Time-of-Flight Mass Spectrometry for the Structural Analysis of Biological Molecules Robert J. Cotter Middle Atlantic Mass Spectrometry Laboratory Department of Pharmacology and Molecular Sciences The Johns Hopkins University School of Medicine Baltimore, MD 21205
Time-of-flight (TOF) mass spectrometers are relatively simple, inexpensive instruments with high sensitivity a n d virtually unlimited m a s s
TOFMS is not a new technique. In 1955 Wiley and McLaren (1) published a design for a TOF mass spectrometer t h a t was commercialized by Bendix Corporation and is still available from CVC Products (Rochester, NY). TOF mass spectrometers were used with pulsed lasers in the 1960s for rapid vaporization and analysis of coals, elemental analysis, isotope r a tio measurements, and pyrolysis (2). The laser microprobe was developed in 1975 (3) and commercialized by Leybold-Hereaus (Kôln, Germany)
INSTRUMENTATION range. There has been great interest in exploiting this mass analyzer for the structural analysis of biological macromolecules such a s proteins, carbohydrates, and oligonucleotides. 0003-2700/92/0364-1027A/$03.00/0 © 1992 American Chemical Society
and Cambridge Instruments (Cambridge, U.K.). Resonance ionization mass spectrometers have been used in a number of laboratories for isotopic analy-
sis, a n d TOF m a s s spectrometers have been used to study multiphoton ionization (MPI) processes of organic molecules. For the most part, these instruments have been used to record ions of relatively low m a s s . T h e plasma desorption (PD) mass spectrometer, introduced in 1974 by Macfarlane and co-workers (4), was perhaps t h e first instrument intended specifically for the analysis of biological macromolecules. Commercialized by Bio-Ion Nordic AB (Uppsala, Sweden) in 1984, it is used in more than 50 laboratories worldwide. The early successes of plasma desorption mass spectrometry (PDMS) encouraged the development of other desorption/ionization techniques, inc l u d i n g fast a t o m b o m b a r d m e n t (FAB), laser desorption (LD), a n d electrospray ionization (ESI). Collectively, these techniques provide powerful tools for the structural analysis of peptides a n d proteins, carbohyd r a t e s , glycolipids, phospholipids, and oligonucleotides by MS. Because FAB and ESI produce ions continuously, they were easily retrofitted to m a g n e t i c sector a n d q u a d r u p o l e mass spectrometers, instruments that are more familiar to mass spectroscopists than the TOF mass spectrometer. With t h e concurrent development of four-sector and triple quadrupole instruments, these ionization techniques have been commercially successful. PD and LD have been used almost exclusively with TOF mass analyzers, which can record all t h e ions from each ionization event and thus offer t h e possibility for higher s e n s i t i v i t y t h a n scanning i n s t r u m e n t s . Although TOF m a s s spect r o m e t e r s with o u t s t a n d i n g m a s s resolution have been described, t h e instruments that have used this analyzer in conjunction with PD and LD have, for t h e most part, been lowresolution i n s t r u m e n t s . This h a s hindered their widespread use a n d c o m m e r c i a l i z a t i o n for s t r u c t u r a l analysis. The most recent interest in TOF mass spectrometers can be a t t r i b uted largely to t h e introduction of matrix-assisted laser desorption (MALD) in 1988 by Karas and Hillenkamp (5) and by Tanaka and coworkers (6). Indeed, during the past four years TOF instruments using t h a t technique have been commercialized by Shimadzu (Kyoto, Japan), Vestec (Houston, TX), Finnigan Corporation (Hemmel-Hempsted, U.K.), B r u k e r - F r a n z e n (Bremen, Germany), Linear Instruments (Reno, NV), VG Biotech (Manchester, U.K.), and Kratos Analytical (Manchester, U.K.). MALD is a n extraordinary tech-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992 · 1027 A
INSTRUMENTATION nique that has recorded molecular weights of proteins that exceed 300 kDa. Initial success with oligonucle otide mixtures has encouraged spec ulation about its future role in map ping t h e h u m a n g e n o m e . P D M S continues to play an important role in the structural analysis of proteins and peptides, and a second commer cial instrument has been introduced by PA Electron (Selmi, Ukraine). TOF mass analyzers have been in terfaced to FAB (7) and ESI (8) sources for on-line LC/MS, and an atmospheric pressure ionization (API) TOF instrument has been com mercialized by Sensar (Provo, UT). Considerable advances have been made in the bandwidth, sampling r a t e , and memory lengths of t h e high-speed electronic recorders used to capture TOF mass spectra. We can anticipate the development of TOF instruments with higher mass reso lution and accuracy, and these will be used with a variety of ionization, chromatographic, and electrophoretic techniques. In this INSTRUMENTATION article, we will describe the basic operation of TOF mass spectrometers and dis cuss the factors affecting mass reso lution and accuracy. In this respect, it is primarily the initial conditions of the ionization method and the fate of metastable ions (and not just im
(a)
Source extraction
0-
provements in ion optics) that must be addressed to improve the perfor mance of TOF instruments for the analysis of large molecules. We will also describe strategies for the anal ysis of biological molecules and ap plications aimed at elucidating pro tein structure and post-translational processing.
