MALDI-TOF/TOF Mass Spectrometer

Perhaps no tool has been as instrumental to the proteomics revolution as the mass spectrometer. With the ability to deconvolute highly complex mixtures over a wide range of abundance levels, these machines enable researchers to identify and quantify proteins and to determine if and how those proteins have been post-translationally modified.The basic mass spectrometer measures an ion's mass-to-charge (m/z) ratio only.

By | April 12, 2004

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Perhaps no tool has been as instrumental to the proteomics revolution as the mass spectrometer. With the ability to deconvolute highly complex mixtures over a wide range of abundance levels, these machines enable researchers to identify and quantify proteins and to determine if and how those proteins have been post-translationally modified.

The basic mass spectrometer measures an ion's mass-to-charge (m/z) ratio only. This enables peptide mass fingerprinting, which is the identification of a protein based on the specific group of peptide masses it produces. But tandem devices, the so-called MS/MS instruments, can provide peptide sequence information as well. The key components shown on these pages demonstrate one of these instruments: the 4700 Proteomics Analyzer made by Applied Biosystems of Foster City, Calif., which features a MALDI (matrix-assisted laser desorption ionization) source and tandem time-of-flight (TOF/TOF) mass analyzers.

1 The process typically begins either with a protein spot extracted from a 1-D or 2-D protein gel, or with liquid chromatography fractions. The proteins are enzymatically treated (e.g., with trypsin), mixed with matrix (typically alpha-cyano-4-hydroxycinnamic acid or dihydrobenzoic acid), arrayed on a metal target plate, and allowed to dry. The plates enable high-throughput research, sometimes holding several hundred samples at once. Equipping the instrument with an autoloader increases the level of walk-away automation.

2 Once an X-Y translation stage has positioned a specific spot, an ultraviolet laser (a nitrogen laser firing at 337 nm or a Nd:YAG laser at 355 nm, for instance) strikes the sample. The matrix material absorbs this light energy, generating enough heat to vaporize and ionize the peptide sample, generally with a charge of +1.

3 As an ion races down the flight tube, its speed is a function of its m/z ratio. Hence, a particle's m/z ratio is measured based upon its time of flight.

4 In tandem MS (MS/MS) mode, the ions pass through a gauntlet of components that select specific ions for further analysis, slow them down, fragment them, and then re-accelerate them. (In standard MS mode, these four components are not used.) Of the two ions shown (blue and green) in the original sample, only the blue ions are selected for fragmentation, known as collision-induced dissociation (CID) analysis.

5 Mass resolution improves with the length of the flight path, so many time-of-flight mass spectrometers contain a reflectron, which functionally elongates the tube without physically doing so. The reflectron also serves to energy-focus ions, correcting their velocity spread to further improve resolution. For simple applications, however, users can forego the reflectron and use the linear detector instead.

6 The detectors measure each ion collision, producing a graph, or spectrum, of m/z versus intensity.

7 Comparing the resulting constellation of m/z values against a database identifies the peptide, and by extension, the protein from which it derives. Tandem MS/MS analysis of fragmented peptides produces an actual sequence, providing a more accurate protein ID. Today's mass spectrometers have high enough resolution and mass accuracy to distinguish peptides bearing only subtle differences, resulting in confident and thorough identifications of proteins from extremely complex mixtures.

Jeffrey M. Perkel can be contacted at jperkel@the-scientist.com.

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