Mass spectrometry

From Freepedia

Mass spectrometry is a technique for separating ions by their mass-to-charge (m/z) ratios. It is used to find the composition of a physical sample. It has two broad applications:

  1. identifying compounds by the mass of one or more elements in a compound
  2. determining the isotopic composition of one or more elements in a compound

A mass spectrometer is a device used for mass spectrometry, and produces a mass spectrum of a sample to find its composition. This is normally achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. A typical mass spectrometer comprises three parts: an ion source, a mass analyzer, and a detector.

Contents

How it works: in layman terms

Different molecules have different masses, and this fact is used in a mass spectrometer to determine what molecules are present in a sample. For example, table salt (NaCl), is vaporized (turned into gas) and broken down (ionized) into electrically charged particles, called ions, in the first part of the mass spectometer. The sodium ions and chloride ions have specific molecular weights. They also have a charge, which means that they will be moved under the influence of an electric field. These ions are then sent into an ion acceleration chamber and passed through a slit in a metal sheet. A magnetic field is applied to the chamber, which pulls on each ion equally and deflects them (makes them curve instead of travelling straight) onto a detector. The lighter ions deflect further than the heavy ions because the force on each ion is equal but their masses are not (this is derived from the equation <math>F=ma</math> which states that if the force remains the same, the mass and acceleration are inversely proportional). The detector measures exactly how far each ion has been deflected, and from this measurement, the ion's 'mass to charge ratio' can be worked out. From this information it is possible to determine with a high level of certainty what the chemical composition of the original sample was.

This example was of a sector instrument, however there are many types of mass spectrometers that not only analyze the ions differently but produce different types of ions; however they all use electric and magnetic fields to change the path of ions in some way.

Instrumentation

Ion source

The ion source is the part of the mass spectrometer that ionizes the material under analysis (the analyte). The ions are then transported by magnetic or electrical fields to the mass analyzer.

Techniques for ionization have been key to determining what types of samples can be analyzed by mass spectrometry. Electron ionization and chemical ionization are used for gases and vapors. Two techniques often used with liquid and solid biological samples include electrospray ionization (due to John Fenn) and matrix-assisted laser desorption/ionization (MALDI, due to M. Karas and F. Hillenkamp). Inductively coupled plasma sources are used primarily for metal analysis on a wide array of samples types. Others include fast atom bombardment (FAB), thermospray, atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS) and thermal ionisation.

Mass analyzer

The mass analyzer is the most flexible part of the mass spectrometer. It uses an electric and/or magnetic field to affect the path and/or velocity of the charged particles in some way. The force exerted by electric and magnetic fields are defined by the Lorentz force law:

<math>\mathbf{F} 
 = q (\mathbf{E} + \mathbf{v} \times \mathbf{B}),</math>

where E is the electric field strength, B is the magnetic field induction, q is the charge of the particle, v is its current velocity (expressed as a vector), and × is the cross product. All mass analyzers use the Lorentz forces in some way either statically or dynamically in mass-to-charge determination.

Besides the original magnetic-sector analyzers, several other types of analyzer are now more common, including time-of-flight, quadrupole ion trap, quadrupole and Fourier transform ion cyclotron resonance mass analyzers. In addition, there are many more experimental mass analyzers and exotic combinations of analyzers.

As shown above, sector instruments change the direction ions are flying through the mass analyzer. The ions enter a magnetic or electric field which bends the ion paths depending on their mass-to-charge ratios (m/z), deflecting the more charged and faster-moving, lighter ions more. The ions eventually reach the detector and their relative abundances are measured. The analyzer can used to select a narrow range of m/z's or to scan through a range of m/z's to catalog the ions present.

Perhaps the easiest to understand is the Time-of-flight (TOF) analyzer which is typically integrated with MALDI ion sources. It boosts ions to the same kinetic energy by passage through an electric field and measures the times they take to reach the detector. Although the kinetic energy is the same, the velocity is different so the lighter more highly charged ion will reach the detector first.

Quadrupole mass analyzers and quadrupole ion traps [QIT] use oscillating electrical fields to selectively stabilize or destabilize ions falling within a narrow window of m/z values.

Fourier transform mass spectrometry measures mass by detecting the image current produced by ions cyclotroning in the presence of a magnetic field.

The best mass analyzer for an experiment depends upon the type of information the experimenter wants to learn.

