Time-of-flight mass spectrometry for the structural analysis of

w

1

mm

II

Il;me-of=FlightMass Spectrometry for the Structural Analysis of Biological Molecules

Time-of-flight (TOF) mass spectrometers are relatively simple, inexpensive instruments with high sensitivity and virtually unlimited mass

TOFMS is not a new technique. In 1955 Wiley and McLaren (1) published a design for a TOF mass spectrometer that 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 ratio measurements, and pyrolysis (2). The laser microprobe was developed in 1975 (3)and commercialized by Leybold-Hereaus (Koln, Germany)

range. There has been great interest in exploiting this mass analyzer for the structural analysis of biological macromolecules such as proteins, carbohydrates, and oligonucleotides.

and Cambridge Instruments (Cambridge, U.K.). Resonance ionization mass spectrometers have been used in a number of laboratories for isotopic analy-

Robert J. Cotter Middle Atlantic Mass Spectrometry Laboratory Department of Pharmacology and Molecular Sciences The Johns Hopkins University School of Medicine Baltimore, MD 21205

0003-2700/92/0364-1027A/$03.00/0 0 1992 American Chemical Society

sis, and TOF mass 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 mass. The plasma desorption (PD) mass spectrometer, introduced in 1974by Macfarlane and co-workers (41, was perhaps the 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, including fast atom bombardment (FAB), laser desorption (LD), and electrospray ionization (ESI). Collectively, these techniques provide pow erful tools for the structural analysis of peptides and proteins, carbohydrates, glycolipids, phospholipids, and oligonucleotides by MS. Because FAB and ESI produce ions continuously, they were easily retrofitted to magnetic sector and quadrupole mass spectrometers, instruments that are more familiar to mass spectroscopists than the TOF mass spectrometer. With the 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 the ions from each ionization event and thus offer the possibility for higher sensitivity than scanning instruments. Although TOF mass spectrometers with outstanding mass resolution have been described, the instruments that have used this analyzer in conjunction with PD and LD have, for the most part, been lowresolution instruments. This h a s hindered their widespread use and commercialization for structural analysis. The most recent interest in TOF mass spectrometers can be attributed largely to the 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 that technique have been commercialized by Shimadzu (Kyoto, Japan), Vestec (Houston, TX),Finnigan Corporation (Hemmel-Hempsted, U.K.), Bruker - Franzen (Bremen, Germa ny), Linear Instruments (Reno, NV), VG Biotech (Manchester, U.K.), and Kratos Analytical (Manchester, U.K.). MALD is an extraordinary tech-

ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1,1992 * 1027 A

INSTRUMEN7A7VON nique that has recorded molecular weights of proteins that exceed 300 kDa. Initial success with oligonucleotide mixtures has encouraged spec ulation about its h t u r e role in mapping t h e human genome. PDMS continues to play an important role in the structural analysis of proteins and peptides, and a second commercial instrument has been introduced by PA Electron (Selmi, Ukraine). TOF mass analyzers have been interfaced to FAB (7) and ESI (8) sources for on-line LC/MS, and an atmospheric pressure ionization (API) TOF instrument has been commercialized by Sensar (Provo, UT). Considerable advances have been made in the bandwidth, sampling rate, and memory lengths of the high-speed electronic recorders used to capture TOF mass spectra. We can anticipate the development of TOF instruments with higher mass resolution 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 discuss the factors affecting mass resolution 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-

provements in ion optics) that must be addressed to improve the performance of TOF instruments for the analysis of large molecules. We will also describe strategies for the analysis of biological molecules and applications aimed at elucidating protein structure and post - translational processing. The TOF mass spectrometer In the TOF mass spectrometer (Figure la), ions are formed in a short source region (s) in the presence of an electrical field (E) that 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 (where e is the charge on an electron and z is the number of charges), but they will have velocities (u)

that depend on their mass (m).The time (t)required to traverse the drift region (3)

also depends on the mass of the ion,

Figure 1. MS with a linear TOF instrument. (a) Basic components of the linear TOF mass spectrometer. (b) PD mass spectrum of the endoproteinase GIu-C peptide RDASSDEEE from the assembly protein obtained from simian cytomegalovirus, showing the H’ and Na+ peaks used to calibrate the mass scale.

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1,1992

so that the time spectrum can be converted directly to a mass spectrum

as shown in Figure lb. To obtain timing information, the time of ion formation (or extraction) must be known. Thus TOF mass spectrometers generally use pulsed ionization or, in the case of PD, random events that can be detected. Certain instruments employ ionization in the gas phase and desorption from surfaces. TOF mass spectrometers differ in their method of data recording. Those with low secondary ion yields generally measure the time interval between ionization and ion detection, whereas larger secondary ion (analog) signals are captured on fast analog- to - digital converters (ADCs). Linear TOF mass spectrometers are the simplest instruments, because the ions move in a straight line through the flight tube to the detector. Other instruments use additional electric fields that change the direction of the ions to correct for their initial kinetic energy distributions and to improve mass resolution. PD and time interval measurements. In the PD technique (Figure 2a), ions are formed by one of two high-energy (50- 100 MeV) fission fragments emitted in opposite directions from a radioactive 252Cfsource. A start detector placed behind the source detects one of the fission fragments. The second fission fragment strikes the sample, which is coated onto a n aluminized Mylar foil, releasing from 1to 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 determine 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 histogram, which is then converted into a mass spectrum (Figure lb). PD mass spectra generally are accumulated for lo6- lo’ ionization events (or more, if needed). For a 10microcurie source, measurement times may be a few minutes but can be as long as several hours if ion intensities 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 exactly the same TOF are recorded as a single stop event, which limits the

dynamic range. In addition, there is a significant dead time following each stop pulse. If two ions of the same mass arrive a t slightly different 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 digitized by a high-speed ADC or transient recorder. A single transient represents a time (or mass) spectrum. Thus, for example, it is possible to produce a mass spectrum from a single laser pulse. Generally, the results of several transients a r e added together by using an integrating transient recorder (7, 10). For a number of years, transient (or waveform) recorders have been limited to sampling rates of 100 Msampleds, 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

