Product
Review
MALDI-TOFMS Pulses Ahead Matrix-assisted laser desorption/ionization (MALDI) is a "soft" ionization technique for introducing very large delicate molecules such as proteins into a mass spectrometer without fragmentation. Michael Karas, Doris Bachmann, and Franz Hillenkamp coined the term "matrix-assisted" in 1985, when they reported the use of tryptophan and nicotinic acid as matrix compounds to permit LD of nonvolatile amino acids and peptides. MALDI of larger biomolecules was presented at a 1987 conference by Koichi Tanaka of Shimadzu uapan), who used a matrix of ultrafine metal powder dispersed in glycerol to desorb 70,000-Da proteins. Hillenkamp's group refined its organic matrix method and published spectra of proteins up to 100,000 Da in 1987 and 1988. Their approach has since been adopted by the mainstream. MALDFs applications have multiplied so rapidly that in only a few years of commercial availability, it has become a substantial and very "hot" subfield in MS. MALDI works by irradiating a sample with a short laser pulse or "shot" and extracting the ionized molecules from the
MALDI methods are rapidly expanding for time-of-flight and other MS systems
stream, so mass-scanning instruments such as quadrupole and double sector mass spectrometers would necessarily miss some of the masses in each packet. TOFMS provides nearly simultaneous detection of all the masses in an extensive mass range, so it takes full advantage of MALDI's capabilities for large-molecule work. Conversely, TOF works best with pulsed ion sources and, until the advent plume that bursts up from the probe surof MALDI, had limited routine applicaface into the mass spectrometer. The sam- tions. ple is mixed with a specially selected We asked Franz Hillenkamp of the Uni"matrix" compound such as sinapinic acid versity of Muenster (Germany) and Robor dihydroxybenzoic acid and (usually) ert Cotter of Johns Hopkins University for is allowed to dry as a crystalline or semitheir comments on current trends in crystalline coating on a probe tip. The ma- MALDI-TOFMS and their advice for intrix compound, which is chosen to abstrument buyers. "The short answer to sorb light strongly at the laser wave'What has changed in the pastfiveyears?' length, appears to protect the analyte by is, everything," says Hillenkamp. Altransferring kinetic energy to it in a way though most of the commercial instruthat allows desorption and ionization with- ments sold for MALDI-MS are time-ofout significant fragmentation. flight (TOF) mass spectrometers, the field is beginning to diversify with the introThe marriage of MALDI with time-ofduction of MALDI-FTMS systems. New flight (TOF) MS is a natural one. Unlike matrices, lasers, components, and configumost ionization methods, MALDI is a pulsed method that produces ions in dis- rations are being explored for TOF and other MS systems. Not all of these crete packets rather than in a continuous Analytical Chemistry, August 1, 1995 497 A
Product
Review
Table 1 . Summary of representative products
Product Company
Reflex II Bruker Analytical Systems Fortune Drive, Manning Park Billerica, MA 01821 508-667-9580
RTOF-1M Comstock 1005 Alvin Weinberg Drive Oak Ridge, TN 37380 615-483-7690
Vision 2000 Finnigan MAT 355 River Oaks Pkwy. San Jose, CA 95134 408-433-4800
Price range MS type Source Lasers
$230,000-$300,000 Linear and reflectron
$119,000-$230,000 Linear and reflectron
$250,000-$300,000 Linear and reflectron
N2; Nd:YAG, IR lasers optional
N2; Nd:YAG
N?, 250 μϋ; Nd:YAG, 1 m j N,, 3 ns; Nd:YAG, 5 ns N?, 50-150 μπη; Nd:YAG, 50-100 μπι N2, nominal 100% transmission down to < 5%; Nd:YAG, to 3%; both continuously variable under software control
N2, 200 μϋ N2, 3 ns; Nd:YAG, 5 ns 50-2000 μηι Continuously variable under servo control
