ORTHOGONAL

Instrumentation

ORTHOGONAL

Orthogonal injection provides a highefficiency interface for transferring ions from a continuous beam to a pulsed mode and makes it easier to obtain high resolution.

Igor V. Chernushevich SCIEX

Werner Ens Kenneth G. Standing University of Manitoba 452 A

|^"j ver the past ten years, MS has J emerged as a major analytical tool in '^J biochemistry and biotechnology. This is primarily a result of the development of two new ionization methods, electrospray ionization (ESI) (1,2) and matrix-assisted laser desorption/ionization (MALDI) (3). Using these techniques, it is possible to form intact molecular ions with molecular weights up to hundreds of kilodaltons, which places increased demands on the corresponding methods of mass measurement. Because the ion beams produced by the two methods have very different characteristics, it has been customary to examine them in different types of mass analyzers MALDI ions are normally produced by a pulsed laser, so MALDI ion sources can be conveniently coupled to time-of-flight (TOF) mass spectrometers, which require a well-defined start time. TOF mass analyzers have several features that are particularly useful for the analysis of biomolecules. First, apart from detection problems, the m/z range is effectively unlimited. Second, no defining slits are needed, and ions can be detected over the whole m/z range at the same time without scanning, so ion transmission is high. Early TOF spectrometers suffered from poor resolution, but electrostatic reflectors (4) and the redis(5) of the benefits of delayed extraction (6) have made substantial ments to this property. Moreover developments in fast electronics have removed

Analytical Chemistry News & Features, July 1, 1999

earlier limitations in the recording of TOF spectra and have allowed the instrument's rapid response to be exploited more fully. Consequently, TOF instruments now provide, in many cases, an optimum combination of resolution, sensitivity, and fast response, particularly under conditions in which the entire mass spectrum is required. In contrast with MALDI, ESI produces a continuous beam of ions. Like other continuous ion sources, it is most compatible with mass spectrometers that operate in a similarly continuous fashion, such as quadruple mass filters. The combination of an ESI source and a quadrupole mass filter has become a popular configuration, but it has some defects. First, most commercial quadrupoles are restricted to m/z values below —4000. Although this is not a problem when examining electrosprayed ions from most species because of the high charge states typically produced, some entities, notably noncovalent complexes, may have m/z values of 10 000 or more. Second the quadrupole mass filter is a scanning device; it examines ion species in the spectrum one at a time which implies a reciprocal relationship between resolution and sensitivity Thus an increase in sensitivity requires a decrease in resolution a significant handicap when the whole mass specHp PYnminpH from a limited amount of sample

Coupling an ESI source to a TOF spectrometer might overcome these problems.

-INJECTION TOFAS FOR ANALYZING BIOMOLECULES

However, such a configuration has its own problems, because there are difficulties in coupling any continuous source to a TOF instrument. The most straightforward technique is to open a gate periodically to admit pulses of ions into the mass analyzer along the z axis, but this procedure is extremely inefficient (7). The need to produce wellseparated ion packets imposes a serious limitation on the duty cycle, which, at best, is the ratio of the gate duration to the time between adjacent gates. Fortunately, TOF instruments can tolerate a relatively large spatial or velocity spread of the ions in a plane perpendicular to

the spectrometer axis, which can be exploited by injecting electrospray ions into the TOF instrument perpendicular to the z axis, that is, "orthogonal injection" (Figure 1). Such a geometry provides a high-efficiency interface for transferring ions from a continuous beam to a pulsed mode. Another advantage is the small velocity spread in the z direction that is usually observed, making high resolution easier to obtain. A short history In 1964,0'Halloran et al. reported the first use of orthogonal injection into a TOF instrument. In their case, the injection came from

an atmospheric-pressure plasma ion source (8). Unfortunately, the work went largely unnoticed by most, so the technique was independently reinvented by many groups 20-25 years later using various types of continuous ion sources (9-16). The earliest of these is the most relevant to the present discussion: Dodonov et al. were the first to couple an ESI source to an orthogonal injection TOF instrument which made it particularly useful for observing biomolecules. They were also the first to use a reflecting geometry with orthogonal injection (9 17-19) which improved resolution Their ion source stemmed from Russian