conversion. Currently, however, low cost 1Gsample/s digital oscilloscopes with bandwidths on the order of 500 MHz a r e available from LeCroy (Spring Valley, NY) and Tektronix (Beaverton, OR). Transient recorders that can accommodate 200 Msample/s are available from Precision Instruments (Knoxville, TN) as expansion boards for 386/486 - based PCs, and ILYS Software (Pittsburgh, PA) offers a TOF acquisition and processing software package that is compatible with most commercial transient recorders and digital oscilloscopes. Transient recorders may be triggered from the same electronic pulse used to initiate ionization. However, for instruments employing laser ionization, the trigger pulse is generally obtained by a high-speed optical detector (photodiode, Figure 2b) intercepting 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 extraction to study metastable fragmentation o r to allow the use of broad or even continuous ionization sources such as ESI. Mass calibration. If the accelerating voltage (V = Es) and drift length are known, Equation 4 can be used to determine the mass - to -charge ratio (mlz) directly. However, masses gen-

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.)

erally are obtained from the empirical equation

m

-=at2+b 2

(5)

where the constants a and b are determined by measuring the flight times of two known masses (such as H+, Na+, and ) ' K t h a t appear in desorption mass spectra (Figure lb). Low-mass atomic ion peaks generally 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 extrapolation 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 common for sector and quadrupole instruments. Even when the mass scale is accurately 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 region. In the MALD technique, peptide and protein molecular ions are desorbed with initial velocities comparable 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 In addition to mass, the time axis in the TOF mass spectrometer reflects the initial conditions of the ions (temporal, spatial, and kinetic energy distributions), their fate during acceleration (metastable fragmentation), and properties of the recording system (jitter, sampling rate, bandwidth, 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 a t different times. Because they have the same velocities, the time interval remains constant as they exit the drift tube and are recorded by the detector. From Equation 4, mass resolution (Am/m) for the TOF mass spectrometer is Am 2At --(6) m t Thus, if peak widths (Af) remain con-

ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1,1992 * 1029 A

INSrRUMEN 7ATlON stant, mass resolution can be improved by extending the flight time ( t ) . This can be accomplished by reducing the accelerating voltage or increasing the drift tube length. Because high accelerating 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 (1)used a broad (1-5 ps) electron beam pulse for ionization, and temporal focusing was achieved by pulsed extraction. The fission fragments from a 252Cf source produce ions in < lo-’ s, and nitrogen lasers are now available with 300-600-ps pulse widths. In addition, jitter (triggering), bandwidth, detector response, and sampling 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 accelerated to a higher kinetic energy than the ion formed near the front of

the source. The extraction field can be adjusted so that these ions will be coincident at a space focus plane (SFP). At the SFP the spatial distribution is converted to a kinetic energy distribution, which can be corrected by using a reflectron or another energy -focusing device. The position of the SFP is independent of mass, but the arrival times of ions at the SFP are mass dependent. Space focusing is achieved by using relatively low (< 1 kV) extraction voltages, whereas energy focusing is improved by high (20-30 kV) accelerating voltages (see below). It is difficult to achieve space and energy focusing simultaneously. However, the spatial distribution is minimized in PD and LD instruments, because ions generally are desorbed from an equipotential surface. Kinetic energy distribution. In Figure 3c two ions of the same mass are formed in the same location but with different kinetic energies. The difference in their arrival times at the detector increases with flight tube length but can be minimized by using high accelerating voltages. Alternatively, reflectrons are used to compensate for the differences in kinetic energy. In Figure 3d two ions of the same mass have the same initial kinetic energy, but their velocities are in opposite directions. The ion moving away from the source exit will travel against the field, stop, turn around, and be accelerated to the same energy as the ion moving initially toward t h e source exit. These two ions will have the same velocity and will, therefore, maintain a constant difference i n arrival times. Similar to the temporal distribution, the turnaround time problem can be minimized by using long flight tubes and high extraction fields. This problem occurs primarily for gas-phase ionization and is more relevant to electron impact or ESI instruments. Ionization in the gas phase. It is possible to derive an equation (1, 14)for the TOF of an ion that reflects its time of formation (to),its location in the extraction field (so), and its initial kinetic energy (Uo)as

is independent of whether the ion had an initial velocity component toward or away from the source exit. The time at which the ion leaves the source does, however, depend on the initial velocity direction; thus, 7Uo reflects the turnaround time. The position in the extraction field at which the ion is formed (so) determines its energy due to acceleration (eEso) and thus its velocity as well as flight time in both the source and drift regions. The second term represents the time spent in the drift region. It is independent of the turnaround time but reflects differences in initial kinetic energy (Uo) and position (so). The third term (to)represents uncertainties in the time of ion formation and factors that affect time measurement accuracy. Desorption from surfaces. I n instruments using desorption from surfaces, eEso is constant and equal to eV, where V is the accelerating voltage. If the drift region is much larger than the source region (i.e., D>>s), the TOF can be approximated by

where the resolution is primarily affected by the initial kinetic energy distribution. The difference in arrival times for an ion with no initial kinetic energy and one with an initial kinetic energy of Uois v2

x

At=.(:’,

Because mass resolution is Am/m

[(eV+ uo)v2 - (ev)””]

-A=m2

(mV2

m

Equation 10 can be approximated as

m or

(a) Two ions formed at different times. (b) Two ions formed at different locations in the ion extraction field. (c) Two ions formed with different kinetic energies. (d) Two ions with the same initial kinetic energy that have initial velocities in opposite directions.

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The first term represents the time spent in the ionization/accelerating region, ( Uo+ eEs0)”’ is the velocity of the ion when it leaves the source and


-

Awz - UO

(12) el/ These equations suggest that high accelerating voltages can be used to minimize the energy spread problem. This result is interesting, because mass resolution does not appear to depend on the length of the flight tube but requires only that it be much greater than the accelerating region. In practice, the length has

m

Figure 3. Effects of initial time, space, and kinetic energy distributions on mass resolution.

