Selected reaction monitoring
Encyclopedia
Selected reaction monitoring (SRM) is a method used in tandem mass spectrometry
in which an ion
of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection.
by mass spectrometry
. Following ionization
in an electrospray
source
, a peptide
precursor is first isolated to obtain a substantial ion
population of mostly the intended species. This population is then fragmented
to yield product ions whose signal abundances are indicative of the abundance of the peptide in the sample. This experiment is primarily performed on triple quadrupole mass spectrometers, where mass-resolving Q1 isolates the precursor, q2 acts as a collision cell, and mass-resolving Q3 is cycled through the product ions which are detected upon exiting the last quadrupole
by an electron multiplier
. A precursor/product pair is often referred to as a transition. Much work goes into ensuring that transitions are selected that have maximum specificity. By spiking in heavy-labeled (e.g., D
, 13C
, or 15N) peptides to a complex matrix as concentration standards
, SRM can be used to construct a calibration curve
that can provide the absolute quantification (i.e., copy number per cell
) of the native, light peptide, and by extension, its parent protein
.
Tandem mass spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.-Tandem MS instruments:...
in which an ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection.
SRM in proteomics
SRM can be used for targeted quantitative proteomicsQuantitative proteomics
The aim of quantitative proteomics is to obtain quantitative information about all proteins in a sample. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about differences between samples. For example, this approach can be used...
by 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...
. Following 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...
in an 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...
source
Ion source
An ion source is an electro-magnetic device that is used to create charged particles. These are used primarily to form ions for mass spectrometers, optical emission spectrometers, particle accelerators, ion implanters and ion engines.- Electron ionization :...
, a peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
precursor is first isolated to obtain a substantial ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
population of mostly the intended species. This population is then fragmented
Fragmentation (chemistry)
Fragmentation is a type of chemical dissociation. Fragmentation of a molecule can take place by a process of heterolysis or homolysis.It is a phenomenon observed in mass spectrometry where it is used as a tool to find the structural formula of a molecule, process called structural elucidation.It...
to yield product ions whose signal abundances are indicative of the abundance of the peptide in the sample. This experiment is primarily performed on triple quadrupole mass spectrometers, where mass-resolving Q1 isolates the precursor, q2 acts as a collision cell, and mass-resolving Q3 is cycled through the product ions which are detected upon exiting the last 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...
by an electron multiplier
Electron multiplier
An electron multiplier is a vacuum-tube structure that multiplies incident charges. In a process called secondary emission, a single electron can, when bombarded on secondary emissive material, induce emission of roughly 1 to 3 electrons...
. A precursor/product pair is often referred to as a transition. Much work goes into ensuring that transitions are selected that have maximum specificity. By spiking in heavy-labeled (e.g., D
Deuterium
Deuterium, also called heavy hydrogen, is one of two stable isotopes of hydrogen. It has a natural abundance in Earth's oceans of about one atom in of hydrogen . Deuterium accounts for approximately 0.0156% of all naturally occurring hydrogen in Earth's oceans, while the most common isotope ...
, 13C
Carbon-13
Carbon-13 is a natural, stable isotope of carbon and one of the environmental isotopes. It makes up about 1.1% of all natural carbon on Earth.- Detection by mass spectrometry :...
, or 15N) peptides to a complex matrix as concentration standards
Standard solution
In analytical chemistry, a standard solution is a solution containing a precisely known concentration of an element or a substance i.e, a known weight of solute is dissolved to make a specific volume. It is prepared using a standard substance, such as a primary standard. Standard solutions are used...
, SRM can be used to construct a calibration curve
Calibration curve
In analytical chemistry, a calibration curve is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration...
that can provide the absolute quantification (i.e., copy number per cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
) of the native, light peptide, and by extension, its parent protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
.