Analytical Chemistry News & Features, July 1, 1999 453 A

Instrumentation ESI work (2), carried out independently at about the same time as the work of Fenn (1) in the United States. The Russsans were able to obtain a spectrum of multiply charged ions of insulin (see Ref. 7). Their results were first reported in 1987 (9), but were not widely known until sometime later (17,18). Recently, numerous other ESI-TOF in-

struments have been constructed (see Ref. 7). Important developments have included collisional cooling, tandem instruments, and the somewhat surprising application of similar techniques to MALDI. In addition to electrospray, orthogonal injection has been used with other types of continuous ionization, including ions from flames (10), electron ionization (11,14), liquid SIMS

(12,15), a cluster ion source (13), atmospheric-pressure ionization (16,17), and inductively coupled plasma (20). TOF configuration

ATOF mass spectrometer with orthogonal ion injection consists of a continuous ion source (ESI, electron ionization, atmospheric-pressure ionization, etc.), an ion modulator, a drift region, a detector, and fast electronics for recording data. Figure 1 (21-23) shows an instrument wiih an ESI source and a single-stage electrostatic ion mirror, which defines the spectrometer's z axis. The ion source continuously injects a slow, nearly monoenergetic ion beam (tens of electronvolts DPI" charge) along they axis into a storage region in the modulator between a flat plate and a grid Initially that region is field-free so ions continue to move in their original direction in the gap When a voltage pulse is applied to the plate, an ion packet is pushed out of the region in a direction nearly parallel to the z axis and accelerated to an energy of several kiloelectronvolts per charge by a uniform dc electric field in the second stage of the modulator. Normally, the voltage pulses are applied at a frequency of a few kilohertz, thus injecting a series of ion packets at that frequency into the field-free drift region of the TOF instrument and providing a maximum flight time of a few hundred microseconds between pulses. The electrostatic mirror and the ion detector are similar to those used with pulsed ionization sources apart from differences associated with a larger beam size (usually a few ppntimetpni in the v Hirprtion Hptpr-

mined by the modulator aperture) The dc acceleration potential, which is normally several kilovolts, must be connected to the electrospray source or the drift tube. Although placing the drift tube at high voltage requires an extra shield within the TOF chamber, it puts the source close to ground potential, which eliminates any possibility of discharges in the most dangerous region of pressure (0.01-1 Torr), and simplifies coupling to other instruments. Figure 1 . Diagram of an ESI-TOF instrument.

Collisional cooling

The conducting liner was subsequently added so that the ion source could be held at ground potential. For clarity, the coordinate system has been translated along the z axis from its true position in line with the electrosprayed beam. The inset shows a portion of the deconvoluted mass spectrum of bovine insulin obtained with this instrument. (Adapted with permission from Refs. 21 and 23.)

Until recently, ions have been introduced into the TOF spectrometer more or less directly from the ion source through an

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Analytical Chemistry News & Features, July 1, 1999

interface that merely provided collimation to define the beam shape and by differential pumping to allow ions to be transferred from the source at atmospheric pressure to high vacuum in the spectrometer. As a result, instrument performance has been defined primarily by the characteristics of the ion beam produced by the source. Because the ions usually diverge from the source, significant losses due to collimation are necessary to produce the narrow beam cross section required for high resolution, or else resolution mnv have to be sacrificed for sensitivity. Another limitation of direct introduction arises from the velocity distribution of the ions in the direction of injection which may lead to significant m/z discrimination It is possible to overcome these limitations by collisional damping of the ions in an rf-quadrupole ion guide at relatively high pressure as introduced first for quadrupole spectrometers (24,25) and more recently forTOF instruments (22,23). The rf field in the quadrupole creates a twodimensional potential well in which the ions oscillate, while collisions with the molecules of the ambient gas reduce their velocities to near-thermal values (23). This produces a beam with a small spatial spread in the z direction and small velocity spreads in both the y and z directions and these properties are almost independent of the original parampffTS of the beam delivered by the source Improved performance is obtained; indeed the collisional cooling inn oiiide