=

2At/t, Equation 9 can be rewritten as Equation 10.

been dictated by a combination of the accelerating voltage, mass range, and time resolution of the digitizer. For example, most LD instruments have incorporated flight tube lengths of -1 m, and mass range is recorded over a 100-ps interval. The 100 Msample/s transient recorders commonly used on these instruments have time resolutions of 10 ns; and because several samples are required to define a peak, the smallest width that can be recorded is 30-50 ns. Thus, time resolution a t 50 ps is about 1/1000, which corresponds to a mass resolution of around 1/500. The Bio-Ion PD instrument, however, uses a flight tube that is only 15 cm long and records 8-16-ps time intervals with a l-ns TDC. Mass resolution is similar, around 1/300 to 1/500. The recent availability of 1 Gsample/s digitizers suggests possibilities for improving mass resolution and/or decreasing the physical size of the instrument, provided that the effects of initial kinetic energy on

I

peak widths can be minimized. Reflectrons and other energy-focusing devices In 1973 Mamyrin and co-workers (15)introduced the reflectron (Figure 4a) as a means for correcting the effects of initial kinetic energy distributions. The reflectron, located a t the end of the flight tube, consists of a series of rings and/or grids with voltages that increase (linearly in the simplest case) up to a value slightly greater than the voltage at the ion source. The ions penetrate the reflectron until they reach zero energy, turn around, and are reaccelerated back through the reflectron, exiting with energies identical to their incoming energy but with velocities in the opposite direction. Ions with larger energies will penetrate the reflectron more deeply and will have longer flight paths, arriving at the detector at about the same time as less energetic ions. In the Mamyrin design, the reflec-

source

I

Reflected ion detector

Reflectron

I

I

!

'"

IIIIIIIIII!

I I

CR

I I I I I I

I I I

I I

p+ I

Figure 4. Reflectron designs. (a) Two-stage Mamyrin reflectron. (b) Coaxial reflectron.

I I

I I

I 1

I

tron is set a t a small angle with respect to the axis. The exiting ions do not follow the same flight path as incoming ions and can be recorded by a detector placed adjacent to t h e source. The reflectron does not diminish the energy spread of the ions; it corrects for the effects of the energy spread on their arrival times at the detector. In addition, the reflectron increases the pathlength. Thus mass resolution is improved both by decreasing At and by increasing t. The most commonly used reflectrons are two-stage devices (Figure 4a). Ions penetrate the first grid, whose potential is the same as that of the flight tube (generally ground potential). The second grid, located at about 10% of the depth of the reflectron, is placed at about twothirds of the potential of the ion acceleration voltage. I n this short distance the ions lose about twothirds of their kinetic energy. The voltage on the third grid is adjusted slightly above the accelerating volt age to provide different penetration depths over the longer stage. The grid at the back end of the reflectron permits the use of a detector a t this location to record linear spectra when the reflectron is turned off. When the reflectron is turned on, this detector can be used to record a spectrum of neutral species because they are unaffected by the field. Energetic neutral species result primarily from metastable dissociation of ions occurring in the flight tube. Because these neutral species maintain the same flight time as their precursor ions, Della-Negra and Le Beyec (16) as well as Standing and coworkers (17)developed a method for correlating neutral species with their associated reflected ion fragments to produce product ion mass spectra. In Standing's instrument (171,a singlestage reflectron (or ion mirror) is used and time dispersion over the range of product ion masses (which all have the same velocity) occurs in the long reflecting field. Because grids necessarily reduce ion transmission, cause localized field distortion, and produce surfaceinduced dissociation, Wollnik et al. (18)created designs for gridless reflectrons. Della-Negra and Le Beyec (16) described a coaxial reflectron in which exiting ions travel back along the same flight axis but diverge slightly so that they can be detected by an annular multichannel plate detector located at the exit of the ion accelerating region (Figure 4b). The MALD TOF instrument designed by Tanaka et al. (6) also uses a coaxial


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INS'TRUMEN7ArION reflectron whose entrance is located at the SFP close to the ion source. In this case, ions of the same mass enter the reflectron at the same time and spend all their flight time in the reflectron. In addition to reflectrons, electro static energy analyzers (ESAs), similar to those used in double -focusing mass spectrometers, have been used. These include the Poschenroeder ESA (19) and a quadruple ESA arrangement developed by Sakuri et al. (20). Both designs compensate for flight time and energy. Molecular ions and metastable fragmentation Methods such as PDMS and LDMS that use TOF mass spectrometers produce abundant molecular ion peaks in their mass spectra, whereas

Figure 5. Mass spectra of phospholipids desorbed from bacterial cells. (a) FAB mass spectrum. (b) PD mass spectrum. (c) Time-delayedliquid secondary ion TOF mass spectrum. (d) Time-delayed IR LD TOF mass spectrum.

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fragment ion peaks that might be used for structural or sequence information are absent. Figure 5a shows the FAB mass spectrum of lysed E. coEi cells obtained with a doublefocusing mass spectrometer. The protonated molecular ion peaks at mlz 704 and 732 (and the surrounding peaks) correspond to phosphati dylethanolamine species containing different compositions of fatty acyl groups. The groups of peaks at mlz 563 and 591 correspond to the loss of t h e phosphoethanolamine h e a d group (141 amu) and reflect the same fatty acyl composition. The PD mass spectrum of the same sample (Figure 5b) reveals only molecular ions, and these are poorly resolved. FAB sources generally are used with sector or quadrupole mass analyzers. Ions are formed in a field-free region and spend a considerable time in the source before entering the mass analyzer. In the PD mass spectrometer, ions are formed directly in the extraction field. Ion extraction and acceleration are intentionally prompt to maintain the timing integrity necessary for accurate TOF measurements. Ions will spend less time in the ion source before entering the drift region, where much of their fragmentation will occur. Because the drift region is field-free, fragment ions will continue at the same velocity and will be recorded at the same time as their molecular precursors. Thus, as shown in Figure 5b, fragmentation will not be observed. Fragmentation can also occur while the ions are being accelerated. They will have a large time spread, contributing to the baseline noise below the molecular ion peak or to the peak width itself and reducing both mass resolution and mass measurement accuracy. The absence of fragmentation is primarily a function of prompt extraction rather than the mode of ionization. Delayed ion extraction. In our laboratory we have developed TOF instruments that desorb ions in a source that is field-free before the application of an extraction pulse. The extraction pulse is applied 10100 ps after the ionization pulse, permitting fragmentation before ion acceleration. Figure 5c shows the liquid secondary ion TOF mass spectrum of lysed E. coEi cells, obtained by extracting the ions 50 ps after the primary ion gun pulse. Fragment ions are observed, and the spectrum is similar to that obtained on the sector instrument. Figure 5d is the IR LD TOF mass spectrum of the same sample. The molecular ions are MK'

ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992

ions, which lead to the same set of fragment ions following time- de layed extraction. Fragmentation of heavy ions. In general, as molecular weight increases, the time frame for fragmentation increases (21,22), and molecular ions become t h e only i o n s observed even after considerable time delays. The internal energy imparted by the desorption process is spread over many more bonds, reducing the rate constants for fragmentation. For peptides in t h e 800010,000-Da range, fragmentation is not observed in sector instruments. For those in the 1000-3000-Da range, fragmentation generally must be induced by collisions. The absence of fragmentation in PD and LD mass spectra is attributable in part to the TOF analyzer and in part to the fact that such techniques have been used to record much larger ions. Dissociation of molecular ions in the flight tube of a TOF mass spectrometer produces fragment ions with the same velocity, but not the same kinetic energy, as their molecular precursors. Thus their arrival times will be affected differently by additional electric fields. For exam ple, the Bendix instrument used a retarding grid to reveal metastable dissociations. When a potential was applied to this grid, neutral products, intact precursor ions, and fragment ions could be observed at slightly different flight times. Similar effects occur for postacceleration detectors. Because these detectors are used to improve detection at high mass, molecular ions and their products may not be resolved. Thus peak broadening as well as reduced mass resolution and accuracy may result (23). Detection efficiency for large ions can best be improved by increasing ion energy during initial acceleration rather than postacceleration. The reflectron is also a retarding field that will affect precursor ions, product ions, and neutral species differently. Molecular ions will be well focused, resulting in better resolved mass spectra. The metastable product ions enter the reflectron at the same velocity as their precursor ions, but because their kinetic energy is considerably less, they do not pene trate the retarding field as deeply before being turned around. They emerge from the reflectron with the same velocity as the reflected precursor ions but arrive sooner because they spend less time in the reflectron. They are more poorly focused, particularly if they are reflected

within the first stage of a two-stage reflectron. Neutral products are not reflected, and fragmentation occurring in the reflectron itself produces ions that are not focused. Reflecting mass spectrometers may be considerably less sensitive when there is substantial metastable fragmentation. Correlated techniques. DellaNegra and Le Beyec (16)introduced a correlated reflex technique in which the reflectron is used for focusing the fragments resulting from metastable decomposition in the flight tube. Reflected ions are recorded only in those cycles in which a neutral species is recorded within a preset time window by a detector placed behind the reflectron. Because the velocity of the neutral species is identical to that of its precursor ion, only the products of metastable decomposition from a specific mass - selected precursor will be recorded. For example, it is possible to obtain separate product ion mass spectra for molecular ions of different species in a mixture. Generally, product ions with masses only slightly lower than the molecular ion mass will be focused. Those of lower mass have much lower energies, do not penetrate the second stage of the reflectron, and are poorly resolved. These ions can be focused by lowering the reflectron voltage, and different regions of the product ion spectrum can be examined (24). Correlated reflex spectra can be obtained only on instruments that use TDCs to record single events. This technique is used primarily for PDMS or for static secondary ion MS methods with low secondary ion yields. For techniques such as LD that produce many ions in each TOF cycle, product ions cannot be correlated with a specific metastable event. Approaches to high and low mass measurements Approaches to instrument design depend largely on the mass range of the ions to be measured. For MALD of proteins in the 10-100-kDa range, linear instruments with high (generally 20-30 kV) accelerating voltages have been most common. High accelerating voltages minimize the effects of initial kinetic energy spread, increase ion transmission, and place the flight times within a time frame that is convenient for existing transient recorders or digital oscilloscopes. Mass resolutions generally are about one part in 500, and peak widths are (at best) around 20-30 ns. Thus 400 Msample/s recorders

(2.5-11s resolution) provide better time centroids than 100 Msample/s recorders, whereas the more expensive 1 Gsample/s recorders may not provide an advantage. Reflectron instruments are important for recording lower mass ions. In an instrument currently being developed in our laboratory (25),we have used relatively low-voltage (4-5 kV) extraction with a two-stage reflectron to obtain MALD laser desorption mass spectra of a series of small peptides. Figure 6 shows the mass spectrum of methionine enkephalin, where the molecular ion (MNa') at m/z 596.2 is recorded at a mass resolution of 2370. A single peak a t m/z 164.0 from the sinnapinic acid matrix was used to calibrate the spectrum with an accuracy of 0.1 m u . Its peak width is 8 ns, whereas that of the sodium ion is 4.5 ns. In this case, the use of a 1Gsample/s (1.0-ns time resolution) recorder is essential. Pulsed, orthogonal extraction and %continuousionization Pulsed ion extraction enables the use of broad ionization pulses or continuous ionization. The Wiley-McLaren i n s t r u m e n t , for example, used 1-10-ps electron beam pulses but achieved ion focusing by using an extraction pulse with a rise time of

4 ns. In most desorption techniques, ions have considerable initial kinetic energies with initial velocities directed along the TOF axis. Thus there are advantages to extracting ions at right angles with respect to their desorption direction. In 1988 we described a liquid secondary ion mass spectrometer that used a logs, 5-keV Ar' primary ion pulse to desorb ions from a glycerol matrix (Figure 7a). The ions are desorbed orthogonal to the TOF direction and drift toward the extraction region, with the more energetic ions arriving first. By using a suitable time delay, we extracted ions with relatively low kinetic energies (26). Dodenov et al. (8) used a similar approach to extract continuously by ESI (Figure 7b). Ions produced by this technique have a broad distribution of kinetic energies but, when extracted orthogonally, have a low kinetic energy distribution in the TOF direction. Dodenov et al.'s ESI TOF mass spectrum of bovine insulin (Figure 7c) shows that TOF mass spectra can indeed be obtained from continuous (and pulsed) ionization techniques.

