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In
mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
, matrix-assisted laser desorption/ionization (MALDI) is an
ionization Ionization, or Ionisation is the process by which an atom or a molecule acquires a negative or positive charge by gaining or losing electrons, often in conjunction with other chemical changes. The resulting electrically charged atom or molecule i ...
technique that uses a laser energy absorbing matrix to create
ion An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...
s from large
molecule A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...
s with minimal fragmentation. It has been applied to the analysis of
biomolecule A biomolecule or biological molecule is a loosely used term for molecules present in organisms that are essential to one or more typically biological processes, such as cell division, morphogenesis, or development. Biomolecules include large ...
s (
biopolymer Biopolymers are natural polymers produced by the cells of living organisms. Like other polymers, biopolymers consist of monomeric units that are covalently bonded in chains to form larger molecules. There are three main classes of biopolymers, cl ...
s such as DNA,
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s,
peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. A ...
s and
carbohydrate In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or ma ...
s) and various organic molecules (such as
polymer A polymer (; Greek '' poly-'', "many" + ''-mer'', "part") is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic a ...
s,
dendrimer Dendrimers are highly ordered, branched polymeric molecules. Synonymous terms for dendrimer include arborols and cascade molecules. Typically, dendrimers are symmetric about the core, and often adopt a spherical three-dimensional morphology. The ...
s and other
macromolecule A macromolecule is a very large molecule important to biophysical processes, such as a protein or nucleic acid. It is composed of thousands of covalently bonded atoms. Many macromolecules are polymers of smaller molecules called monomers. The ...
s), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is similar in character to
electrospray ionization Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules becaus ...
(ESI) in that both techniques are relatively soft (low fragmentation) ways of obtaining ions of large molecules in the gas phase, though MALDI typically produces far fewer multi-charged ions. MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed
laser A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The fir ...
irradiates the sample, triggering
ablation Ablation ( la, ablatio – removal) is removal or destruction of something from an object by vaporization, chipping, erosion, erosive processes or by other means. Examples of ablative materials are described below, and include spacecraft materi ...
and
desorption Desorption is the physical process where a previously adsorbed substance is released from a surface. This happens when a molecule gains enough energy to overcome the activation barrier of the bounding energy that keeps it in the surface. There ...
of the sample and matrix material. Finally, the analyte molecules are ionized by being
protonated In chemistry, protonation (or hydronation) is the adding of a proton (or hydron, or hydrogen cation), (H+) to an atom, molecule, or ion, forming a conjugate acid. (The complementary process, when a proton is removed from a Brønsted–Lowry acid, i ...
or
deprotonated Deprotonation (or dehydronation) is the removal (transfer) of a proton (or hydron, or hydrogen cation), (H+) from a Brønsted–Lowry acid in an acid–base reaction.Henry Jakubowski, Biochemistry Online Chapter 2A3, https://employees.csbsju.edu ...
in the hot plume of ablated gases, and then they can be accelerated into whichever mass spectrometer is used to analyse them.