in the instrument in Fieriire 1

reduced m/z discrimination in that

'instrument significantlv and it also improved both sensitivity and resolution Resolution In general, resolution is limited by the initial spatial and velocity spreads of the ions in the z dimension. For a pure spatial spread, ions originating from different z positions in the storage region are focused onto a plane parallel to the xy plane somewhere beyond the acceleration region by the fields in the modulator; the ions exchange their original spatial spread for an energy distribution on this plane. In a linear TOF instrument, the focal plane can be arranged to coincide with the plane of the detector by adjusting the relative ampli-

tudes of the injection pulse and the dc acceleration field in the modulator (6). Better resolution is obtained in a reflecting spectrometer, as in Figure 1, by placing the focal plane close to the modulator (21); it then serves as the object plane for the mirror. The ions often have a considerable energy spread in this plane (up to 10% of their energy, because of the range of energies gained during acceleration), but the spread can be corrected to first order by a single-stage mirror. By making a nonuniform field in the modulator, or by using a multistage mirror, it is possible to adjust voltages so as to obtain second-order (or

For smaller ions, resolution decreases but remains sufficiently high to resolve two or more peaks with the same nominal mass. higher) time focusing (4). However, resolution will then be limited in most practical situations by other factors, such as turnaround time (6) or the temporal characteristics of the detector. Detailed calculations of resolution in an ESI-TOF mass spectrometer have been reported (18). The instrument in Figure 1 has a resolving power (Af/AM^jJ of 8000-10,000 for peptides with masses of 1000-6000 Da (inset). (The resolution was at least 20% lower before collisional cooling was introduced.) This resolution is sufficient to distinguish separate isotopic peaks for most peptides, and, hence, to determine their individual charge states. Such a capability is particularly useful when interpreting complicated spectra resulting from mixtures such as tryptic digests or the fragmentation of multiply charged ions (26). For smaller ions, the resolution decreases, but it remains sufficiently high to resolve two or more peaks having the same nominal mass (7). This is an advantage of

TOF mass spectrometers over quadrupoles, in which the resolution usually decreases linearly with decreasing mass in the low mass range. For heavy ions (m >10,000 Da) the peak shape is determined mostly by the isotopic envelope. m/z discrimination In the geometry of the instrument in Figure 1, the ion beam has to be directed at the proper angle (a few degrees from the z axis) after leaving the acceleration region so that the reflected ions will strike the detector. If the initial angle is wrong, it can be corrected by subsequent electrostatic deflection. However, deflection by a simple pair of deflection plates introduces a time spread because it causes the plane of the ion packet to tilt by approximately the deflection angle (18 27). The deflection should therefore be kept to a minimum for optimum resolution. The need for deflection after acceleration is avoided altogether if the ions leave the modulator with the correct ratio of velocity components in the y and z directions. This ratio may depend on m/z. Because all m/z ions ideally acquire the same energy during acceleration in the modulator, they have a z component of velocity (v ) that is inversely proportional to (m/z)v\ However, their ;y component of velocity (v ) is the velocity with which they entered the storage region, and this will often have a different m/z dependence. In particular, ions entering from a supersonic jet as in electrosprav all have the same jet velocity to a first approximation As a result ions injected directly from the jet will reach the detector plane with a y position depending on m/z and many will miss the detector Here collisional cooling makes an important contribution. After the ions are cooled to near-thermal velocities, they can be reaccelerated to the energy required to strike the detector, defining the velocity component IL, which is again inversely proportional to (m/z)'/2. Thus, ,ons leave the modulator at the correct angle and are independent of m/z, and mass discrimination is avoided. In the instrument in Figure 1, it was possible to observe ions over a mass range from ~ 1300 Da to more than 1 MDa at the same time (m/z range from -600 to >12 000) .23) after collisional cooling was introduced

Analytical Chemistry News & Features, July 1, 1999 4 5 5 A

Instrumentation An alternative solution to the deflection problem is to use a deflector composed of several bipolar deflection plates (N) (28))

in which trie effect of deflection on resolution is reduced by a factor of TV. In that case, the mass spectrometer can be constructed with a coaxial geometry (29).