N2; optional Er:YAG, switchable via computer N2, 300 μJ; Er:YAG, 20 mJ N2, < 5 ns; Er:YAG, 70 ns N2, 100 μηι Continuously variable under software control
Optional
Yes
Variable extraction field
1 grid with Multiprobe source; 0 with Scout χ,κ stage source ±30kV
2-stage
Multistage gridless
± 20 kV; ± 30 kV in linear mode
±30kV
Einzel lens 1.55 m linear 3 m reflectron
Second-order energy focusing 1.2 m linear 2.15 m reflectron
Cylindrical multielement 0.8 m linear 1.7 m reflectron
2 dual-channel MCDs standard; hybrid detector optional for polymers
2 dual-channel MCDs
Dual SEMs; 25-kV conversion dynode for reflectron and PSD mode
2-staqe qridless with ion refocusinq element Standard Optional with precursor ion selector
2-stage gridless Standard Optional with ion gate
Multistage gridless Standard Optional with precursor ion selection for analysis of mixtures
Range 1.00-30 kV; typically 15-20 steps, dependinq on mass range SunSparc 5 workstation with UNIX Motif/XII graphical user interface; multi user and multitasking software with icon/window user interface
Computer-controlled voltage to ±30kV Pentium/90 MHz with LabViewbased software; 500 MHz dualchannel digitizer
25%/step
Sample probes
26-position targets for x,y sample stage with viewing optics; smooth targets and custom plates for membranes
Multisample 1-axis sample stage
Special features
Optional CID gas cell; optional sample visualization
Options
Optional CID accessory for high-energy fragmentation; ion refocusing element in reflectron for maximum ion transmission; modular design; high-resolution viewing optics with > 100X magnification CID
2-axis x,y sample stage
Reader Service Number E-mail reflector keyword
401 ac MMS622
402 ac MMS623
Max. laser energy Pulse width range Focused spot size range Attenuator range and control Ion extraction Split extraction geometry No. stages/grids Total acceleration potential Mass spectrometer Ion optics Length of flight tube Effective pathlength Detectors
Reflectron Type Standard or coaxial PSD analysis
Voltage control Data system
NA = Not applicable ΙΝΑ = Information not available at press time
498 A
Analytical Chemistry, August 1, 1995
Pentium/75 MHz PC with Windowsbased user interface and software for instrument control, data acquisition, analysis, and protein database searching 28-position probe with x,y positioning; accommodates liquid matrices for IR applications and direct desorption of biopolymers from membranes 50-μηι resolution zoom microscope and CCD camera for sample viewing; reflectron operates over full mass range; differential pumping; positive and negative ion detection Bioworks software, synthetic polymer software package 403 ac MMS624
TofSpec-SE Fisons/VG Organic 55 Cherry Hill Dr. Beverly, MA 01915 508-524-1000
IG 2025 LD-TOF Hewlett Packard 1601 California Rd. Palo Alto, CA 94304 415-857-8975
$235,000-$350,000 Linear; reflectron option
$95,000 Linear
N2; optional Nd:YAG or Er;YAG; optional 2nd laser bench mounting N2, 180μϋ N2, 4 ns Fixed at - 175 μπι Continuously variable with variable iris and neutral-density filter under software control
Corrected N 2
Variable extraction field
I Voyager-Elite PerSeptive Biosystems/Vestec Biospectrometry Products 500 Old Connecticut Path Framingham, MA 01701 800-899-5858 $225,000-$300,000 Free-standing reflectron
I Kompact MALDI III Shimadzu Scientific Instruments 7102 Riverwood Dr. Columbia, MD 21046 410-381-1227 $149,950 Linear and reflectron benchtop
N2
N2; Nd:YAG optional; expansion ports for additional lasers N2, -200 μϋ N2, 3 ns ΙΝΑ, manually adjustable 1000-step fully variable laser intensity under software control
200-300 μϋ 3 ns Adjustable down to 20 μιτι Variable in 180 steps under software control
Focusing repeller geometry with variable extraction field 2-stage
Fully variable field strength under software control 2-stage grid