History

The term matrix-assisted laser desorption ionization (MALDI) was coined in 1985 by
Franz Hillenkamp Franz Hillenkamp (March 18, 1936 – August 22, 2014) was a German scientist known for his development of the laser microprobe mass analyzer and, with Michael Karas, matrix-assisted laser desorption/ionization (MALDI). Early life and education Fr ...
,
Michael Karas Michael Karas (born 1952) is a German physical chemistry scientist and Professor, known for his researches on matrix-assisted laser desorption/ionization (MALDI), a technique in mass spectrometry. Michael Karas studied Chemistry at the University ...
and their colleagues. These researchers found that the
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
alanine Alanine (symbol Ala or A), or α-alanine, is an α-amino acid that is used in the biosynthesis of proteins. It contains an amine group and a carboxylic acid group, both attached to the central carbon atom which also carries a methyl group side c ...
could be ionized more easily if it was mixed with the amino acid
tryptophan Tryptophan (symbol Trp or W) is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α- carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic ...
and irradiated with a pulsed 266 nm laser. The tryptophan was absorbing the laser energy and helping to ionize the non-absorbing alanine. Peptides up to the 2843 Da peptide
melittin Melittin is the main component (40–60% of the dry weight) and the major pain producing substance of honeybee (''Apis mellifera'') venom. Melittin is a basic peptide consisting of 26 amino acids. Function The principal function of melittin a ...
could be ionized when mixed with this kind of "matrix". The breakthrough for large molecule laser desorption ionization came in 1987 when
Koichi Tanaka is a Japanese electrical engineer who shared the Nobel Prize in Chemistry in 2002 for developing a novel method for mass spectrometric analyses of biological macromolecules with John Bennett Fenn and Kurt Wüthrich (the latter for work in NMR ...
of Shimadzu Corporation and his co-workers used what they called the "ultra fine metal plus liquid matrix method" that combined 30 nm
cobalt Cobalt is a chemical element with the symbol Co and atomic number 27. As with nickel, cobalt is found in the Earth's crust only in a chemically combined form, save for small deposits found in alloys of natural meteoric iron. The free element, pr ...
particles in
glycerol Glycerol (), also called glycerine in British English and glycerin in American English, is a simple triol compound. It is a colorless, odorless, viscous liquid that is sweet-tasting and non-toxic. The glycerol backbone is found in lipids known ...
with a 337 nm
nitrogen laser A nitrogen laser is a gas laser operating in the ultraviolet rangeC. S. Willett, ''Introduction to Gas Lasers: Population Inversion Mechanisms'' (Pergamon, New York,1974). (typically 337.1 nm) using molecular nitrogen as its gain medium, pum ...
for ionization. Using this laser and matrix combination, Tanaka was able to ionize biomolecules as large as the 34,472 Da protein carboxypeptidase-A. Tanaka received one-quarter of the 2002
Nobel Prize in Chemistry ) , image = Nobel Prize.png , alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
for demonstrating that, with the proper combination of laser wavelength and matrix, a protein can be ionized. Karas and Hillenkamp were subsequently able to ionize the 67 kDa protein albumin using a nicotinic acid matrix and a 266 nm laser. Further improvements were realized through the use of a 355 nm laser and the
cinnamic acid Cinnamic acid is an organic compound with the formula C6H5-CH=CH- COOH. It is a white crystalline compound that is slightly soluble in water, and freely soluble in many organic solvents. Classified as an unsaturated carboxylic acid, it occurs n ...
derivatives
ferulic acid Ferulic acid is a hydroxycinnamic acid, an organic compound with the formula (CH3O)HOC6H3CH=CHCO2H. The name is derived from the genus ''Ferula'', referring to the giant fennel (''Ferula communis''). Classified as a phenolic phytochemical, ferulic ...
,
caffeic acid Caffeic acid is an organic compound that is classified as a hydroxycinnamic acid. This yellow solid consists of both phenolic and acrylic functional groups. It is found in all plants because it is an intermediate in the biosynthesis of lignin, one ...
and
sinapinic acid Sinapinic acid, or sinapic acid (Sinapine - Origin: L. Sinapi, sinapis, mustard, Gr., cf. F. Sinapine.), is a small naturally occurring hydroxycinnamic acid. It is a member of the phenylpropanoid family. It is a commonly used matrix in MALDI mass ...
as the matrix. The availability of small and relatively inexpensive
nitrogen laser A nitrogen laser is a gas laser operating in the ultraviolet rangeC. S. Willett, ''Introduction to Gas Lasers: Population Inversion Mechanisms'' (Pergamon, New York,1974). (typically 337.1 nm) using molecular nitrogen as its gain medium, pum ...
s operating at 337 nm wavelength and the first commercial instruments introduced in the early 1990s brought MALDI to an increasing number of researchers. Today, mostly organic matrices are used for MALDI mass spectrometry.