Strategies and applications for protein analysis Despite advances in the development

163.1

MNa+

I I

596.2

At= 9.5 ns t445.040ps

m h = 2370

Figure 6. MALD mass spectrum of methionine enkephalin (MW 573.1) obtained on a high-resolution instrument developed in our laboratory. (Adapted with permission from Reference 26.)

ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1 , 1992

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INSTRUMEN TATION of TOF instruments with improved mass resolution, the recovery of fragmentation information from delayed extraction or correlated techniques, and the use of continuous ionization sources, the instruments likely to be available to the biologist or the biochemist provide (primarily) lowresolution molecular weight measurements. However, one cannot overestimate the value of such instruments in the protein laboratory, where their high sensitivity permits the analysis of biological samples available in very small quantities. Although TOF instruments usually are not used to obtain amino acid se-

I 1 I

probe

quences directly, numerous strate gies use molecular weight information to elucidate protein structure and post - translational processing. Often, a protein's full (or partial) amino acid sequence can be inferred from the nucleotide sequence of the gene encoding the protein. The relatively simple, straightforward molecular weight measurements obtained with TOF instruments can provide critical information about the final protein product. In our laboratory PD a n d / o r MALD molecular weight measurements have been used to characterize the N-linked glycopeptides in bovine fetuin (27),to assess

Deflectors

pulse

Channelplate detector

-

a

r,

-. '1

I

11 Electrospray source

Figure 7. Pulsed ion extraction. (a) Diagram of the pulsed, orthogonal extraction, liquid secondary ion mass spectrometer described by Olthoff et ai. (Adapted with permission from Reference 26). (b) Diagram of the ESI instrument described by Dodenov et al. (Adapted with permission from Reference 8). (c) ESI TOF mass spectrum of bovine insulin obtained by Dodenov et ai. (Adapted with permission from Reference 8.)

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carbohydrate heterogeneity in trypanosomes (28) and phosphate heterogeneity in the riboflavin binding pep tide (29),to determine cleavage sites in the processing of the amyloid precursor protein (30) and the assembly protein from simian cytomegalovirus (31), and to verify the structures of Staphylococcus nuclease insertion products (32). In many cases, partial amino acid sequence information has been obtained by in situ digestion with carboxy- or aminopeptidases, followed by molecular weight analysis of the truncated peptides (30,31).The three examples below illustrate our methods and strategies for TOF analysis, which can be carried out on commercially available instruments. Constitutive cleavage site of the amyloid precursor protein. Amyloid precursor proteins (APPs) are 695-770 amino acid (110-130 kDa) membrane- bound glycoproteins that are encoded by alternatively spliced transcripts derived from a single gene on chromosome 21 (Figu r e 8a). A short 4-kDa peptide known as P-amyloid (PA141 is the principal component of fibrillar extracellular deposits (plaques) found in the brains of Alzheimer's disease patients. I t encompasses 14-15 amino acids of the transmembrane domain and 28 amino acids of the extracellular region of APP. Sisodia et al. (33) showed that normal processing of the APP includes proteolytic cleavage in the extracellular portion and within the PA/4 region, releasing a soluble peptide (SAPP); however, the exact cleavage site was unknown. Soluble APP-770, expressed in a Chinese hamster ovary (CHO) cell line, was purified; digested with cyanogen bromide (CNBr), which cleaves at methionine; and fractionated by reversed-phase HPLC. Two fractions, found to contain the C-terminal peptide, were examined by PDMS (Figures 8b and 8c) to determine the cleavage site (30).Approximately 5 pmol of each peptide were deposited on nitrocellulose foils and inserted into the mass spectrometer. In Figure 8b, the peak at mlz 1955.6 corresponds to the MH' ion for a 16 amino acid peptide with the sequence DAEFRHDSGYEVHHQK (MW = 1953.9). I n Figure 8c, the peak at mlz 1826.9 corresponds to the MH' ion for the 15 amino acid peptide DAEFRHDSGYEVHHQ (calculated MW = 1825.8). Peaks at m/z 1827.1 and 1955.6 in Figures 8b and 8c, respectively, reflect the incomplete chromatographic

separation of these two peptides. Negative-ion PDMS spectra were also obtained and are shown in the inserts. The peaks at mlz 1953.7 and 1824.5 in Figures 8b and 8c, respectively, are (M-H)- ions of these two peptides and confirm the interpretation of the peaks in the positive-ion mass spectra as MH' ions, and not (for example) MNa' ions. Thus cleavage occurs on either side of the lysine (residue 687 in APP-770). The 16 amino acid peptide was then removed from the mass spectrometer and digested directly on the

sample foil for 3 h using 400 ng of carboxypeptidase Y. The sample foil was rinsed to remove the enzyme and reinserted into the mass spectrometer. The spectrum in Figure 8d shows protonated molecular ions for the intact 16 amino acid peptide DAEFRHDSGYEVHHQK (mlz 1956) as well as two truncated peptides, DAE FRHDSGYEVHHQ (mlz 1827) and DAEFRHDSGYEVHH (mlz 16991, confirming the C-terminal amino acids as lysine and glutamine (34). Synthetic 16 amino acid peptide DAEFRHDSGYEYHHQK was also

