Tandem mass spectrometry
Encyclopedia
Tandem mass spectrometry, also known as MS/MS or MS2, involves multiple steps of mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...

 selection, with some form of fragmentation occurring in between the stages.

Tandem MS instruments

Multiple stages of mass analysis separation can be accomplished with individual mass spectrometer elements separated in space or using a single mass spectrometer with the MS steps separated in time.

Tandem in space

In tandem mass spectrometry in space, the separation elements are physically separated and distinct, although there is a physical connection between the elements to maintain high vacuum
Vacuum
In everyday usage, vacuum is a volume of space that is essentially empty of matter, such that its gaseous pressure is much less than atmospheric pressure. The word comes from the Latin term for "empty". A perfect vacuum would be one with no particles in it at all, which is impossible to achieve in...

. These elements can be sectors, transmission quadrupole
Quadrupole mass analyzer
The quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry. As the name implies, it consists of 4 circular rods, set parallel to each other. In a quadrupole mass spectrometer the quadrupole is the component of the instrument responsible for filtering sample ions, based on...

, or time-of-flight
Time-of-flight mass spectrometry
Time-of-flight mass spectrometry is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined via a time measurement. Ions are accelerated by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has...

.

Tandem in time

By doing tandem mass spectrometry in time, the separation is accomplished with ions trapped in the same place, with multiple separation steps taking place over time. A quadrupole ion trap
Quadrupole ion trap
A quadrupole ion trap exists in both linear and 3D varieties and refers to an ion trap that uses constant DC and radio frequency oscillating AC electric fields to trap ions. It is commonly used as a component of a mass spectrometer...

 or FTMS
Fourier transform ion cyclotron resonance
Fourier transform ion cyclotron resonance mass spectrometry, also known as Fourier transform mass spectrometry, is a type of mass analyzer for determining the mass-to-charge ratio of ions based on the cyclotron frequency of the ions in a fixed magnetic field...

 instrument can be used for such an analysis. Trapping instruments can perform multiple steps of analysis, which is sometimes referred to as MSn (MS to the n). Often the number of steps, n, is not indicated, but occasionally the value is specified; for example MS3 indicates three stages of separation.

Notation

For tandem mass spectrometry in space, the different elements are often noted in shorthand.
QQuadrupole mass analyzer
Quadrupole mass analyzer
The quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry. As the name implies, it consists of 4 circular rods, set parallel to each other. In a quadrupole mass spectrometer the quadrupole is the component of the instrument responsible for filtering sample ions, based on...

qRadio frequency
Radio frequency
Radio frequency is a rate of oscillation in the range of about 3 kHz to 300 GHz, which corresponds to the frequency of radio waves, and the alternating currents which carry radio signals...

 collision quadrupole
TOFTime-of-flight
Time-of-flight mass spectrometry
Time-of-flight mass spectrometry is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined via a time measurement. Ions are accelerated by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has...

 mass analyzer
B – Magnetic sector
E – Electric sector


The notation can be combined to indicate various hybrid instrument, for example
QqQTriple quadrupole mass spectrometer
Triple quadrupole mass spectrometer
A triple quadrupole mass spectrometer is a tandem mass spectrometer consisting of two quadrupole mass spectrometers in series, with a radio frequency only quadrupole between them to act as a collision cell for collision-induced dissociation...

QTOF – Quadrupole time-of-flight mass spectrometer (also QqTOF)
BEBE – Four-sector (reverse geometry) mass spectrometer

Tandem MS experiments

There are a number of different tandem MS experiments and all have their own applications and offer their own information. An instrument equipped for tandem MS can still be used to run MS experiments. Tandem MS can be done in either time or space. Tandem MS in space involves the physical separation of the instrument components (QqQ or QTOF), tandem MS in time involves the use of an ion trap
Ion trap
An ion trap is a combination of electric or magnetic fields that captures ions in a region of a vacuum system or tube. Ion traps have a number of scientific uses such as mass spectrometery and trapping ions while the ion's quantum state is manipulated...

.

Tandem MS modes

There are four main scan experiments possible using MS/MS.
  • Precursor ion scan
    • The product ion is selected in the second mass analyzer, and the precursor masses are scanned in the first mass analyzer. A precursor ion scan cannot be done with time based MS instruments. Note that precursor ion is synonymous with parent ion and product ion with daughter ion; however the use of these anthropomorphic terms is discouraged.