Software-selectable extraction at 20 kV or 5 kV 1 -stage grid
± 30 kV linear; ± 25 kV reflectron
±30kV
±30kV
±20kV
Lenses for source focusing 1.6 m linear 3.5 m reflectron
Orthogonal pulsed-field mass filter 1.0 m 1.0 m
Einzel lens < 1 m linear < 2 m reflectron
Hybrid detector for linear mode; additional MCD for reflectron mode
Dual-channel MCD with secondary ion generator
Guide wire 2.0 m linear; expandable to 4.2 m 3.0 m reflectron; expandable to 6.5 m 2 dual-channel MCDs; optional hybrid MCD-SEM for linear detector Single-stage grid Coaxial Standard with timed ion selection for precursor ions
2-stage grid Standard Not standard; upgradable to MALDI IV with curved-field reflectron for scanless PSD analysis at full accelerating voltage NA
2-stage gridless
200 μϋ 3 ns Eiliptical,100x300um Gradient neutral-density filter with minimum energy 0.1 μϋ; 0.1 μϋ/ step with software control
1-stage gridded NA Coaxial NA. Optional with Bradbury-Nielson ion NA gate for precursor ion selection and grounded collision cell for CID Up to 12 variable steps; mass range up to 40%/step under software control AlphaStation 200 with open VMS/ Motif windows platform and optional data analysis, MaxEnt deconvolution, and biopolymer software packages 15-position target standard, 130position target optional; accommodates 70 χ 70 mm gels
NA
Range 0-30 kV; typically - 7 steps, depending on mass range Vectra 586 computer with Windows- 486/66 MHz PC with Windowsbased control and analysis soft based control and analysis soft ware; smart control of applied laser ware; metastable fragment analy sis software for protein sequencenergy and sample position; autoing; 500 MHz digitization mated data acguisition 100-position reusable sample ΙΝΑ plate; x,y stage controlled with joystick and video viewing camera
Differential vacuum pumping
Vacuum-induced sample prepara tion for homogeneous crystalliza tion; optional 1-gigasample/s digitizer; calibration standards and matrices for MALDI CCD camera sample viewing optics; ΙΝΑ CID cell; 2nd laser mounting bench 404 ac MMS625
405 ac MMS626
Delayed ion extraction; optional CID cell; high-resolution camera optics with 100X magnification
2 high dynamic range SEMs
SunSparc multiprocessing work station; software for chromatography, membrane analysis, peptides and polymers, enzyme digests, and database searching Slides for 10, 20, and 30 samples; continuous membrane slides for gel electrophoresis work User-selectable mass range of 250,000 Da or 1,000,000 Da
Expanded flight path for enhanced Upgrade to Kompact MALDI IV with curved-field reflectron mass resolution; range of sample plate formats 407 406 ac MMS628 ac MMS627
Analytical Chemistry, August 1, 1995 499 A
Product
Review
are nearing commercialization just yet, he says, but MALDI-MS is changing so rapidly that some of the advances reported at this year's ASMS conference (see Focus, p. 4931 A) may well be on the market in two or three years. Table 1 presents a variety of commercial MALDI-TOF mass spectrometers. Although the table features only one instrument for each company, most of the companies offer a range of MALDI-TOF instruments at different levels of complexity and cost, and many of the instruments can be upgraded or modified. With this issue, Analytical Chemistry introduces an e-mail reflector that offers supplementary information provided by each company. Users can request a single document or several by sending one e-mail message to
[email protected]. Type the keyword phrase that appears at the bottom of the table on the subject line to receive one document; leave the body of the message blank. To receive several documents, type each keyword phrase on a separate line in the body of the message. Each phrase must appear at the beginning of a new line.