prepared, and its retention time on reversed- phase HPLC was compared with that of the putative C-terminal peptide from SAPP. The PD mass spectrum of the synthetic peptide (Figure 8e) shows a protonated molecular ion a t mlz 1955.8, a doubly charged molecular ion (MHP) at mlz 978.6, and a series of fragment ions that reveal most of the amino acid sequence. As is often the case, synthetic peptides that are available at higher purity and in larger amounts than those peptides obtained from biological samples produce sequence- specific fragmentation comparable to that observed in FABMS on sector instruments. Although the reasons for this are not clear, it is possible that larger samples result in desorption of peptides that are not weakly bound by direct hydrophobic interactions with the nitrocellulose foil. The resulting ions would have higher internal energy and fragment more promptly. Staphylococcus nuclease inser tion protein. MALD was used to verify the insertion of a glycine residue in StaphyZococcus nuclease (MW 16,801 Da) by mapping the unfractionated endoproteinase Arg-C digests of the wild-type and insertion peptides (32).This approach provides better specificity than measurements of intact molecular weights, because only one of the enzymatic fragments should show a change in mass. Table I shows the peptides expected from the endoproteinase ArgC digest of the wild-type StaphyZococcus nuclease. This enzyme cleaves at arginine residues, and we also observed cleavages a t adjacent lysine residues. The glycine insertion is between residues 126 and 127. Figure 9 shows the MALD mass spectrum of the glycine insertion peptide digest (9).The peak a t mlz 2634.8 corresponds to the protonated molecular ion of the peptide [lo-111 with a n additional 56.7 mass units, corresponding to the glycine insertion. Processing of the assembly protein from simian cytomegalovirus. B -capsids of cytomegalovirus contain a phosphorylated 37-kDa protein, known as the assembly protein (AP), which is involved in capsid assembly and maturation. The genomic region for the AP from simian cytomegalovirus consists of four nested genes that result in four independently transcribed 3'coterminal RNAs coding for four carboxycoterminal peptides. One of these proteins, the preassembly protein (PAP), is a 310 amino acid protein whose sequence can be inferred from

-

0

1400

--

I

1827

m/z MH+

195k8

.

.

.

Figure 8. Determination of the constitutive cleavage site of the amyloid precursor protein (APP). (a) APP structure showing the P-amyloid peptide that spans the transmembrane region and is found in extracellular plaques of Alzheimer's disease patients. (b and c) PD mass spectra of the C-terminal 16 amino acid and 15 amino acid peptides, respectively, resulting from constitutive cleavage of the amyloid precursor protein. (d) PD mass spectrum of the on-foil digest of the 16 amino acid C-terminal peptide. (e) PD mass spectrum of synthetic amyloid,-,,.


1035 A

INS'TRUMEN'TA7lON

GLAYIYADGKMVNEALVh

KEKLNIWSEDNADSGQ KSEAQAKKEKLNIWSED

the gene sequence. The PAP is the precursor for the AP and contains the same amino terminus. PDMS was used to determine its cleavage site (31). The AP obtained from human foreskin fibroblast cells was purified by HPLC and digested with endoproteinase Lys-C and endoproteinase

Glu-C. Both digests were fractionated by HPLC, and PD mass spectra were obtained for all the major fractions. In both cases, all the peptides from t h e amino terminus to the cleavage site were accounted for. The cleavage site was determined from the measured masses of the carboxyterminal peptides.

1635.7

I

I

1612.5

\

I

551.0

/

I I

1579.8

\,

1484.2

21p0.6

1

3085.0 1m.3

2634.8 12855.0

Figure 9. MALD mass spectrum of Staphylococcus nuclease containing a glycine insertion. 1036 A * ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER l,1992

For the endoproteinase Lys-C digest, the peak observed a t mlz 903.3 (Figure loa) corresponds to the protonated molecular ion of the carboxyl- terminal peptide SAERGWNA terminating at the alanine-277 residue. For the endoproteinase Glu-C digest, peaks observed at mlz 744.5 and 616.3 (Figure lob) correspond to protonated molecular ions of peptides ERGWNA and RGWNA, respectively. Both fractions were then digested with carboxypeptidase P. The mass spectrum of the digested endoproteinase G l u - C f r a c t i o n showed a peak at mlz 462.2, corresponding to the peptide SAER. The mass spectrum of the digested endoproteinase Lys - C fraction showed a peak a t mlz 547.6, corresponding to t h e peptide RGVVN. Thus, t h e cleavage site for the 310 amino acid preassembly protein was firmly established as the alanine-277 residue. Protein structure using TOFMS. TOF mass spectrometers play a crucial role in our laboratory for the structural analysis of proteins and peptides (Figure 11).MALD can be used to obtain t h e molecular weight of the intact protein with far better accuracy than can be obtained using polyacrylamide gel electro phoresis (SDS-PAGE). For glycoproteins, the molecular weights of the native and the deglycosylated protein determined by MALD can be used to assess overall carbohydrate content and heterogeneity. Although much attention has been focused on the use of MALD to measure the molecular weights of intact proteins, in our laboratory we have used both MALD and PDMS to ana-

lyze their enzymatic and chemical digests. Molecular weights of t h e smaller peptides derived from digestion can be determined with far more accuracy than that of the intact protein. TOF mass spectrometers are particularly well suited for mapping such digests before fractionation by HPLC, because they can record the entire mass range simultaneously. At this level of analysis they may provide much higher sensitivity than techniques that use scanning instruments. Also, because both MALD and PDMS produce (primarily) singly charged molecular ions, it is much easier to interpret the mass spectra of these complex mixtures than it is to interpret ESI mass spectra. For a large protein, our protocol begins with chemical digestion with CNBr, which cleaves at methionine residues and converts the C- termi nal methionines on the resultant fractions to homoserine or homoserine lactone residues. Methionine residues are not abundant in proteins; thus, the resultant peptides are large and most easily mapped by

using MALD (35).Often, cleavage is not complete, and peaks representing overlapping fragments can be used to establish their order. In addition, when all the cleavage products are accounted for, t h e sum of their masses can provide a more accurate molecular weight than can be obtained from the intact protein. The intact protein or its CNBr fragments can be digested with trypsin, which cleaves at arginine and lysine residues. The unfractionated digest can then be mapped directly by obtaining either MALD or PD mass spectra. When the protein sequence is unknown, the individual peptides must, of course, be purified and sequenced by using the Edman procedure or tandem MS. When the sequence is known, the masses of peptides expected from tryptic digestion can be calculated and compared with those observed. Much information thus can be obtained directly from the peptide map. Disulfide bonds result in a peak whose mass corresponds to t h e masses of two tryptic peptides minus