Detector

The final element of the mass spectrometer is the detector. The detector records the charge induced or current produced when an ion passes by or hits a surface. In a scanning instrument the signal produced in the detector during the course of the scan versus where the instrument is in the scan (at what m/z) will produce a mass spectrum, a record of how many ions of each m/z are present.

Typically, some types of electron multiplier is used, though other detectors (such as Faraday cups) have been used. Because the number of ions leaving the mass analyzer at a particular instant is typically quite small, significant amplification is often necessary to get a signal. Microchannel Plate Detectors are commonly used in modern commercial instruments. In FTMS, the detector consists of a pair of metal plates within the mass analyzer region which the ions only pass near. No DC current is produced, only a weak AC image current is produced in a circuit between the plates.

Kinds of MS

Gas chromatography/MS

see also the main article on Gas chromatography-mass spectrometry

A common form of mass spectrometry is gas chromatography-mass spectrometry (GC/MS or GC-MS). In this technique, a gas chromatograph is used to separate compounds. This stream of separated compounds is fed on-line into the ion source, a metallic filament to which voltage is applied. This filament emits electrons which ionize the compounds. The ions can then further fragment, yielding predictable patterns. Intact ions and fragments pass into the mass spectrometer's analyser and are eventually detected.

Liquid chromatography/MS

see also the main article on Liquid chromatography-mass spectrometry

Similar to gas chromatography MS (GC/MS), liquid chromatography mass spectrometry (LC/MS or LC-MS) separates compounds chromatographically before they are introduced to the ion source and mass spectrometer. It differs from GC/MS in that the mobile phase is liquid, usually a combination of water and organic solvents, instead of gas. Most commonly, an electrospray ionization source is used in LC/MS.

MS for large molecules

For large molecules typical of biological applications, special techniques are used. The ion source subjects a sample of material to an Electric charge that causes the material to be ionized. Types of ion sources include electrospray ionization (ESI), chemical ionization (CI), fast atom bombardment (FAB), matrix-assisted laser desorption ionization (MALDI), Thermal ionisation (TI), Secondary ionisation (SI), and inductively coupled plasma ICP-MS.

Chemical ionization MS

In chemical ionization MS, the analyte is ionized by chemical ion-molecule reactions during collisions in the source.

One form of chemical ionization is atmospheric pressure chemical Ionization (APCI) which allows for the high flow rates typical of HPLC to be used directly, often without diverting the larger fraction of volume to waste. Typically the mobile phase containing eluting analyte is heated above 400 degrees Celsius, sprayed with high flow rates of nitrogen and the entire aerosol cloud is subjected to a corona discharge that creates ions. Often APCI can be performed in a modified ESI source.

Several techniques use ions created in a dedicated ion source injected into a flow tube or a drift tube: selected ion flow tube (SIFT-MS), and proton transfer reaction (PTR-MS), are variants of CI dedicated for trace gas analysis of air, breath or liquid headspace using well defined reaction time allowing calculations of analyte concentrations from the known reaction kinetics without the need for internal standard or calibration.

Tandem MS

Tandem mass spectrometry involves multiple steps of mass selection or analysis, usually separated by some form of fragmentation. A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry. For example, one mass analyzer can isolate one peptide from many entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyzer then catalogs the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD).

Isotope ratio MS

Mass spectrometry is also used to determine the isotopic composition of elements within a sample. Differences in mass among isotopes of an element are very small, and the less abundant isotopes of an element are typically very rare, so a very sensitive instrument is required. These instruments are called isotope ratio mass spectrometers (IR-MS) and usually use a single magnet to bend a beam of ionized particles towards a series of Faraday cups which convert particle impacts to electric current. A fast on-line analysis of deuterium content of water can be done using Flowing afterglow mass spectrometry, FA-MS.

Fourier tranform mass spectrometry

see also the main article on Fourier transform ion cyclotron resonance

Instead of measuring the deflection of ions with a detector such as a electron multiplier, the ions are injected into a Penning trap (a static electric/magnetic ion trap) where they effectively form part of a circuit. Detectors at fixed positions in space measure the electrical signal of ions which pass near them over time producing cyclical signal. Since the frequency of the ions' cycling is determined by its mass to charge ratio, this can be deconvoluted by performing a Fourier transform on the signal. FTMS has the advantage of improved sensitivity (since each ion is 'counted' more than once) as well as much higher resolution and thus precision.

