MALDI-TOF Goes Mainstream: Laser Desorption Mass Spectrometers For Multisample Analysis

MALDI-TOF Table Micromass' laser-addressable sample array target for MALDI Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry has become, in recent years, a tool of choice for large-molecule analyses, especially for proteins. Published applications address protein and nucleic acid sequence, structure, purity, heterogeneity, cleavage, posttranslational modification, and a host of other molecular characteristics that are often difficult to study by other means. MALDI-TOF is also being used as a QC tool to verify peptide, protein, and DNA syntheses. With state-of-the-art machines claiming megadalton capabilities and femtomolar sensitivities, MALDI-TOF and related techniques seem set to play an increasingly important role in protein and proteome analysis. It's commonly said that a mass spectrometrist figures out what something is by smashing it to bits with a hammer and examining the individual pieces. The various types of mass spectrometry (MS) use different and specialized methods for smashing and ionizing the target molecules and for analyzing the results, but they are all essentially the same technique. The "hammer" is whatever energy source is selected to ionize the molecules of interest. There are lots of ways to ionize a molecule, including bombardment with an electron beam, high-energy ions, or a laser. Ionization charges some of the sample molecules, which can either remain intact or fragment into a variety of charged and neutral particles. The ions are accelerated by an electrostatic or magnetic field in the mass analyzer and separated by deflection or time of flight to the detector. Some mass analyzers can differentiate between oxygen at 15.999 Da and the similarly sized NH2 ion at 16.021 Da. Mass accuracy is generally cited in parts per million (ppm), and many systems claim mass accuracies of ~100-200 ppm. Although much of the technology has improved in the last decade, C. Brunee's 1987 search for the perfect mass analyzer still provides an excellent survey of many different strategies.1 There are essentially two types of ion detectors: electron multipliers and microchannel plates. While it might be argued that electron multipliers, which consist of several layers of charged dynodes, are more stable to high ion flux, the two technologies are both well suited to ion detection. The first widely available configurations for MS included an electron beam ionization source, a scanning quadrupole mass filter, and a multidynode ion detector and were suited primarily for analysis of smaller molecules. MS first became useful for protein research when fast atom bombardment (FAB) ionization sources were designed to smash larger molecules (including proteins and peptides up to ~10 kDa) into manageable pieces. Electron spray ionization (ESI) increased the protein mass range to ~100 kDa. Quadrupole and magnetic sector ESI MS became very valuable tools. For proteins, ESI MS has in many ways been superseded by MALDI as the hammer and by time-of-flight mass analyzer tubes as the detector. FAB uses a high-energy (5-10 keV) stream of inert gas particles to "ballistically ionize" the sample. It is limited by a relatively poor efficiency of target ionization and can lead to high backgrounds when the ionizing particles themselves break up, ionize, and impact the detector. ESI uses a high electric field to aerosolize a solution of the target analyte; the droplets subdivide until they contain a single analyte molecule that carries a residual charge. Often, ESI-produced ions carry multiple charges, which can be a benefit or a problem, depending on your instrument and application. Neither FAB nor ESI is suited to working with samples in bulk form or on a solid support. MALDI uses pulses of laser light to desorb the analyte from a solid phase directly to an ionized gaseous state. Pulsed lasers had been used to ionize proteins prior to 1988, but the technique was limited due to protein light absorption. A metal powder matrix for laser desorption and ionization of analytes was first presented in 1987 by Koichi Tanaka and colleagues.2 The more common MALDI method using an organic photoactive compound was published in 1988 by Michael Karas and Franz Hillenkamp3 and has been more recently reviewed by Ronald Beavis and Brian Chait.4 In MALDI, the protein is embedded in a solid medium by cocrystallization with a photoactive compound such as gentisic acid, 4-HCCA ( alpha-cyano-4-hydroxycinnamic acid), or dithranol. The matrix compound absorbs the light and uses the energy to eject and ionize the embedded protein molecules. As the protein does not fragment during desorption, MALDI is often referred to as being a "soft" ionization technique. The list of suitable matrix compounds for MALDI is extensive. Many are also available as precoated single-use MALDI target foils from Lab Connections (www.labconnections.com). Although other options are available, most MALDI techniques illuminate at about 20 mJ cm-2 using nitrogen lasers (337 nm) or Q-switched neodymium:yttrium-aluminum-garnet (Nd-YAG) lasers with frequency tripled to 354 nm or quadrupled to 266 nm. Longer wavelengths are favored for protein work because they are less readily absorbed. Magnetic sector and quadrupole mass spectrometers work by accelerating a stream of ionized sample along a vacuum tube toward an electrostatic or magnetic field that deflects or filters particles based on momentum or mass-to-charge ratio (m/z). A good review of MS detectors can be found on the JEOL Web site at www.jeol.com/ms/ms_analyzers.html. In TOF MS, the ionized analyte molecules and fragments are accelerated in an electrostatic field to a common kinetic energy. If all the ions have the same initial