an overall reduction in performance. The ion trap is typically filled with helium to a pressure of about 1 mtorr. Collisions with helium dampens the kinetic energy of the ions and serve to quickly contract trajectories toward the center of the ion trap, enabling trapping of injected ions. Trapped ions are further focused toward the center of the trap through the use of an oscillating potential, called the fundamental rf , applied to the ring electrode. An ion will be stably trapped depending upon the values for the mass and charge of the ion, the size of the ion trap (r), the oscillating frequency of the fundamental rf ( w), and the amplitude of the voltage on the ring electrode ( V). The dependence of ion motion on these parameters is described by the dimensionless parameter qz,

qz = 4eV/mr2w2 Equation 1

Ion Trap Glossary

ac voltage: also called supplementary or auxiliary potential, is a voltage placed on the endcap electrodes. It is used to induce resonance excitation and resonance ejection.

bath gas, damping gas, target gas: helium gas in the trapping volume at a pressure of about 1 mtorr serves to cool ion kinetic energies and focus ion trajectories into a tight packet at the center of the trap.

fundamental rf: a (typically) 1.1 MHz potential applied to the ring electrode. The amplitude of this potential deter mines the range of m/z values that can be trapped and is ramped to eject ions.

high resolution: an experiment where peaks corresponding to carbon isotopes may be resolved. The mass scan speed of the instrument is reduced, resulting in an increase in the density of data points per unit m/z, thus increasing mass resolution.

qz: a dimensionless parameter that determines stability of ion trajectories depending upon their mass-to-charge ratio, the size of the ion trap, and the amplitude and frequency of the fundamental rf.

resonance: an ac voltage is applied to the endcap and the q z value of an ion of interest is changed until the secular frequency of the ion matches the frequency of the applied ac voltage. When resonance occurs, the amplitudes of ion trajectories linearly increase with time. A high-amplitude ac voltage will cause resonance ejection, while a low -amplitude ac voltage will cause resonance excitation.

secular frequency: the frequency, dependent upon the q z value, with which an ion oscillates in the trap.

space-charge effects: too many ions in the trap distort the electric fields, leading to significantly impaired perfor mance.

tickle voltage: an ac voltage applied to the endcap electrodes during an excitation period. The amplitude of the voltage is generally small so as to enable fragmentation of the ions by collisions with the helium damping gas rather than ejection.

For the case of existing commercial ion traps, r = 1 cm, w/2p = 1.1 MHz, and V ranges from 0 to 7,500 V 0-p. In addition, a dc potential, U, can be placed on the ring electrode and can also affect the stability of the ion trajectories via a parameter a z that depends upon the amplitude of U.


Figure 2 shows the "stability diagram", a region where radial and axial stability overlap. Depending upon the amplitude of the voltage placed on the ring electrode, an ion of a given m/z will have a qz value that will fall within the boundaries of the stability diagram, and the ion will be trapped. If the q z value at that voltage falls outside of the boundaries of the stability diagram, the ion will hit the electrodes and be lost.

Figure 2: Diagram showing the regions of stability in the quadrupole ion trap parameterized in terms of the operating voltages and frequencies.

Ion Excitation and Ejection Ions oscillate with a frequency, known as the secular frequency, that is determined by the values for az and qz and by the frequency of the fundamental rf. Resonance conditions are induced by matching the frequency of a supplementary potential applied to the endcap electrodes to the secular frequency of the ion. Structural information is obtained by the application of a low-amplitude ac resonance signal across the endcap electrodes causing the ion kinetic energies to increase and leads to ion dissociation due to many collisions with the helium damping gas. This process causes random fragmentation along the peptide backbone in a manner analogous to that obtained using a triple quadrupole mass spectrometer. A mass spectrum is generated by sequentially ejecting fragment ions from low m/z to high m/z by choosing amplitudes of the fundamental rf potential that sequentially make ion trajectories unstable. Ions are ejected through holes in the endcap electrode and detected using an electron multiplier.

More Detailed Examples

Ion Trapping Ions of different m/z values may have stable orbits at the same time, as shown in Figure 3. Because ion trajectories become unstable when qz = 0.908 (see Figure 2), a well-defined low-mass cutoff is created for a given value of the amplitude of the applied rf voltage, V. No ions below that mass will be trapped, but ions above that mass will be trapped with trapping efficiency decreasing for larger m/z values. Low-mass cutoffs for various amplitudes of the applied fundamental rf voltage are listed in Figure 3.

Figure 3: Relative positions of ions with three different mass-to -charge ratios along the mass-selective instability line, a z = 0. The effect of increasing the amplitude of the fundamental rf voltage is shown in panels (a) through (c).

