Tandem mass spectrometry
- 1 Tandem MS instruments
- 2 Notation
- 3 Tandem MS experiments
- 4 Fragmentation in tandem mass spectrometry
- 5 Peptide fragmentation
- 6 Oligosaccharide fragmentation
- 7 Oligonucleotide fragmentation
- 8 Newborn screening
- 9 See also
- 10 References
- 11 External links
- 12 Bibliography
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. These elements can be sectors, transmission quadrupole, or time-of-flight. When using multiple quadrupoles, they can act as both mass analyzers and collision chambers.
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 or FTMS 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.
For tandem mass spectrometry in space, the different elements are often noted in shorthand.
- Q – Quadrupole mass analyzer
- q – Radio frequency collision quadrupole
- TOF – Time-of-flight mass analyzer
- B – Magnetic sector
- E – Electric sector
The notation can be combined to indicate various hybrid instrument, for example
- QqQ – Triple quadrupole mass spectrometer
- 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.
Tandem MS modes
There are four main scan experiments possible using MS/MS:
- product 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.
- precursor 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.
Often, the ionization process is sufficiently violent to leave the resulting ions with sufficient internal energy 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 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 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 (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.
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:
where represents the photon absorbed by the ion. Ultraviolet lasers can be used, but can lead to excessive fragmentation of biomolecules. Infrared photons will heat the ions and cause dissociation if enough of them are absorbed. This process is called infrared multiphoton dissociation (IRMPD) and is often accomplished with a carbon dioxide laser and an ion trapping mass spectrometer such as a FTMS. Blackbody radiation can also be used in a technique known as blackbody infrared radiative dissociation (BIRD). In the BIRD method, the entire mass spectrometer vacuum chamber is heated to create infrared radiation.
A peptide sequence tag 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 high energy dissociation conditions. These include the side chain loss ions d, v, w and immonium ions and additional sequence-specific fragment ions associated with particular amino acid residues.
Oligosaccharides may be sequenced using tandem mass spectrometry in a similar manner to peptide sequencing. Fragmentation generally occurs on either side of the glycosidic bond (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.
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Newborn screening is the process of testing newborn babies for treatable genetic, endocrinologic, metabolic and hematologic 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.
- Accelerator mass spectrometry
- Bottom-up proteomics
- Charge remote fragmentation
- Cross section (physics)
- Hybrid mass spectrometer
- Mass-analyzed ion kinetic energy spectrometry
- Shotgun proteomics
- Top-down proteomics
- Triple quadrupole mass spectrometer
- Unimolecular ion decomposition
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