The TOF mass spectrometer In the TOF mass spectrometer (Fig ure la), ions are formed in a short source region (s) in the presence of an electrical field (E) t h a t accelerates the ions into a longer, field-free drift region (D). Ideally, all ions enter the drift region with the same kinetic energy (KE) KE = zeEs (1) (where e is the charge on an electron and ζ is the number of charges), but they will have velocities (v)
..(*&)"
(2)
that depend on their mass (m). The time (t) required to traverse the drift region , . (jS-)"D \2zeEs)
(3)
also depends on the mass of the ion,
Drift region
Detector
m
©— Qh (+>-* Θ— Θ-* &*
1
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En=0
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2,500,000 2,000,000
c 1,500,000 1,000,000 MH+
500,000 0
pAlAVijlh f
200
r
400 600 mlz
1000
1400
Figure 1. MS with a linear TOF instrument. (a) Basic components of the linear TOF mass spectrometer, (b) PD mass spectrum of the endoproteinase Glu-C peptide RDASSDEEE from the assembly protein obtained from simian cytomegalovirus, showing the H* and Na+ peaks used to calibrate the mass scale. 1028 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
so that the time spectrum can be con verted directly to a mass spectrum
7— (if
(4)
as shown in Figure lb. To obtain timing information, the time of ion formation (or extraction) m u s t be known. Thus TOF m a s s spectrometers generally use pulsed ionization or, in the case of PD, r a n dom events t h a t can be detected. Certain instruments employ ioniza tion in the gas phase and desorption from surfaces. TOF mass spectrome ters differ in their method of data re cording. Those with low secondary ion yields generally m e a s u r e t h e time interval between ionization and ion detection, whereas larger second ary ion (analog) signals are captured on fast analog-to-digital converters (ADCs). Linear TOF mass spectrom eters are the simplest instruments, because the ions move in a straight line through the flight tube to the de tector. Other instruments use addi tional electric fields that change the direction of the ions to correct for their initial kinetic energy distribu tions and to improve mass resolu tion. P D and time interval m e a s u r e ments. In the PD technique (Figure 2a), ions are formed by one of two high-energy (50-100 MeV) fission fragments emitted in opposite direc tions from a radioactive 252 Cf source. A start detector placed behind t h e source detects one of the fission frag ments. The second fission fragment strikes the sample, which is coated onto an aluminized Mylar foil, r e leasing from 1 to 10 sample ions. The sample ions are registered by a stop detector placed at the end of the drift region. A multistop time-to-digital converter (TDC) is then used to de termine the time interval between each stop pulse and the start pulse. This information is downloaded to a computer, and the cycle is repeated. The results from many TOF cycles are accumulated into a time histo gram, which is then converted into a mass spectrum (Figure lb). PD mass spectra generally are ac cumulated for 1 0 6 - 1 0 7 ionization events (or more, if needed). For a 10m i c r o c u r i e source, m e a s u r e m e n t times may be a few minutes but can be as long as several hours if ion in tensities are especially low. TDCs have excellent time resolution (1 ns to 78 ps), and multistop versions can record all the ions in each cycle. However, coincident ions with ex actly the same TOF are recorded as a single stop event, which limits the
conversion. Currently, however, lowcost 1 Gsample/s digital oscilloscopes with bandwidths on the order of 500 MHz are available from LeCroy (Spring Valley, NY) and Tektronix (Beaverton, OR). Transient recorders that can accommodate 200 Msample/s are available from Precision In struments (Knoxville, TN) as expan sion boards for 386/486-based PCs, and ILYS Software (Pittsburgh, PA) offers a TOF acquisition and process ing software package that is compat ible with most commercial transient recorders and digital oscilloscopes. Transient recorders may be trig gered from the same electronic pulse used to initiate ionization. However, for instruments employing laser ion ization, the trigger pulse is generally obtained by a high-speed optical de tector (photodiode, Figure 2b) inter cepting a portion of the laser beam. Alternatively, timing may be based on pulsed ion extraction rather than on ionization. This permits a time delay between ion formation and ex traction to study metastable frag mentation or to allow the use of broad or even continuous ionization sources such as ESI. Mass calibration. If the acceler ating voltage (V = Es) and drift length are known, Equation 4 can be used to determine the mass-to-charge ratio (m/z) directly. However, masses gen