Duty cycle To avoid overlap of consecutive spectra, an injection pulse cannot be applied until the slowest ion from the previous pulse has reached the detector. For instruments without deflection the v of ion in the fllght tube is the same as its velocity in the storage region Thus the slowest ions enterino* the storage resion be s ad distance time the next injectinn mike arrives if

the storage region is 1

U

U

long enough, where D is the separation in the v direction between the modulator and detector centers. Only a finite slice of this beam, of length A/e can be accelerated and detected (A/ is determined by tures ir the dotector and.or emo u a-

Figure 2. ESI-TOF spectra of citrate synthase and trp repressor. (a) Spectrum of recombinant E. coli citrate synthase obtained under denaturing conditions (pH ~2.5). (b) Spectrum of citrate synthase obtained from 5 mM ammonium acetate buffer (pH —6). In (a) and (b), portions of the deconvoluted spectrum are shown as insets, (c) Spectrum of the trp repressor (20 uM) in the presence of a mixture of 10 pM each DNA1 (consensus spacing 4 bp), DNA2 (2 bp spacing), and DNA3 (6 bp spacing). Only the specific operator, DNA1, forms a complex. 456 A

Analytical

Chemistry

News & Features, July 1, 1999

rives. Thus, the duty cycle is proportional to (m/z)1/2, and there is systematic discrimination against \ovf-m/z ions. Nevertheless, this discrimination is predictable, and signals at low m/z are normally stronger than at high m/z, so, in most cases, this discrimination is preferable to the cut-off at high-m/z values found in quadrupole mass filters. In most ESI-TOF instruments, the duty cycle is 5-50%, depending on the m/z of the ion and the instrumental parameters. For example, the spectrometer in Figure 1 has a maximum duty cycle of -20%. In another instrument, an attempt has been made to improve the duty cycle by trapping ions in a two-dimensional ion trap, and then gating them in short bursts into tiie TOF spectrometer (30). With this method, a 100% ddty cycle was obtained over a range of several hundred m/z, but the efficiency outside this ran P'P w*is significantly reduced. Ion detection

The TOF detector normally consists of two large-area microchannel plates in a chevron configuration. Signals may be recorded by either digital or analog techniques, each of which has its own advantages. In the digital technique used in the instrument in Figure 1, the detector signal passes to a preamplifier and a constant-fraction discriminator and is then registered in a pulse-counting mode by a time-to-digital converter (TDC). The TDC can record hundreds of stops for each extraction pulse, but it can read only one stop per extraction pulse for a given m/z species that is within the "dead time" of the TDC which is tvnically a few nanoseconds For high counting rates this may cause distortion but in the experiments described here the average counting rate has hfpn in most cases much less than injec-

JJCL (111111 \Jll L11C U U L y

tion nntap nf*r inn QnpHpQ Qn distortinn of

value as Al/D. If the incoming beam is monoenergetic, vy is inversely proportional to (m/z)1/2, so lighter ions would be spread out over a distance larger than D when the next pulse ar-

this kind is neriigible Mass spectra can also be recorded using analog detection with an integrating transient recorder. This method is more tolerant of high counting rates, but it is more limited in resolution and/or repetition rate. At present, it is therefore necessary to choose between a lower counting rate or lower time resolution. Note that existing transient recorders are well-suited for MALDI (in the axial geometry) because

that technique typically operates at a much lower repetition rate (a few hertz), whereas a high repetition rate in the range of sev-

eral kilohertz is necessary to obtain a reasonable duty cycle in the TOF instruments previously described. ESI-TOF of noncovalent complexes

As stated earlier, a large m/z range is needed to study noncovalent biomolecular complexes (31, 32), which play key roles in biological phenomena involving molecular recognition. The structure of such complexes often makes many of the protonation sites inaccessible, so the complex may have a much higher m/z value than the constituent monomers.