Matrix

The
matrix Matrix most commonly refers to: * ''The Matrix'' (franchise), an American media franchise ** ''The Matrix'', a 1999 science-fiction action film ** "The Matrix", a fictional setting, a virtual reality environment, within ''The Matrix'' (franchis ...
consists of
crystal A crystal or crystalline solid is a solid material whose constituents (such as atoms, molecules, or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. In addition, macros ...
lized molecules, of which the three most commonly used are
sinapinic acid Sinapinic acid, or sinapic acid (Sinapine - Origin: L. Sinapi, sinapis, mustard, Gr., cf. F. Sinapine.), is a small naturally occurring hydroxycinnamic acid. It is a member of the phenylpropanoid family. It is a commonly used matrix in MALDI mass ...
, α-cyano-4-hydroxycinnamic acid (α-CHCA, alpha-cyano or alpha-matrix) and
2,5-dihydroxybenzoic acid Gentisic acid is a dihydroxybenzoic acid. It is a derivative of benzoic acid and a minor (1%) product of the metabolic break down of aspirin, excreted by the kidneys. It is also found in the African tree ''Alchornea cordifolia'' and in wine. Pro ...
(DHB). A
solution Solution may refer to: * Solution (chemistry), a mixture where one substance is dissolved in another * Solution (equation), in mathematics ** Numerical solution, in numerical analysis, approximate solutions within specified error bounds * Soluti ...
of one of these molecules is made, often in a mixture of highly purified water and an organic
solvent A solvent (s) (from the Latin '' solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a solution. A solvent is usually a liquid but can also be a solid, a gas, or a supercritical fluid. Water is a solvent for ...
such as acetonitrile (ACN) or ethanol. A counter ion source such as
trifluoroacetic acid Trifluoroacetic acid (TFA) is an organofluorine compound with the chemical formula CF3CO2H. It is a structural analogue of acetic acid with all three of the acetyl group's hydrogen atoms replaced by fluorine atoms and is a colorless liquid with a ...
(TFA) is usually added to generate the +Hions. A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN:water:TFA (50:50:0.1). The identification of suitable matrix compounds is determined to some extent by trial and error, but they are based on some specific molecular design considerations. They are of a fairly low molecular weight (to allow easy vaporization), but are large enough (with a low enough vapor pressure) not to evaporate during sample preparation or while standing in the mass spectrometer. They are often acidic, therefore act as a proton source to encourage ionization of the analyte. Basic matrices have also been reported. They have a strong optical absorption in either the UV or IR range, so that they rapidly and efficiently absorb the laser irradiation. This efficiency is commonly associated with chemical structures incorporating several
conjugated double bond In theoretical chemistry, a conjugated system is a system of connected p-orbitals with delocalized electrons in a molecule, which in general lowers the overall energy of the molecule and increases Chemical stability, stability. It is Resonance ...
s, as seen in the structure of
cinnamic acid Cinnamic acid is an organic compound with the formula C6H5-CH=CH- COOH. It is a white crystalline compound that is slightly soluble in water, and freely soluble in many organic solvents. Classified as an unsaturated carboxylic acid, it occurs n ...
. They are functionalized with polar groups, allowing their use in aqueous solutions. They typically contain a
chromophore A chromophore is the part of a molecule responsible for its color. The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
. The matrix solution is mixed with the analyte (e.g.
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
-sample). A mixture of water and organic solvent allows both
hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, th ...
and water-soluble (
hydrophilic A hydrophile is a molecule or other molecular entity that is attracted to water molecules and tends to be dissolved by water.Liddell, H.G. & Scott, R. (1940). ''A Greek-English Lexicon'' Oxford: Clarendon Press. In contrast, hydrophobes are no ...
) molecules to dissolve into the solution. This solution is spotted onto a MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize, leaving only the recrystallized matrix, but now with analyte molecules embedded into MALDI crystals. The matrix and the analyte are said to be co-crystallized. Co-crystallization is a key issue in selecting a proper matrix to obtain a good quality mass spectrum of the analyte of interest. In analysis of biological systems, inorganic salts, which are also part of protein extracts, interfere with the ionization process. The salts can be removed by solid phase extraction or by washing the dried-droplet MALDI spots with cold water. Both methods can also remove other substances from the sample. The matrix-protein mixture is not homogeneous because the polarity difference leads to a separation of the two substances during co-crystallization. The spot diameter of the target is much larger than that of the laser, which makes it necessary to make many laser shots at different places of the target, to get the statistical average of the substance concentration within the target spot. The matrix can be used to tune the instrument to ionize the sample in different ways. As mentioned above, acid-base like reactions are often utilized to ionize the sample, however, molecules with conjugated pi systems, such as naphthalene like compounds, can also serve as an electron acceptor and thus a matrix for MALDI/TOF. This is particularly useful in studying molecules that also possess conjugated pi systems. The most widely used application for these matrices is studying
porphyrin Porphyrins ( ) are a group of heterocyclic macrocycle organic compounds, composed of four modified pyrrole subunits interconnected at their α carbon atoms via methine bridges (=CH−). The parent of porphyrin is porphine, a rare chemical com ...
-like compounds such as
chlorophyll Chlorophyll (also chlorophyl) is any of several related green pigments found in cyanobacteria and in the chloroplasts of algae and plants. Its name is derived from the Greek words , ("pale green") and , ("leaf"). Chlorophyll allow plants to a ...
. These matrices have been shown to have better ionization patterns that do not result in odd fragmentation patterns or complete loss of side chains. It has also been suggested that conjugated porphyrin like molecules can serve as a matrix and cleave themselves eliminating the need for a separate matrix compound.