  • Product ion scan
    • A precursor ion is selected in the first stage, allowed to fragment and then all resultant masses are scanned in the second mass analyzer and detected in the detector that is positioned after the second mass analyzer. This experiment is commonly performed to identify transitions used for quantification by tandem MS.

  • Neutral loss scan
    • The first mass analyzer scans all the masses. The second mass analyzer also scans, but at a set offset from the first mass analyzer. This offset corresponds to a neutral loss that is commonly observed for the class of compounds. Neutral loss scans cannot be done with time based MS instruments. In a constant-neutral-loss scan, all precursors that undergo the loss of a specified common neutral are monitored. To obtain this information, both mass analyzers are scanned simultaneously, but with a mass offset that correlates with the mass of the specified neutral. Similar to the precursor-ion scan, this technique is also useful in the selective identification of closely related class of compounds in a mixture.

  • Selected reaction monitoring
    • Both mass analyzers are set to a selected mass. This mode is analogous to selected ion monitoring for MS experiments. A very selective analysis mode, which can increase sensitivity.

Fragmentation in tandem mass spectrometry

Fragmentation of gas-phase ions is essential to tandem mass spectrometry and occurs between different stages of mass analysis. There are many methods used to fragment the ions and these can result in different types of fragmentation and thus different information about the structure and composition of the molecule.

In-source fragmentation

Often, the ionization
Ionization
Ionization is the process of converting an atom or molecule into an ion by adding or removing charged particles such as electrons or other ions. This is often confused with dissociation. A substance may dissociate without necessarily producing ions. As an example, the molecules of table sugar...

 process is sufficiently violent to leave the resulting ions with sufficient internal energy
Internal energy
In thermodynamics, the internal energy is the total energy contained by a thermodynamic system. It is the energy needed to create the system, but excludes the energy to displace the system's surroundings, any energy associated with a move as a whole, or due to external force fields. Internal...

 to fragment within the mass spectrometer. If the product ions persist in their non-equilibrium state for a moderate amount of time before auto-dissociation this process is called metastable fragmentation. Nozzle-skimmer fragmentation refers to the purposeful induction of in-source fragmentation by increasing the nozzle-skimmer potential on usually electrospray
Electrospray ionization
Electrospray ionization is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized...

 based instruments. Although in-source fragmentation allows for fragmentation analysis, it is not technically tandem mass spectrometry unless metastable ions are mass analyzed or selected before auto-dissociation and a second stage of analysis is performed on the resulting fragments. In-source fragmentation is often used in addition to tandem mass spectrometry (with post-source fragmentation) to allow for two steps of fragmentation in a pseudo MS3-type of experiment.

Post-source fragmentation

Post-source fragmentation is most often what is being used in a tandem mass spectrometry experiment. Energy can also be added to the ions, which are usually already vibrationally excited, through post-source collisions with neutral atoms or molecules, the absorption of radiation, or the transfer or capture of an electron by a multiply charged ion. Collision-induced dissociation
Collision-induced dissociation
In Mass spectrometry, Collision-induced dissociation , referred to by some as collisionally activated dissociation , is a mechanism by which to fragment molecular ions in the gas phase. The molecular ions are usually accelerated by some electrical potential to high kinetic energy and then allowed...

 (CID), also called collisionally activated dissociation (CAD), involves the collision of an ion with a neutral atom or molecule in the gas phase and subsequent dissociation of the ion. For example, consider

where the ion collides with the neutral species M and subsequently breaks apart. The details of this process are described by collision theory
Collision theory
Collision theory is a theory proposed by Max Trautz and William Lewis in 1916 and 1918, that qualitatively explains how chemical reactions occur and why reaction rates differ for different reactions. For a reaction to occur the reactant particles must collide. Only a certain fraction of the total...

.

If an electron
Electron
The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...

 is added to a multiply charged positive ion, the Coulomb energy
Coulomb's law
Coulomb's law or Coulomb's inverse-square law, is a law of physics describing the electrostatic interaction between electrically charged particles. It was first published in 1785 by French physicist Charles Augustin de Coulomb and was essential to the development of the theory of electromagnetism...

 is liberated. Adding a free electron is called electron capture dissociation
Electron capture dissociation
Electron-capture dissociation is a method of fragmenting gas phase ions for tandem mass spectrometric analysis . ECD involves the direct introduction of low energy electrons to trapped gas phase ions...