kinetic energy, lighter ions travel faster and heavier ions--with the same momentum--travel more slowly. The ionized particles enter at one end of the time-of-flight tube, (basically a long, empty tube for free flight), and the number of ions reaching a detector at the other end is recorded in a time-dependent manner. Assuming all the ions have the same electrical charge, the lightest ions reach the detector first and the heaviest arrive last. The entire mass spectrum is recorded in a fraction of a second as ion flux versus time. For TOF to work, the time at which the ions leave the source must be precisely controlled and defined. While MALDI ionization techniques have been coupled with quadrupole ion and magnetic sector mass analyzers, the commonest modern combination is with time-of-flight tubes, because the ionization event automatically provides the start pulse for the clock. The short duration of laser pulsing makes MALDI a particularly suitable match for TOF MS. Typically, flight-tube lengths are a couple of meters and flight times are ~100 ms--thousands of times longer than the nanosecond laser pulses. The mass range of a TOF instrument is generally limited by the detector technology employed. The high m/z ions end up traveling very slowly and are very poorly detected by conventional detectors. GSG Analytical Instruments' Future MALDI-TOF spectrometer extends the mass range of MALDI-TOF out to 1,000,000 Da with the help of a two-stage detector that captures these behemoths more effectively and a fast (1GHz) digitizer to increase resolution. The simplest TOF instruments have a linear configuration, with the detector placed at the end of the flight tube. All companies that offer MALDI-TOF instruments supply instruments with linear flight paths. During sample desorption and ionization, analyte particles can leave the surface of the protein-matrix cocrystal with a small but variable amount of kinetic energy in addition to the energy imparted by the acceleration process. This variable kinetic energy has the effect of "smearing" the mass-to-charge ratio of a specific analyte fragment over a small time range, decreasing the signal-to-noise ratio and broadening the analyte bands, but it can be largely eliminated in a couple of ways. The first is time lag focusing or delayed extraction,5,6 in which newly formed ions are held close to the surface of the protein-matrix cocrystal with a low voltage (generally 1 keV or so) pulse before applying the main acceleration pulse (generally 20-30 keV). Most instruments now incorporate this feature. The second way to focus an ion band is to change the TOF geometry by adding a reflectron to the end of the flight tube and moving the detector(s). A reflectron or "ion mirror" consists of a series of electrostatic and magnetic fields that collect and redirect the ions in a controlled manner. Ions with a given m/z slow down as they approach the reflectron mirror, focus into a tighter packet, and are then repelled either at an angle toward a detector at the end of a second stage of flight tube or backward along the same tube to a detector placed near the ion source. For many applications, reflectron-based TOF tubes give sharper signals by reducing the effects of initial kinetic energy differences. Because reflectrons effectively increase--almost double--the TOF free-flight path, they increase resolution and therefore improve mass accuracy. Reflectron technology also allows researchers to study molecular structure of ions via postsource decay, in which ionized fragments decompose further in the flight tube and the secondary products provide additional information about the structure of the original ion. The information gained from postsource decay detection is similar to that provided by tandem MS (MS/MS), where ions are intentionally refragmented after passage through a mass analyzer and the secondary fragmentation products are examined in a second mass analyzer. Most companies produce reflectron-based MALDI-TOF instruments in addition to their linear instruments. Comstock's RTOF-260 instrument, for example, is a reflectron-based version of its LTOF-160. PerSeptive Biosystems (a division of PE Biosystems) offers the Voyager DE™ workstation, and Micromass and Kore produce the TofSpec-2E and R-500 TOF MS, respectively. Bruker Daltonics offers two reflectron systems: the customizable REFLEX III system and the BIFLEX III system for high-end research. Kratos Analytical's KOMPACT™ Probe Kratos Analytical, a Shimadzu company, manufactures two reflectron-based instruments: the Kompact DISCOVERY and the Kompact SEQ. Kratos' reflectrons have a unique design incorporating a curved field rather than stepped or tiered linear fields. In normal ion reflection configurations, many of the postsource decay ions are "out of time-focus" and are therefore lost. Most instruments collect only about 10 percent of the range of postsource decay particles, necessitating repeated experiments at different collection points. The curved field allows collection of the entire range of postsource decay products from one laser pulse without rastering or scanning and eliminates the need to compile data from sequential experiments. A limitation in MALDI is the extensive and exacting sample preparation required. The protein and the matrix must be cocrystallized, a step that requires the protein and the matrix to be codissolved in the same solvent at some point. The single-use precoated target foils limit this requirement to some degree, but the protein must be prepared in a solvent that allows the matrix to dissolve locally as the protein is added. BioMolecular Instruments' Dynamo Multisample target formats are becoming more important to users, and many companies have started to offer them. For example, BioMolecular