Ion Ejection Depicted in Figure 3 is an example of the relative positions of three ions of differing m/z values on the line a z = 0. Three different amplitude values of the fundamental rf signal are given. As the voltage is increased, the qz value for the ion also increases. Figure 3(c) shows that at 6000 V, the ion of m/z 500 has been ejected from the ion trap. At the maximum amplitude of 7,500 V, the qz value for m/z 1,500 has only reached 0.404, thus that ion cannot be ejected from the ion trap. The amplitude of the rf has not been increased above 7,500 V due to practical difficulties encountered when interfacing high voltages to electronic circuitry.

An applied oscillating signal may be used to increase ion kinetic energies and excite ion trajectories (6). If the amplitude of the signal is large enough, ions will be ejected from the trap rather than undergo fragmentation. This technique, termed resonance ejection, allows ejection to occur at voltages lower than those required for ejection at qz of 0.908, extending the nominal mass range of the ion trap. Conceptually, this may be viewed as creating a "hole" in the stability diagram. This effect is illustrated in Figure 4 where an ellipse represents a resonance (or instability) point that extends the mass range by a factor of 4. At 1,000 V none of the ions has a qz value approaching that of the resonance point so the ions remain inside the ion trap. At 3,000 V, m/z 500 has been ejected and m/z 1,000 is in the process of being ejected. The q z value for m/z 1,500 is smaller than 0.227, thus that ion will not be ejected. At 6,000 V, the q z values for all the ions are greater than 0.227, the q z value of the resonance point. This example shows that when resonance ejection is used and the amplitude of the voltage is ramped from low to high amplitudes, all the ions "fall through the hole" and are ejected from the trap and detected. A combination of forward and reverse resonance ejection ramps may be used to isolate ions in the trap, as shown in Figure 5.

Figure 4: The same conditions as in Figure 3 except a resonance point at qz = 0.227 has been imposed to increase the effective mass range by a factor of 4. A region of instability is created that affords the ejection of ions at lower voltages than would normally be required, therefore ions of large m/z can be ejected from the ion trap and detected.

Figure 5: Reverse-then-forward scanning of the amplitude of the fundamental rf voltage in conjunction with the application of an auxiliary signal to create an instability point affords ion isolation. (i) Reverse scanning resonantly ejects ions from high to low m/z. (ii) Forward scanning resonantly ejects ions from low to high m/z. (iii) Resultant isolation of one value of m/z.

An Example of the Application of the Technique

Micro-electrospray ionization coupled to ion trap mass spectrometry was applied to the analysis of a peptide mixture resulting from the enzymatic digestion of recombinant tissue plasminogen activator (tPA), a mixture containing 51 expected tryptic peptides (7). Results are illustrated in Figure 6. The entire digest (0.5 pmol/ml) was infused into the mass spectrometer using a flowrate of 50 nl/min. The hump in the background of the MS spectrum is due primarily to the presence of large numbers of ions in the mixture. An ion at m/z 879 was isolated utilizing reverse-then-forward isolation. The resolution of the ion trap was increased by slowing the mass scan speed using the method of Schwartz et al. (8). This creates a small range of rf voltages that are scanned, thus a small mass window that is ejected. The same number of data points are maintained for the smaller mass window as for the full mass range, therefore the resolution is increased because the number of data points per unit m/z is increased. The distance (about 1 u) between the isotope peaks indicates the ion is singly-charged. The amino acid sequence of the peptide is shown. MS/MS was performed using resonance excitation, and the presence of several sequence ions was sufficient to identify the peptide. Random fragmentation along the peptide backbone creates a number of different types of sequence ions. Nomenclature depends upon the site of cleavage and the location of the retained charge. In the figure, underscoring of an amino acid residue indicates observation of a y-type ion with charge retained on the carboxyl-terminus, while overscoring indicates a b-type ion with charge retained on the amino-terminus. Ions in the middle of the peptide were not observed due to suppression of fragmentation following the G residue. Less than 2.5 pmol of material (less than 5 ml) were required to stabilize and optimize the microspray and obtain all the data. The four mass spectra shown represent the consumption of less than 12 fmol of material, indicative of the combined sensitivity of microspray and quadrupole ion traps. Most of the sample was consumed while stabilizing the spray due to our lack of proficiency using this new ionization source.