 (ECD), and is represented by


for a multiply protonated molecule M. Adding an electron through an ion-ion reaction is called electron transfer dissociation
Electron transfer dissociation
Electron-transfer dissociation is a method of fragmenting ions in a mass spectrometer. Similar to electron-capture dissociation, ETD induces fragmentation of cations by transferring electrons to them. It was invented by Donald F. Hunt, Joshua Coon, John E. P...

 (ETD), and is represented by


Such fragmentation can also occur with a deprotonated species, in which an electron is transferred from the specie to an cationic reagent in a negative electron transfer dissociation (NETD) event:


The energy required for dissociation can be added by photon
Photon
In physics, a photon is an elementary particle, the quantum of the electromagnetic interaction and the basic unit of light and all other forms of electromagnetic radiation. It is also the force carrier for the electromagnetic force...

 absorption, resulting in ion photodissociation
Photodissociation
Photodissociation, photolysis, or photodecomposition is a chemical reaction in which a chemical compound is broken down by photons. It is defined as the interaction of one or more photons with one target molecule....

 and represented by


where represents the photon absorbed by the ion. Ultraviolet lasers can be used, but can lead to excessive fragmentation of biomolecules. Infrared
Infrared
Infrared light is electromagnetic radiation with a wavelength longer than that of visible light, measured from the nominal edge of visible red light at 0.74 micrometres , and extending conventionally to 300 µm...

 photons will heat the ions and cause dissociation if enough of them are absorbed. This process is called infrared multiphoton dissociation
Infrared multiphoton dissociation
Infrared multiphoton dissociation is a technique used in mass spectrometry to fragment molecules in the gas phase usually for structural analysis of the original molecule. -How it works:...

 (IRMPD) and is often accomplished with a carbon dioxide laser
Carbon dioxide laser
The carbon dioxide laser was one of the earliest gas lasers to be developed , and is still one of the most useful. Carbon dioxide lasers are the highest-power continuous wave lasers that are currently available...

 and an ion trapping mass spectrometer such as a FTMS
Fourier transform ion cyclotron resonance
Fourier transform ion cyclotron resonance mass spectrometry, also known as Fourier transform mass spectrometry, is a type of mass analyzer for determining the mass-to-charge ratio of ions based on the cyclotron frequency of the ions in a fixed magnetic field...

. Blackbody radiation can also be used in a technique known as blackbody infrared radiative dissociation
Blackbody infrared radiative dissociation
In mass spectrometry blackbody infrared radiative dissociation is a method for fragmenting gas phase ions for tandem mass spectrometric analysis . BIRD uses the light from black body radiation to thermally excite the ions until a bond breaks...

 (BIRD). In the BIRD method, the entire mass spectrometer vacuum chamber is heated to create infrared radiation.

With surface-induced dissociation (SID), the fragmentation is a result of the collision of an ion with a surface under high vacuum.

Peptide fragmentation

A peptide sequence tag
Peptide sequence tag
A peptide sequence tag is a piece of information about a peptide obtained by tandem mass spectrometry that can be used to identify this peptide in a protein database.-Mass spectrometry:...

 obtained by tandem mass spectrometry can be used to identify a peptide in a protein database. A notation has been developed for indicating peptide fragments that arise from a tandem mass spectrum. Peptide fragment ions are indicated by a, b, or c if the charge is retained on the N-terminus and by x, y or z if the charge is maintained on the C-terminus. The subscript indicates the number of amino acid residues in the fragment. Superscripts are sometimes used to indicate neutral losses in addition to the backbone fragmentation, * for loss of ammonia and ° for loss of water. Although peptide backbone cleavage is the most useful for sequencing and peptide identification other fragment ions may be observed under certain conditions. These include the side chain loss ions d, v, w and immonium ions.

Oligosaccharide fragmentation

Oligosaccharide
Oligosaccharide
An oligosaccharide is a saccharide polymer containing a small number of component sugars, also known as simple sugars...

s may be sequenced using tandem mass spectrometry in a similar manner to peptide sequencing. Fragmentation generally occurs on either side of the glycosidic bond
Glycosidic bond
In chemistry, a glycosidic bond is a type of covalent bond that joins a carbohydrate molecule to another group, which may or may not be another carbohydrate....