For example, Figures 2a and 2b show TOF mass spectra of E. coli ciirate synthase (33), the enzyme that initiates the citric acid cycle. Under denaturing conditions (Figure 2a), a wide distribution of charge states centered around m/z —1100 is observed, corresponding to the monomer. The corresponding mass distribution, which was obtained by "deconvoluting" the m/z distribution (34), is shown as an inset. When the pH is raised closer to physiological values narrow charge distributions responding to the noncovalent complexes of dimer (m/z —5300) and hexamer (m/z —9000) (Figure 2b); again the responding deconvoluted mass distributions are shown as insets Resolution was good enough to resolve individual additions of the (

665 T\~\ anrl tVinc in f^vnlam fViA

mechanism of inhibition and determine the eqiiilibrium constants (??> assuming that the electrospray measurements are correlated to solution conditions (31). A second example is the noncovalent complex formed between the trp repressor protein and its specific DNA operator (35). Figure 2c shows the spectrum obtained from a mixture of the trp repressor and three DNA species, which differ in the spacing between the two binding sites. Only the DNA with the correct spacing formed a complex, a strong indication that the binding is specific. Although the ions of interest in this case had m/z —4000 or less, the large m/z range of the TOF instrument was still useful in ruling out the presence of other complexes at higher m/z. Nearly all complexes studied until now have had m/z values of 10,000 or less, but some clusters have been observed above that value, and one example of a peak at m/z —27,000 has been tentatively identified as an intact virus of 4.65 MDa (32) Tandem MS

Figure 3. (a) Schematic of the tandem QqTOF mass spectrometer prototype, and (b) a portion of the MS/MS spectrum of a protein. The measured mass differences between the fragments correspond to the masses of the amino acid residues indicated to within 0.01 Da, except for the very weak line at m/z -733.4, where the differences are 0.03 Da. For example, the mass difference between the two labeled peaks is 115.03 Da, corresponding to the mass of an aspartic acid residue D. (F is phenylalanine, G is glycine, E is glutamic acid, and N is asparagine.)

Tandem MS, or MS/MS, has become a valuable means for determining molecular structure (36). In such measurements, a "parent ion" is selected in one mass spectrometer and then broken up, usually by collisions with the ambient gas in a collision cell. The resulting "daughter ions" are examined in a second mass analyzer. The

Analytical Chemistry News & Features, July 1, 1999 457 A

Instrumentation most popular instrument of this type is the triple quadrupole QqQ, in which both mass analyzers, designated Q, are quadrupole massfilters,and the collision cell lies within an rf quadrupole, designated q. A quadrupole mass filter must be scanned to obtain a complete mass spectrum, but this is unnecessary in the first quadrupole, because it merely selects a given parent ion. Indeed, a quadrupole is well-suited to this role, because it couples efficiently to the collision cell. However, the entire mass spectrum of the daughter ions is usually of interest and must be obtained by scanning the final quadrupole, which considerably reduces either sensitivity or resolution. This suggests that it would be worthwhile to replace the third quadrupole with aTOF spectrometer in

which case the entire daughter ion spectrum can be measured in parallel. The first instrument of this type (with Einzel lenses instead of a q) was reported by Glish et al. (37, 38)) but the ions were injected into the TOF spectrometer along the axis, so its performance was limited. The resulting duty cycle was —0.5%, and the mass resolution was below 100 fwhm. Again, orthogonal injection provides a remedy for the problem. Recently, two such tandem spectrometers with orthogonal injection have been reported. One has the collision cell in anrfhexapole (QhTOF) (39) and the other has the cell in an rfquadrupole (QqTOF) (26) The latter instrument is shown in Figure 3a and was constructed by combining the front section of a triple quadrupole with

spectrometer in Figure 1 (26). Commercial versions of both instruments are now available. The tandem QqTOF mass spectrometer in Figure 3a consists of three quadrupoles—q0, Qj, and q2—followed by the TOF spectrometer. The additional quadrupole, q0, is used for the collisional cooling of the ions entering the instrument, so it is always operated in the rf-only mode, as is the collision cell q2. For single-MS TOF measurements, the mass filter Q1 is operated in the rf-only mode, so it serves merely as a transmission element. Under this condition (q0, Q1( and q2 in the rf-only mode) ions covering approximately tor of 10 in TH/z are transmitted simultaneously into the TOF instrument If a wider m/z range is required the rf voltage is modulated (stepped between two or more rf-levpIO during spectnim accumulation providing a larger averaged over time Single-MS spectra can be acquired either with or without collision gas in o In the former rase lV i energv is kept belo 10 V r' avoid fragmentation. For tandem MS, Qx is operated in the mass filter mode to select the parent ion. The ion is then accelerated to an energy of 40-180 eV before it enters the collision cell. The pressure in the collision cell is maintained at about 10 mTorr to provide collisional damping of the fragment ions and the remaining parent ions. As a result, ,he ions are thermalized, and both spatial and energy spreads are reduced, providing better transmission into and through the TOF spectrometer. After leaving the collision cell, ions are reaccelerated to 10 eV per charge and focused by ion optics into a parallel beam which enters the ion storage modulator The selected ratio of velocities in the two orthogonal directions allows ions to reach the TOF fWopfrv*- without oVflectirm