Figure 6: Results from microspray/ion trap analysis of a tryptic digest from recombinant tPA. The top panel displays the mass spectrum of the digest. Isolation of m/z 879 was accomplished using the reverse-then-forward scanning technique depicted in Figure 5. The isolated ion was ejected with attenuated scan speeds to obtain the high resolution mass spectrum. The spacing between the isotopes indicates the ion is singly-charged. The isolated ion was fragmented by applying a resonance signal to the endcap electrodes. The duration of the excitation pulse was 30 msec, and the qz value for the ion was 0.306.

The pulsed nature of the quadrupole ion trap, due to the opening and closing of the ion gate, makes it particularly well -suited for pulsed ionization techniques such as MALDI. A MALDI-ion trap that has been described previously (9) was used for mapping the tryptic peptides from tPA. One pmol of the digest was loaded onto a probe tip and co-crystallized with 1 ml of a saturated solution of a-cyano-4-hydroxycinnamic acid in 1:1 0.1% trifluoroacetic acid:acetonitrile. The MALDI-generated mass spectrum of the digest is shown in Figure 7. Several of the peaks corresponding to tryptic peptides are labeled. Approximately 75% of the expected peptides falling within the measurement mass range were detected. The analytical potential of MALDI-ITMS is continuing to be explored and shows great promise for application to biological molecules.

Figure 7: MALDI-ITMS mass spectrum of a tryptic digest from recombinant tPA. Several of the tryptic peptides are labeled.

The New Generation of Ion Traps

In the past, the ITMS has not been an instrument well -suited for the robust and routine analyses required by biochemists and biologists. High performance innovations to the ITMS developed over the last several years have been used to build a new generation of ion trap mass spectrometer. This instrument has been carefully designed to interface with atmospheric pressure ionization techniques that are optimal for the analysis of biomolecules. Unit mass resolution, or the ability to separate an m/z value of 1,500 from 1,501, is maintained over the 2,000 dalton mass range with a mass accuracy of 0.015%. These figures of merit are comparable with the performance of current triple quadrupoles. It is expected that the mass range of the instrument will increase to 5,000 daltons in the next year or two. A limitation of the ion trap is that the alternate scan modes of triple quadrupole mass spectrometers, such as precursor ion and neutral loss scans, are currently not possible. These scan modes are particularly useful for identifying the presence of trace components in complex mixtures.

The most striking feature of the new ion trap is the software control of instrument operation. An Ion Trap instrument Control Language (ITCL) was developed to control all the elements of an experiment, requiring limited user interaction. All parameters needed to perform a given experiment are automatically set using one ITCL command, compared with the necessity to manually set a number of parameters using the ITMS software found on the older generation of commercial ion traps. The ITCL language also enables the user to perform data-dependent experiments. For example, a command such as "hires mass(1)" would perform a high resolution mass scan on the most intense ion detected in the previous mass scan. This experiment allows the determination of the charge state of multiply-charged ions by expanding a region of the mass range near an ion of interest so the distribution of carbon isotopes may be resolved. Charge -state determination is not possible using a triple quadrupole mass spectrometer under the conditions normally used for high sensitivity peptide analysis. Very complicated data-dependent routines such as "on-the-fly" tandem mass spectrometry can be performed by stringing together commands in the form of a computer program. A graphical user interface is employed to simplify the use of the ITCL language and to edit the type of experiment desired during the course of an analysis. No user intervention in the process is required except for the initial setup of the analysis. This level of control is unprecedented in mass spectrometry. In fact, the reliance on embedded software control is so great that instrument upgrades will essentially require downloading software from a CD-ROM to change operational parameters, obviating the need for expensive additions of hardware.

An example of the application of these experiments is illustrated in Figures 8-10. Figure 8 depicts the charge-state determination of a high-mass, multiply-charged ion from Interleukin 8 (rat), a 7.8 kDa protein, obtained by slowing the mass scan speed of the instrument. Figure 8(a) shows the unit -resolution full-range mass spectrum. The ion at m/z 1,962 was chosen for charge-state determination, and the result of the high resolution scan mode is demonstrated in Figure 8(b). The spac -ing between the isotope peak centroids is about 0.25 u, indicating the ion has a charge state of +4. The width of the peak at half the maximum intensity (FWHM) for the signal at m/z 1,962.1 is 0.1 u, providing a mass resolution of 19,600 for that ion.

Figure 8: Charge state determination for a high-mass ion from Interleukin 8 (rat). Sample at a concentration of 10 pmol/ ml was infused into an atmospheric pressure ionization source. (a) Unit -resolution full-range mass spectrum. (b) High resolution scan to resolve the isotopic peaks. The approximately 0.25 u distance between the peaks indicates the ion has a charge state of +4.