616.3

. * h d . w A c (

Figure 10. PD mass spectra of the C-terminal peptides from the simian , cytomegalovirusassembly protein, following digestion with (a) endoproteinase Lys-C and (b) endoproteinase GIu-C.

the mass of two hydrogen atoms. These can be verified by reducing the peptide mixture with dithiothreitol (DTT) and obtaining a new spectrum. Glycosylation sites can be determined by comparing the tryptic map with one obtained for a digest treated with a glycosidase. A phosphorylation site produces a peak whose mass is 80 Da greater than that predicted for its tryptic peptide. For a protein with a known sequence, there is usually a target peptide that contains a glycosylation or a phosphorylation site, a point mutation, a cleavage site, or some other post - translational modification. The target peptide is purified by HPLC fractionation of the enzyme digest and analyzed by MALD or PDMS to verify its mass. PD is advantageous because the peptide is bound to a nitrocellulose-coated sample foil. The sample can be removed from the instrument, treated directly on the foil with aminopeptidase or carboxypep tidase, washed to remove the enzyme, and reinserted into the mass spectrometer. The resultant mass spectrum can reveal portions of the amino - (or carboxyl -) terminal se quence and the exact site of modification from the masses of the truncated peptides observed i n t h e spectrum. Because fragmentation is not observed on TOF instruments, information on the protein structure, post- translational modifications, and cleavage sites is obtained from molecular weights of cleaved or truncated peptides and relies on the skillful use of multiple enzyme reactions. Several enzymatic approaches are used to provide better specificity. Be cause trypsin may result in a large number of small peptide fragments, endoproteinase Arg-C and endoproteinase Lys - C-which cleave at argi nine and lysine residues, respective ly-can be used t o reduce t h e number of peptides. Alternatively, endoproteinase Glu-C, which cleaves at glutamic acid, may be used to isolate a peptide that is then digested further with trypsin. Although such approaches are cumbersome to the mass spectroscopist, they are familiar to protein chemists who may ultimately comprise the major user group for TOF instruments, and they provide a highly sensitive alternative to tandem MS.

Conclusions How far have we come? In their 1973 paper, Mamyrin and co-workers (15) reported a TOF mass spectrum of the trimer of rhenium bromide whose


1037 A

INS7RUMENTArION isotopic cluster from mlz 1270 to 1286 was resolved to one part in 3500, an extraordinary resolution by today's standards. In 1988 Wollnik et al. (18) measured the molecular ion of triazine at mlz 866 with a resolving power of one part in 15,000, demonstrating that TOF mass spectrometers can indeed compete with sector instruments. Bergmann and co workers (36)developed a TOF instrument using MPI, orthogonal extraction, and hyperbolic quadrupole focusing with a cluster beam source. The instrument can obtain mass resolutions greater than one part in 5000 at molecular weights of 100,000 Da. Such performance cannot be achieved by even the most expensive commercial double - focusing instru ments. What has changed since the early work of Wiley and McLaren (1)is our interest in using TOF mass spec-

Protein

trometers for the analysis of biological molecules that are available in extremely small quantities; the necessity for developing reasonably simple, compact, and inexpensive instrumentation that might be widely used; and the availability of highspeed electronics that may accomplish the task. The instruments described by Wiley and McLaren ( I ) , Mamyrin et al. (15),Wollnik and coworkers (18),and Bergmann and coworkers (36)all use copious samples whose molecular ions can be continuously regenerated. Methodologies that use MPI as a postionization technique for large molecules (37) achieve high mass resolution by ionizing only a portion of the desorbed neutrals having a narrow kinetic energy spread. In contrast, the protein chemist or biochemist needs to collect all ions that can be formed from a picomole or a femtomole sample.

LD mass spectrum MW Assess carbohydrate composition

LD mass spectrum No. of chains

No. of disulfide bonds Cyanogen bromide

fragments

LD mass spectrum MW of CNBr fragments

There is good news for both the present and the future. Although limited in mass resolution, simple linear TOF mass spectrometers using PD and MALD ionization are finding their way into a number of biological laboratories. As suggested by the applications described above, these instruments are being used to solve biological structural problems. High sensitivity, simultaneous recording of the mass range encompassing an unfractionated tryptic digest, and the ability to carry out enzymatic reactions on samples recovered from the instrument (without transferring these from the sample probe) all provide an opportunity for analyzing minute quantities of biological samples. The recent availability of inexpensive, high-speed digitizers, software, and PC interfacing as well as turn-key instruments ensures that many more laboratories will use TOF mass spectrometers. In the future we should see further development of TOF instruments with better mass resolution, and they will be commercially available at reasonable cost. These instruments will also be interfaced to continuous ionization sources such as ESI and will be combined with separation techniques such as microbore LC and capillary zone electrophoresis. Tandem (TOF/TOF) instruments will enable this technology to combine the advantages of simultaneous recording with those enjoyed by both foursector and quadrupole mass spec trometer s. References

etc

-

ion

peptide

Truncated

PDMS or LDMS Mass mapping Location of S S bonds Glycosylation sites Phosphorylationsites

PDMS spectrum Verify target peptide by its mass Glycosyl or phosphoryl microheterogeneity

-

PDMS sDectrum N-terminal sequence C-terminal sequence Location of post-translationalmodification

Figure 11. Methods and strategies for the analysis of peptides and proteins by TOFMS. 1038 A