Quadrupole ion trap mass spectrometry

see also the main article on quadrupole ion trap mass spectrometer

Ions are created and trapped in a mainly quadrupole RF potential and separated by mass, non-destructively or destructively. There are many mass/charge separation and isolation methods but most commonly used is the mass instability mode in which the RF potential is ramped so that the orbit of ions with a mass <math>a>b</math> are stable while ions with mass b become unstable and are ejected on the z-axis onto a detector. The cylindrical ion trap mass spectrometer is a derivative of the quadrupole ion trap mass spectrometer.

Mass spectrometry of proteins

Mass spectrometry is an important emerging method for the characterization of proteins. As indicated in the section MS for large molecules, the two primary methods for ionization are electrospray ionization and matrix-assisted laser desorption ionization (MALDI). In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyser. In the second, proteins are enzymatically digested into smaller peptides using an agent such as trypsin or pepsin. Other proteolytic digest agents are also used. The collection of peptide products are then introduced to the mass analyser. This is often refered to a the "bottom-up" approach of protein analysis.

Whole protein mass analysis is primarily conducted using either time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance. These two types of instrument are preferable here because of their wide mass range, and in the case of FT-ICR, its high mass accuracy. Mass analysis of proteolytic peptides is a much more popular method of protein characterization, as cheaper instrument designs can be used for characterization. Additionally, sample preparation is easier once whole proteins have been digested into smaller peptide fragments. The most widely used instrument for peptide mass analysis is the quadrupole ion trap. Multiple stage quadrupole-time-of-flight and MALDI time-of-flight instruments also find use in this application.

Protein and peptide fractionation coupled with mass spectrometry

Proteins of interest to biological researchers are usually part of a very complex mixture of other proteins and molecules that co-exist in the biological medium. This presents two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture contains roughly equal amounts of constituents, while in biological samples, different proteins tend to be present in widely differing amounts. If such a mixture is ionized using electrospray or MALDI, the more abundant species have a tendency to "drown" signals from less abundant ones. The second problem is that the mass spectrum from a complex mixture is very difficult to interpret due to the overwhelming number of mixture components. This is exacerbated by the fact that enzymatic digestion of a protein gives rise to a large number of peptide products.

To contend with this problem, two methods are widely used to fractionate proteins, or their peptide products from an enzymatic digestion. The first method fractionates whole proteins and is called two-dimensional gel electrophoresis. The second method, high performance liquid chromatography is used to fractionate peptides after enzymatic digestion. In some situations, it may be necessary to combine both of these techniques.

Gel spots identified on a 2D Gel are usually attributable to one protein. If the identity of the protein is desired, the gel spot can be excised, and digested proteolytically. The peptide masses resulting from the digestion can be determined by mass spectrometry using peptide mass fingerprinting. If this information does not allow unequivocal identification of the protein, its peptides can be subject to tandem mass spectrometry.

Characterization of protein mixtures using HPLC/MS is also called shotgun proteomics and mudpit. A peptide mixture that results from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography. The eluent from the chromatography stage can be either directly introduced to the mass spectrometer through electrospray ionization, or laid down on a series of small spots for later mass analysis using MALDI.

Protein identification

There are two main ways MS is used to identify proteins. Peptide mass fingerprinting (mentioned in the previous section) uses the masses of proteolytic peptides as input to a search of a database of predicted masses that would arise from digestion of a list of known proteins. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample.

Tandem MS is becoming a more popular experimental method for identifying proteins. Collision-induced dissociation is used in mainstream applications to generate a set of fragments from a specific peptide ion. The fragmentation process primarily gives rise to cleavage products that break along peptide bonds. Because of this simplicity in fragmentation, it is possible to use the observed fragment masses to match with a database of predicted masses for one of many given peptide sequences. Tandem MS of whole protein ions has been investigated recently using electron capture dissociation and has demonstrated extensive sequence information in principle but is not in common practice. This is sometimes refered to as the "top-down" approach in that it involves starting with the whole mass and then pulling it apart rather than starting with pieces (proteolytic fragments) and piecing the protein back together (bottom-up).

History

The first mass spectrography technique was described in an 1899 article by English scientist J.J. Thomson. The processes that more directly gave rise to the modern version were devised by Arthur Jeffrey Dempster and F.W. Aston in 1918 and 1919 respectively.

In 2002, John Fenn received the Nobel Prize in Chemistry for electrospray ionization. The same year Koichi Tanaka received the Nobel Prize in Chemistry for macromolecule ionization by laser irradiation. However, matrix-assisted laser desorption/ionization (MALDI) was created by M. Karas and F. Hillenkamp.

See also

External links

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