The benefit of performing multiple stages of mass spectrometry is demonstrated in Figure 9 where MS3 was performed on the triply-charged ion from human ACTH 18-39, which has the sequence RPVKVYPNGAEDESAEAFPLEF. The top panel shows the unit-resolution full-range mass spectrum, where the triply-charged ion is the most abundant species. MS /MS on that ion provided the mass spectrum shown in the middle panel. Ions below 270 u were ejected upon the application of the resonance excitation pulse. A third stage of mass spectrometry, depicted in the bottom panel, provides additional low mass sequence ions.

Figure 9: MS3 on the triply-charged ion from ACTH, a 2,467 Da peptide with 22 amino acids. A 2.5 pmol/ml sample solution was infused into an atmospheric pressure ionization source at a flowrate of 1 ml/min. The precursor displayed in the top panel was chosen for fragmentation. A fragment ion at m/z 505, shown in the middle panel, was chosen for a further stage of fragmentation, and the resulting mass spectrum is exhibited in the bottom panel.

A final example shown in Figure 10 demonstrates the application of the instrument to the analysis of components in a complex biological mixture. An enriched periplasmic extract from E. coli was fractionated using ion exchange chromatography (MonoQ). One of the fractions was concentrated and buffer -exchanged using Centricon filters then digested with trypsin. An aliquot was loaded onto a 1 mm C18-packed column and separated by reversed-phase high performance liquid chromatography. Shown in the top panel is the ion chromatogram, where the signal from the most intense peak in each scan is plotted. The middle panel depicts the full-range mass scan corresponding to scan number 1,014 in the chromatogram. A number of co-eluting species are observed. The ion at m/z 763 was chosen automatically for MS/MS, and the results are displayed in the bottom panel. Some selected fragment ions are labeled. The SEQUEST database searching algorithm (10) was used to identify the amino acid sequence of the peptide and determine its origin. The result of the computer analysis indicates that the peptide has the sequence ELNADDVVFSFDR and is derived from a periplasmic dipeptide transport protein.

Figure 10: On-line data-dependent analysis of tryptic peptides from an E. coli periplasmic extract. The ion chromatogram is depicted in the top panel. The full-range mass spectrum from scan number 1,014 is shown in the middle panel. The ion at m/z 763 was automatically chosen for further analysis. MS/MS was performed, and the resulting fragmentation mass spectrum is illustrated in the bottom panel. Computer-aided identification of the peptide indicated it was a fragment from a periplasmic dipeptide transport protein


The quadrupole ion trap is an extremely versatile, yet relatively low-priced mass spectrometer. The sensitivity and performance characteristics of the instrument, especially the automated experiments developed for the newly commercialized ion traps, make quadrupole ion trap mass spectrometry an attractive technique to apply to the analysis of biological and biochemical problems.


The authors would like to thank John Stults of Genentech, Inc. for graciously providing the tPA sample, N. Yates for the MS3 data, and A. Link for the data on the periplasmic protein.


1. Paul, W. (1990) Angew. Chem. Int. Ed. Engl. 29, 739.

2. Cooks, R.G., McLuckey, S.A. and Kaiser, R.E. (1991) Chemical and Engineering News 69 (12), 26-41.

3. Dehmelt, H. (1995) Physica Scripta Volume T T59, 87 -92.

4. Stafford, G.C., Jr., Kelley, P.E., Syka, J.E.P., Reynolds, W.E. and Todd, J.F.J. (1984) International Journal of Mass Spectrometry and Ion Processes 60, 85-98.

5. Louris, J.N., Cooks, R.G., Syka, J.E.P., Kelley., P.E., Stafford, G.C., Jr. and Todd, J.F.J. (1987) Analytical Chemistry 59, 1677-1685.

6. Kaiser, R E., Jr., Cooks, R.G., Stafford, G.C., Jr., Syka, J.E.P. and Hemberger, P.H. (1991) International Journal of Mass Spectrometry and Ion Processes 106, 79-115.

7. Billeci, T.M. and Stults, J.T. (1993) Analytical Chemistry 65, 1709-1716.

8. Schwartz, J.C., Syka, J.E.P. and Jardine, I. (1991) Journal of the American Society for Mass Spectrometry 2, 198 -204.

9. Jonscher, K.R., Currie, G., McCormack, A.L. and Yates, J.R., III. (1993) Rapid Communications in Mass Spectrometry 7, 20-26.

10. Eng, J.K., McCormack, A.L. and Yates, J.R., III. (1994) Journal of the American Society for Mass Spectrometry 5, 976-989.

The authors may be contacted at the University of Washington, Department of Molecular Biotechnology, Box 357730, Seattle, WA 98195-7730.


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Created: 21st September 1996
Last modified: 30th September 1996