Figure 4. Data-dependent LC/MS/MS from a tryptic digest of bovine serum albumin acquired on a QhTOF. The sample was injected onto a trap cartridge, washed to remove salts, and then eluted through a C 18 column using an acetonitrile:water:formic acid gradient, (a) Total ion chromatogram. Spikes below baseline indicate automatic acquisition of MS/MS data, (b) MS/MS spectrum acquired for 5 s from an abundant peptide precursor; 31 such spectra were acquired. (Inset) An indication of the resolution afforded by this instrument is shown using the singly charged m/z 1017.6 ion as an example. (Adapted with permission from Ref. 47.) 458 A

Analytical Chemistry News & Features, July 1, 1999

All mass spectra from the TOF spectrometer (in both the MS and MS/MS modes) are recorded with a IDC. A four-anode detector and four parallel channels of registration are used to improve the dynamic range of die ion-counting technique. The instrument has sensitivity in die subfemtomole range; resolution up to 16,000 fwhm has been measured by Chernushevich in a later version of the instrument.

An alternative configuration for a hybrid instrument has a sector mass analyzer injecting ions into a TOF spectrometer (40). In this case, the parent ion selection is carried out in the sector instrument, and, once again, the injection into the TOF spectrometer is orthogonal. Proposals have also been made to select parent ions within the collision cell. In one, the rf quadrupole is operated close to the Mathieu stability region (low-mass cutoff at q —0.907) (41), thereby achieving resonance excitation and consequent preferential breakup at a selected m/z value (42). In another approach, the desired ion species is excited by the application of a signal at its fundamental frequency (43, 44)) Biological applications

The quadrupole-TOF hybrid spectrometers are particularly useful in proteinsequencing experiments (26, 39, 45, ,6). Their high resolution and mass accuracy have made possible significant improvements in the interpretation of spectra, even for very low sample amounts. Moreover, amino acid residues with the same nominal mass (Q and K with Am = 0.036 6a, F and M with Am = 0.033 Da) )an bb eouttnely distinguished (46). Chernushevich used MS/MS to analyze a small intact protein (MW 4587 Da)) omitting the usual intermediate stage of tryptic digestion. A series of 5+ fragment ions was produced from the soft fragmentation of the 6+ ion of the protein (Figure 3b). Ions with 4 and 6 charges were also present, but a well-resolved isotopic pattern made it possible to distinguish between the charge states. The 5+ series turned out to be easily readable in terms of amino acid residues from m/z = 865.04 down to m/z ~ 740. The absence of abundant peaks in the region just below m/z 740 suggested either glycine or proline as the next residue. Close examination of the area (inset) confirmed that there iis ,ndeed, a peak corresponding to the loss of glycine. The next residue (phenylalanine) was then easy to identify. The resulting seven-residue-long sequence was large enough to search the protein data bases. Together with the highaccuracy mass measurement of the protein (4587.33 ± 0.03 Da), it led to a anique match human fibrinogen B chain.

Figure 5. MALDI ion source constructed for use with the orthogonalinjection TOF instrument shown in Figure 1. The ion guide is somewhat longer than the one used for electrosprayed ions and is segmented for flexibility. A similar source has also been adapted to the QqTOF instrument shown in Figure 3.

Because the TOF analyzer is capable of recording several full spectra per second, quadrupole-TOF spectrometers can also perform on-line LC/MS without sacrificing either sensitivity or resolution. Moreover, MS/MS spectra can be acquired during an LC/MS experiment in an automated, datadependent manner (47). The advantages of this approach have been shown previously with scanning mass spectrometers; however, performance compromises had to be made to balance the data acquisition rate, scan range and sensitivity Figure 4 shows an example in which a QhTOF instrument achieved high resolution (>6000), high mass accuracy (