 (b, c, y and z ions) but also under more energetic conditions through the sugar ring structure in a cross-ring cleavage (x ions). Again trailing subscripts are used to indicate position of the cleavage along the chain. For cross ring cleavage ions the nature of the cross ring cleavage is indicated by preceding superscripts.

Oligonucleotide fragmentation

A notation for gas-phase fragmentation of oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...

 ions has been proposed.

Newborn screening

Newborn screening is the process of testing newborn babies for treatable genetic
Genetic disorder
A genetic disorder is an illness caused by abnormalities in genes or chromosomes, especially a condition that is present from before birth. Most genetic disorders are quite rare and affect one person in every several thousands or millions....

, endocrinologic
Endocrinology
Endocrinology is a branch of biology and medicine dealing with the endocrine system, its diseases, and its specific secretions called hormones, the integration of developmental events such as proliferation, growth, and differentiation and the coordination of...

, metabolic
Inborn error of metabolism
Inborn errors of metabolism comprise a large class of genetic diseases involving disorders of metabolism. The majority are due to defects of single genes that code for enzymes that facilitate conversion of various substances into others...

 and hematologic
Hematology
Hematology, also spelled haematology , is the branch of biology physiology, internal medicine, pathology, clinical laboratory work, and pediatrics that is concerned with the study of blood, the blood-forming organs, and blood diseases...

 diseases. The development of tandem mass spectrometry screening in the early 1990s led to a large expansion of potentially detectable congenital metabolic diseases that affect blood levels of organic acids.

See also

  • Accelerator mass spectrometry
    Accelerator mass spectrometry
    Accelerator mass spectrometry differs from other forms of mass spectrometry in that it accelerates ions to extraordinarily high kinetic energies before mass analysis. The special strength of AMS among the mass spectrometric methods is its power to separate a rare isotope from an abundant...

  • Bottom-up proteomics
    Bottom-up proteomics
    Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry. The proteins may first be purified by a method such as gel electrophoresis...

  • Charge remote fragmentation
    Charge remote fragmentation
    Charge remote fragmentation is a type of covalent bond breaking that occurs in a gas phase ion in which the cleaved bond is not adjacent to the location of the charge. This fragmentation can be observed using tandem mass spectrometry....

  • Cross section (physics)
    Cross section (physics)
    A cross section is the effective area which governs the probability of some scattering or absorption event. Together with particle density and path length, it can be used to predict the total scattering probability via the Beer-Lambert law....

  • Hybrid mass spectrometer
    Hybrid mass spectrometer
    A hybrid mass spectrometer is a device for tandem mass spectrometry that consists of a combination of two or more m/z separation devices of different types.-Notation:...

  • Mass-analyzed ion kinetic energy spectrometry
    Mass-analyzed ion kinetic energy spectrometry
    Mass-analyzed ion-kinetic-energy spectrometry is a mass spectrometry technique by which mass spectra are obtained from a sector instrument that incorporates at least one magnetic sector plus one electric sector in reverse geometry...

  • Shotgun proteomics
    Shotgun proteomics
    Shotgun proteomics is a method of identifying proteins in complex mixtures using a combination of high performance liquid chromatography combined with mass spectrometry. The name is derived from shotgun sequencing of DNA which is itself named by analogy with the rapidly-expanding, quasi-random...

  • Top-down proteomics
    Top-down proteomics
    Top-down proteomics is a method of protein identification that uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry analysis. The name is derived from the similar approach to DNA seqencing...

  • Triple quadrupole mass spectrometer
    Triple quadrupole mass spectrometer
    A triple quadrupole mass spectrometer is a tandem mass spectrometer consisting of two quadrupole mass spectrometers in series, with a radio frequency only quadrupole between them to act as a collision cell for collision-induced dissociation...

  • Unimolecular ion decomposition
    Unimolecular ion decomposition
    Unimolecular ion decomposition is the fragmentation of a gas phase ion in a reaction with a molecularity of one. Ions with sufficient internal energy may fragment in a mass spectrometer, which in some cases may degrade the mass spectrometer performance, but in other cases, such as tandem mass...


External links

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