dynamic range. In addition, there is a significant dead time following each stop pulse. If two ions of the same mass arrive at slightly differ ent times because of differences in kinetic energy, only the first will be recorded. Pulsed ionization and timed amplitude measurements. Most other techniques involve ionization events that are initiated at regular intervals by an electronic pulse that gates or deflects an ion beam onto the sample, or triggers a pulsed laser (Figure 2b) (9). Pulsed ionization techniques generally produce a large number of ions in each cycle, and the entire time/amplitude record is digi tized by a high-speed ADC or tran sient recorder. A single transient represents a time (or mass) spec trum. Thus, for example, it is possi ble to produce a mass spectrum from a single laser pulse. Generally, the results of several transients are added together by using an integrat ing transient recorder (7, 10). For a number of years, transient (or waveform) recorders have been limited to sampling rates of 100 Msamples/s, corresponding to time resolutions considerably lower than those of TDCs (10 ns). In addition, limited bandwidths (50 MHz) and high cost have made such approaches less attractive than time-to-digital
Detector
Detector
(a)
h
Drift region _JL·
20 kV Computer
CFD
Ν
(b)
Stop
Photodiode Nitrogen laser Detector Drift region 30 kV
Computer
Κ
- = at2
+
b
(5)
ζ
where the constants a and b are de termined by measuring the flight times of two known masses (such as H + , Na + , and K+) that appear in desorption mass spectra (Figure lb). Low-mass atomic ion peaks gener ally are well resolved. If their peak widths are on the order of only two or three sampling intervals, their time centroids will be inaccurate and will not produce reliable slopes for ex trapolation into the kilodalton (and higher) mass range. Matrix ions are often used as internal calibration standards in MALD spectra, which certainly improves mass accuracy. External mass calibration is rarely used, although this approach is com mon for sector and quadrupole in struments. Even when the mass scale is accu rately established, mass assignment accuracy is compromised if molecular species (e.g., MH+ and MNa+) and adduct ions cannot be resolved or if peaks are distorted by metastable fragmentation in the accelerating re gion. In the MALD technique, pep tide and protein molecular ions are desorbed with initial velocities com parable to that of the matrix species in which they are entrained (11-13). The initial kinetic energies of these heavier species will be considerably larger than those of the matrix ions; therefore, their flight times will be shorter and their masses will be underestimated. Time and mass resolution
CFD TDC
Start
erally are obtained from the empiri cal equation
Digital oscilloscope
In addition to mass, the time axis in the TOF mass spectrometer reflects the initial conditions of the ions (temporal, spatial, and kinetic en ergy distributions), their fate during acceleration (metastable fragmenta tion), and properties of the recording system (jitter, sampling rate, band width, and detector response). Temporal distribution. Figure 3a illustrates the case in which two ions have the same mass and initial kinetic energy but are formed at dif ferent times. Because they have the same velocities, the time interval re mains constant as they exit the drift tube and are recorded by the detec tor. From Equation 4, mass resolu tion (Am/m) for the TOF mass spec trometer is Am 2At
Figure 2. Block diagrams of TOF mass spectrometers. (a) Bio-Ion PD mass spectrometer. CFD represents the constant fraction discriminator, (b) MALD mass spectrometer developed in our laboratory. (Part b adapted with permission from Reference 9.)
Thus, if peak widths (At) remain con-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992 · 1029 A
INSTRUMENTATION stant, mass resolution can be im proved by extending the flight time (t). This can be accomplished by re ducing the accelerating voltage or in creasing the drift tube length. Be cause h i g h a c c e l e r a t i n g voltages improve ion transmission and energy focusing (see below), longer flight lengths ( 1 - 4 m) have been common. Equally important, however, is the reduction of uncertainties in the time of ion formation (i.e., shorter At). The Wiley-McLaren instrument (2) used a broad ( 1 - 5 μβ) electron beam pulse for ionization, and temporal focusing was achieved by pulsed extraction. The fission fragments from a 252 Cf source produce ions in < 1 0 - 9 s, and nitrogen lasers are now available with 3 0 0 - 6 0 0 - p s pulse widths. In addition, jitter (triggering), band width, detector response, and sam pling rate contribute to the temporal distribution. Spatial distribution. Figure 3b shows two ions of the same mass that have the same initial kinetic energy but are formed in different locations along the direction of the electric field. The ion formed toward the rear of the ionization source falls through a larger electrical potential and is ac celerated to a higher kinetic energy than the ion formed near the front of
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