what they're doing will start adjusting one parameter, then another which throws off the first, and eventually the settings that work best for their applications are lost." Cotter agrees, noting that many users are better off with a system that either has set source parameters or can optimize them automatically. Some systems offer the possibility of interchanging lasers for different applications, either through a single port or through separate ports for different lasers. The standard laser is usually an inexpensive ( ~ $5000) nitrogen laser that operates at 337 nm. Nd:YAG lasers that are tripled or quadrupled to operate at 335 nm or 256 nm, respectively, and EnYAG lasers for IR work cost $20,000-50,000. In addition, some of the more expensive UV lasers are designed to operate at pulse widths narrower than the usual 3-6 ns,
ture is dried before use. However, some instruments have accessories for doing MALDI of proteins separated on a slab gel and transferred to a membrane. Frozen glycerol or ice can be used as IR matrices but require a chilled sample support that can maintain the samples as solids. None of the commercial instruments offered this type of sample probe at press time. Extraction
After desorption and ionization from the sample probe, the gas-phase ions are accelerated or "extracted" into the mass analyzer using an applied electricfield.The extractor consists of the sample probe backplate and one or two fine-mesh metal grids or ring electrodes between the sample probe and the entrance to the mass analyzer. "It is generally assumed that the higher the acceleration potential, the better the performance," Hillenkamp says. "This is certainly correct for linear TOF, and by now most instruments offer up to 30 keV. Many reflectron instruments operate at the same (high) potential in reflectron mode as in linear mode, but for reflectron mode, 10 keV can give as good a result as 30 keV." Sources The most common configuration is exMost MALDI sources are sold as an intetraction in line with desorption from the gral part of an MS system, usually TOF, sample probe, but orthogonal extraction, and the degree of flexibility or user conwhich accelerates the ions at the top and trol varies with the instrument. In general, bottom of the plume at the sametime,is the less expensive instruments are less available in a few commercial instruments complex, have fewer adjustable parameand, says Cotter, was used in some older ters, and offer fewer lasers, sampling opTOF analyzers before the advent of tions, and accessories. says Cotter, and may offer enhanced mass MALDI. Similar geometries are under development for MALDI sources by Kenneth The ionization source consists of a laresolution for TOFMS. IR lasers have ser, focusing optics, an attenuator, a sam- somewhat broader pulse widths of 50-200 Standing and Werner Ens at the University of Manitoba (Canada) and by others. ple stage, and an ion accelerator or "ex- ns and consume more material per shot, tractor" that pulls desorbed ionized mole- but still give better mass resolution than Having more than one grid or ring cules into the mass spectrometer. The two UV lasers for some (i.e., IR) applicaelectrode with independent voltage conmost important factors in evaluating a tions. Hillenkamp adds that the developtrol offers more flexibility for ion extracMALDI source, says Hillenkamp, are the ment of small, good-quality C0 2 lasers, tion techniques, including single- or twoshot-to-shot reproducibility and the de- comparable in size and cost to today's stage extraction. "Something to look out sired degree of flexibility or user conN2 lasers, may eventually make IR MALDI for, particularly in reflectron instrutrol. Shot-to-shot reproducibility depends more affordable. ments, is the ability to operate in either on a number of factors, including sample soft or hard extraction mode; that is, with The specific type of focusing optics preparation and homogeneity, the choice used in the source is generally not as criti- a low or a high electricfieldin front of the of matrix, laser power and spot size, and so cal as the laser beam quality and pulse sample," says Hillenkamp, because field on. "One thing experience has shown us strength determines the time available for energy reproducibility from shot to shot, is that the overall reproducibility of he says, and in most cases the optics are fragmentation, or post-source decay MALDI is very sample dependent," Hillen- fixed. However, the attenuator, which (PSD), of molecular ions after extraction kamp says. into the drift tube. regulates the amount of energy that He admits he's of the school that likes reaches the sample, should be adjustable. The latest development is the use of to be able to adjust everything, so his per- He recommends choosing an attenuator pulsed or delayed ion extraction, a refinethat allows adjustment in small incresonal preference is for the most flexible ment of Wiley and McLaren's 1955 experidesign possible. But high flexibility may ments of a few percent forfinecontrol of ments with "time lag focusing" to imsample exposure to the laser. not be for everyone, he cautions, even in prove mass resolution. At the ASMS Cona research-grade instrument. "What can ference, Vestec/PerSeptive Biosystems The standard sample platform is a happen is that users who don't know presented a prototype design using deprobe on which the sample-matrix mix500 A
