MALDI-TOF-MS

(Bob Sinclair.  The Scientist , 13(12), 1999 )

Laser Desorption Mass Spectrometers for Multisample Analysis

Matrix-assisted laser desorption/ionizationtime 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, post-translational 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 ~100200 ppm. Although much of the technology has improved in the last decade, the search for the perfect mass analyzer still provides an excellent survey of many different strategies. There are essentially two types of ion detectors: electron multipliers and microchannel plates (MCP). 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.

 

Moving up the Mass Scale

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 (510 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 colleagues¹   The more common MALDI method using an organic photoactive compound was published in 1988 by Michael Karas and Franz Hillenkamp² and has been more recently reviewed by Ronald Beavis and Brian Chait.³

 

In MALDI, the protein is embedded in a solid medium by co-crystallization 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. 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.

 

Getting TOF with Ions

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). 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.

 

Linear and Reflectron Instruments

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 co-crystal 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^4,5  in which newly formed ions are held close to the surface of the protein-matrix co-crystal with a low voltage (generally 1 keV or so) pulse before applying the main acceleration pulse (generally 2030 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.

 

1. K. Tanaka et al., Shimadzu Corp., Kyoto, Japan, "Proceedings of the 2nd Japan-China Joint Symposium on Mass Spectrometry," 185, 1987.

2. M. Karas, F. Hillenkamp, "Laser desorption of proteins with molecular masses exceeding 10,000 Daltons," Analytical Chemistry