Instruments, a division of Thermo BioAnalysis, recently introduced the Dynamo, which is highly automated and incorporates a video camera in the ionization chamber for direct sample monitoring. The company has discontinued its Vision 2000 and Lasermat 2000 systems, though it will continue to support both instruments, because users appear to prefer the multisample capabilities of the newer system. The Bruker REFLEX III and BIFLEX III instruments both offer integration with Bruker's SCOUT 384 automated sampler. The SCOUT 384 uses standard microtiter plate formats and an X-Y positioner with 4 mm accuracy for unattended data acquisition from up to 1,536 samples. The limitations imposed by cocrystallization generally require the protein to be relatively pure prior to embedding it in the matrix. Ciphergen Biosystems' ProteinChips and SELDI (surface enhanced laser desorption/ionization) technology reverse the conventional MALDI sample preparation scheme by using an array of addressable protein binding sites on a solid substrate for the sample target. These binding sites are generally chemical or biochemical affinity ligands but could conceivably be any desired method to capture any molecule of choice. The chip is incubated with a crude--or purified to any level--preparation that contains the analyte molecules under investigation. Target analytes are bound by the affinity ligands on the chip. Residual material, including compounds such as DMF or DMSO that might interfere with conventional MALDI-TOF, are washed away. In theory, almost unlimited concentration of a dilute analyte can be achieved and capture specificity can be influenced by wash stringency. Finally, a molecule that absorbs laser energy--the SELDI equivalent of the MALDI matrix--is added to the entire chip. The analyte has been distributed homogeneously across the spot area. Although the process is not well understood and may involve cocrystallization at some scale, the fact that the analyte is bound to the chip indicates that it does not have to be in solution simultaneously with the energy-absorbing molecule. Finally, laser desorption/ionization, mass analysis, and detection of the entire chip on a spot-by-spot basis tells you what you captured. Another company, Intrinsic Bioprobes Intl. (www.inficad.com/~ibi), also sells immobilization chemistries for affinity-based sample selection prior to matrix preparation and MALDI-TOF analysis. MALDI-TOF is an important technique for measuring large molecular masses accurately and studying protein-ligand interactions. But MALDI-TOF also has some limitations: Most notably it is not well suited for quantitative analyses, and its resolving power and mass accuracy are actually quite limited for some applications such as sequencing. And although TOF per se can be paired with chromatography and capillary electrophoresis, MALDI still has not been interfaced successfully with these separation methods. Increased accuracy can be accomplished by a more sophisticated variant of MS called Fourier transform ion cyclotron resonance (FT-ICR), which involves "holding" the ion--even collecting ions from multiple pulses--in a magnetic chamber and analyzing them in a variety of ways. FT-ICR MS has been used by companies such as GSG and IonSpec to increase mass accuracy to sub-ppm levels for large molecules. For sequencing, this can narrow the range of possible sequences for a given signal from hundreds to just four or five. FT-ICR MS is compatible with a MALDI front end. Companies such as Sensar, Micromass, and PerSeptive have concluded that conventional ESI can be modified for compatibility with the short pulse requirements of TOF. Orthogonal acceleration ESI-TOF is one promising approach. In this technique, a continuous flow of ions (either from a static source or from a flowing system such as capillary electrophoresis) is gently accelerated in one direction, resulting in a densely packed but slowly moving analyte ion stream. A second acceleration mechanism that pulses at right angles to this ion stream pushes a well-defined packet of ions toward a detector, which can be almost any kind of mass analyzer, including TOF. Time-of-flight mass analysis, allowing accurate mass determinations of large molecules, is becoming an important part of protein science. Nanosecond-pulsed lasers, coupled with energy-absorbing matrix molecules that prevent excessive light absorption by the analyte, have facilitated the tightly defined ionization times needed for TOF to be an efficient process. MALDI-TOF will undoubtedly play an increasingly prominent role in many areas of large-molecule analysis, especially proteome analysis, but MALDI-FT-ICR MS and orthogonal acceleration ESI-TOF are probably also technologies to watch. C. Brunee, "The ideal mass analyzer: fact or fiction?" International Journal of Mass Spectrometry and Ion Processes, 76:125-237, 1987. K. Tanaka et al., Shimadzu Corp., Kyoto, Japan, "Proceedings of the 2nd Japan-China Joint Symposium on Mass Spectrometry," 185, 1987. M. Karas, F. Hillenkamp, "Laser desorption of proteins with molecular masses exceeding 10,000 Daltons," Analytical Chemistry, 60:2299, 1988. R.C. Beavis, B. Chait, "Matrix assisted laser desorption ionization mass-spectrometry of proteins," Methods in Enzymology, 270:519, 1996. W.C. Wiley, I.H. McLaren, Review of Scientific Instruments, 26:1150-7, 1955. B. Spengler, R.J. Cotter, "Ultraviolet laser desorption/ionization mass spectrometry of proteins above 100,000 Daltons by pulsed ion extraction time of flight analysis," Analytical Chemistry, 62:793-6, 1990. The author, can be contacted at bobsinclair@tech-write-edit.com.

MALDI-TOF Goes Mainstream: Laser Desorption Mass Spectrometers For Multisample Analysis

Interested in reading more?

Become a Member of