High flexibility may not be for everyone, even in a researchgrade instrument
Analytical Chemistry, August 1, 1995
Just Published! layed pulsed extraction, and plans to offer this feature commercially. Cotter notes that his lab and others have used the technique for several years, but his group has also achieved comparable resolution using a narrow-pulse-width laser (600 ps) without pulsed extraction. "It may be that pulsed extraction compensates for the longer laser pulse on an N2 or Nd:YAG laser and therefore provides time rather than energy focusing." TOF configurations
A linear TOF analyzer includes a long field-free drift tube and either a microchannel plate detector (MCD) or a secondary electron multiplier (SEM) detector. MCDs are more commonly used but are more likely to be saturated when the ion current is high. Some TOF designs also include ion optics such as Einzel lenses or a wire ion guide with an applied rf potential that extends down the length of the drift tube to keep the ions focused. A reflectron acts as an ion mirror at the end of the drift tube, focusing the energy of ions with the same mass. It consists of a series of grids and ring electrodes (gridless reflectrons are also in use), generally with linearly increasing voltages. Ions are reflected either at an angle after penetrating the reflectron or, in newer coaxial designs, back along the flight path for detection. "Single-stage reflectrons are simpler and more common, but dualstage reflectrons provide second-order focusing and are physically smaller, which allows for a more compact design," says Cotter. Quadratic mirror reflector designs, for which the flight time depends on mass alone and not on kinetic energy, have been proposed but are difficult to construct in practice. "Research-grade instruments should allow both a linear mode [with reflectron voltage at zero] and a reflector mode. A fast and easy software-controlled toggle between the two should be possible," says Hillenkamp. One detector, placed behind the reflectron in line with the first drift tube, records ions in linear mode and neutrals when the reflectron voltage is on. The neutral fragment flight times are indicative of the precursor ion flight times. A second detector is placed in the ion beam in the postreflectron drift region (or for coaxial instruments, back up near the ion extractor) to detect the refocused ions. Reflectrons also allow MS/MS-like structural characterization of analytes through the detection of product ions from PSD. "Depending on who you talk to, you
may hear that PSD is the most important MALDI application, which I think is exaggerated, but it is certainly important," Hillenkamp adds. Cotter notes that although some biochemists may find PSD analysis more important than mass resolution, there are trade-offs. "In current designs PSD spectra must be acquired in increments by stepping the reflectron voltage," he explains, "because for both single- and dualstage reflectrons, focusing is optimized over a limited mass region for a given voltage. Although these spectral segments can be 'stitched' together to assemble the final PSD spectrum, one loses the important multichannel advantage of TOF." Cotter's laboratory has developed a "curvedfield" reflectron that focuses most of the PSD mass range at full reflectron voltage to provide what he calls "seamless" spectral acquisition for PSD. Shimadzu has licensed this feature for its latest model in the Kompact series.
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TOF instruments are the most widely available for MALDI, but they have lower mass resolution than FTMS and sector mass analyzers. MALDI-FTMS instruments for high mass resolution and accuracy are less common but are available from companies such as Extrel FTMS, Bruker, and lonSpec. Quadrupole ion trap systems are being explored at the R&D level. Fisons offers a MALDI sector-TOF hybrid system, and JEOL USA has recently introduced a MALDI kit with an integrating scanning CCD array detector for sector MS. New instruments offer both electrospray ionization and MALDI. Hillenkamp foresees greater emphasis on PSD techniques, a wider variety of matrices, and an expansion into IR methods as cheaper lasers become available. Cotter's vision of the future includes true MS/MS with collision-induced dissociation for sequencing unfractionable mixtures of peptides, massively parallel DNA sequencing, and diagnostic instruments for clinical screening. Both see a growing trend in simplified, dedicated instruments for routine use. Until these developments bear fruit, Hillenkamp's advice for buying a MALDI instrument is fairly simple: "If I were to buy a MALDI instrument now I would take a bag of samples as they typically appear in my lab, travel to each of the manufacturers or their representatives, and have them analyze the samples in my presence." Deborah Noble
384 pages (1994) Clothbound: ISBN 0-8412-2983-X $39.95 Paperbound: ISBN 0-8412-2984-8 $24.95
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Analytical Chemistry, August 1, 1995 501 A