(1)Wiley, W. C.; McLaren, I. H. Rev. Sci. Instrum. 1955,26,1150. ( 2 ) Conzemius, R.J.; Capellen, J. M. Int. J. Mass Spectrom. Ion Phys. 1980,34, 197. (3) Hillenkamp, F.; K a u f m a n n , R.; Nitsche, R.; Unsold, E. Apjl. Phys. 1975, 8, 341. (4)Macfarlane, R. D.;Skowronski, R. P.; Torgerson, D. F. Biochem. Biophys. Res. Commun. 1974,60,616. ( 5 ) Karas, M.;Hillenkamp, F. Anal. Chem. 1988,60,2299. (6)Tanaka, K.;Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988,2,151. (7) Emary, W. B.; Lys, I.; Cotter, R. J.; Simpson, R.; Hoffman, A. Anal. Chem. 1990,62,1319-24. (8) Dodenov, A. F.; Chernushevich, I. V.; Laiko, V. V. Presented at the 12th International Mass Spectrometry Conference, Amsterdam, The Netherlands, Aug. 26-30, 1991. (9)Chewier, M. R.; Cotter, R. J. Rapid Commncn. Mass Spectrom. 1991,5, 61117. (10)Holland, J. F.;Enke, C. G.; Allison, J.; Stults, J. T.; Pinkston, J. D.; Newcombe, B.; Watson, J. T. Anal. Chem. 1983,55,497A. (11)Beavis, R. C.; Chait, B. T. Chem. Phys.

Lett. 1991,5,479. (12)Huth-Fehre, T.; Becker, C. H. Rapid Commun. Mass Spectrom. 1991, 8, 37882. (13)Pan, Y.; Cotter, R. J. Org. Mass Spectrom. 1992,27,3-8. (14)Cotter, R.J . Biomed. Environ. Mass Spectrom. 1989,18,513-32. (15)Mamyrin, B. A.; Karatajev, V. J.; Shmikk, D. V.; Zagulin, V. A. Soviet Phys. JETP 1973,37,45-48. (16)Della-Negra, S.; Le Beyec, Y. Anal. Chem. 1985,57,2035. (17)Standing, K. G.;Beavis, R.; Bollback, G.; Ens, W.; Lafortune, F.; Main, D.; Schueler, B.; Tang, X.; Westmore, J. B.Ana1. Znstrum. 1987,16,173. (18) Grix, R.; Kutscher, R.; Li, G.; Gruner, U.; Wollnik, H. Rapid Commun. Mass Spectrom. 1988,2,83. (19)Poschenroeder, W. Znt. J. Mass Spectrom. Zon Phys. 1971,6,413. (20)Sakuri, T.; Fujita, Y.; Matsuo, T.; Matsuda, H.; Katakuse, I. Int. J. Mass Spectrom. Ion Proc. 1985,66,283. (21)Chait, B. T. Znt. J Mass Spectrom. Ion Phys. 1983,53,227. (22)Demirev, P.; Olthoff, J. K.; Fenselau, C.; Cotter, R. J . Anal. Chem. 1987, 59, 1951-54. (23)Spengler, B.; Pan, Y.; Cotter, R. J.; Kan, L-S. Rapid Commun. Mass Spectrom. 1990,4,99-102. (24)Brunelle, A.; Della-Negra, S.; Depauw, J.; Joret, H.; Le Beyec, Y. Rapid Commun. Mass Spectrom. 1991,5,40-43. (25)Cornish, T.;Cotter, R. J. Rapid Commun. Mass Spectrom. 1992,6,242-48.

(26)Olthoff, J. K.; Lys, I. A.; Cotter, R. J. Rapid Commun. Mass Spectrom. 1988,2, 171-75. (27)Townsend, R. R.; Alai, M.; Hardy, M. R.; Fenselau, C. Anal. Biochem. 1988, 171,180, (28)Bean, M. F.; Bangs, J. D.; Doering, T. L.; Englund, P. T.; H a r t , G . W.; Fenselau, C.; Cotter, R. J. Anal. Chem. 1989,61,2686-88. (29)Vaughn, V. L.; Wang, R.; Fenselau, C.; White, H. N., I11 Biochem. Biophys. Res. Commun. 1987,147,115. (30)Meschia, J.; Sisodia, S. S.; Wang, R.; Cotter, R. J . J. Biol. Chem. 1991, 25, 16960-16964. (31)Welch, A. R.;Woods, A. S.; McNally, L. M.; Cotter, R. J. Proc. Nat’l. Acad. Sci. 1991,88,10792-10796. (32)Keefe, L. J.;Lattman, E. E.; Wolkow, C.; Woods, A.; Chevrier, M.; Cotter, R. J. J. Appl. Cystallog. 1992,25,205-10. (33) Sisodia, S. S.; Koo, E. H.; Beyreuther, K.; Unterbeck, A.; Price, D.L. Science 1990,248,492-95. (34)Wang, R.; Cotter, R. J.; Meschia, J. F.; Sisodia, S. S. Techniques in Protein Chemisty ZI; Academic Press: New York, 1992;pp. 505-13. (35)Hua, S.; Vestling, M.; Murphy, C.; Fenselau, C.; Spengler, B.; Pan, Y.; Wang, R.; Cotter, R. J.; Theibert, J.; Collins, J. H. Techniques in Protein Chemistry ZI; Academic Press: New York, 1991; pp. 95-106. (36) B e r g m a n n , T.; M a r t i n , T. P.; Schaber, H. Rev. Sci. Znstrum. 1989,60, 792.

(37)Boesl, U.; Weinkauf, R.; Schlag, E. W. Znt. J. Mass Spectrom. Zon Proc. 1992, 112,121-66.

RobertJ. Cotter received his B.S. degree in chemistry fiom the College of the Holy Cross and his Ph.D. in phrsical chemistry fiom The Johns Hopkins University. He is currently professor of pharmacology and molecular sciences at The Johns Hopkins University School of Medicine and director of the Middle Atlantic Mass Spectrometry Laboratory. He has been involved in developing TOF mass spectrometers, including an IR laser desorption instrument, a puked liquid SIMS TOF with a high-speed integrating transient recorder, and matnkassisted laser desorption mass spectrometers using Nd:YAG and pulsed nitrogen lasers.

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Time-of-flight mass spectrometry for the structural analysis of

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