Instrumentation

There are several variations of the MALDI technology and comparable instruments are today produced for very different purposes. From more academic and analytical, to more industrial and high throughput. The mass spectrometry field has expanded into requiring ultrahigh resolution mass spectrometry such as the FT-ICR instruments as well as more high-throughput instruments. As many MALDI MS instruments can be bought with an interchangeable ionization source (
electrospray ionization Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules becaus ...
, MALDI,
atmospheric pressure ionization An ion source is a device that creates atomic and molecular ions. Ion sources are used to form ions for Mass spectrometry, mass spectrometers, optical emission spectrometers, particle accelerators, Ion implantation, ion implanters and Ion thrus ...
, etc.) the technologies often overlap and many times any soft ionization method could potentially be used. For more variations of soft ionization methods see:
Soft laser desorption Soft laser desorption (SLD) is laser desorption of large molecules that results in ionization without fragmentation. "Soft" in the context of ion formation means forming ions without breaking chemical bonds. "Hard" ionization is the formation of ion ...
or
Ion source An ion source is a device that creates atomic and molecular ions. Ion sources are used to form ions for mass spectrometers, optical emission spectrometers, particle accelerators, ion implanters and ion engines. Electron ionization Electron ...
.


Laser

MALDI techniques typically employ the use of UV
lasers A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The fir ...
such as nitrogen lasers (337 nm) and frequency-tripled and quadrupled Nd:YAG lasers (355 nm and 266 nm respectively). Infrared laser wavelengths used for infrared MALDI include the 2.94 μm Er:YAG laser, mid-IR
optical parametric oscillator An optical parametric oscillator (OPO) is a parametric oscillator that oscillates at optical frequencies. It converts an input laser wave (called "pump") with frequency \omega_p into two output waves of lower frequency (\omega_s, \omega_i) by means ...
, and 10.6 μm
carbon dioxide laser The carbon-dioxide laser (CO2 laser) was one of the earliest gas lasers to be developed. It was invented by Kumar Patel of Bell Labs in 1964 and is still one of the most useful types of laser. Carbon-dioxide lasers are the highest-power continuou ...
. Although not as common, infrared lasers are used due to their softer mode of ionization. IR-MALDI also has the advantage of greater material removal (useful for biological samples), less low-mass interference, and compatibility with other matrix-free laser desorption mass spectrometry methods.


Time of flight

The type of a mass spectrometer most widely used with MALDI is the
time-of-flight mass spectrometer Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined by a time of flight measurement. Ions are accelerated by an electric field of known strength. This acceleration resul ...
(TOF), mainly due to its large mass range. The TOF measurement procedure is also ideally suited to the MALDI ionization process since the pulsed laser takes individual 'shots' rather than working in continuous operation. MALDI-TOF instruments are often equipped with a
reflectron A reflectron (mass reflectron) is a type of time-of-flight mass spectrometer (TOF MS) that comprises a pulsed ion source, field-free region, ion mirror, and ion detector and uses a static or time dependent electric field in the ion mirror to reve ...
(an "ion mirror") that reflects ions using an electric field. This increases the ion flight path, thereby increasing time of flight between ions of different m/z and increasing resolution. Modern commercial reflectron TOF instruments reach a resolving power m/Δm of 50,000 FWHM (full-width half-maximum, Δm defined as the peak width at 50% of peak height) or more. MALDI has been coupled with
IMS Ims is a Norwegian surname. Notable people with the surname include: * Gry Tofte Ims (born 1986), Norwegian footballer * Rolf Anker Ims (born 1958), Norwegian ecologist See also * IMS (disambiguation) Ims is a Norwegian surname. Notable people wit ...
-TOF MS to identify
phosphorylated In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, whi ...
and non-phosphorylated peptides. MALDI-
FT-ICR Fourier-transform ion cyclotron resonance mass spectrometry is a type of mass analyzer (or mass spectrometer) for determining the mass-to-charge ratio (''m''/''z'') of ions based on the cyclotron frequency of the ions in a fixed magnetic field. T ...
MS has been demonstrated to be a useful technique where high resolution MALDI-MS measurements are desired.


Atmospheric pressure

Atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) is an ionization technique (ion source) that in contrast to vacuum MALDI operates at normal atmospheric environment. The main difference between vacuum MALDI and AP-MALDI is the pressure in which the ions are created. In vacuum MALDI, ions are typically produced at 10 mTorr or less while in AP-MALDI ions are formed in
atmospheric pressure Atmospheric pressure, also known as barometric pressure (after the barometer), is the pressure within the atmosphere of Earth. The standard atmosphere (symbol: atm) is a unit of pressure defined as , which is equivalent to 1013.25 millibars, 7 ...
. In the past the main disadvantage of the AP-MALDI technique compared to the conventional vacuum MALDI has been its limited sensitivity; however, ions can be transferred into the mass spectrometer with high efficiency and attomole detection limits have been reported. AP-MALDI is used in mass spectrometry (MS) in a variety of applications ranging from proteomics to drug discovery. Popular topics that are addressed by AP-MALDI mass spectrometry include: proteomics; mass analysis of DNA, RNA, PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small molecules and synthetic polymers, similar applications as available also for vacuum MALDI instruments. The AP-MALDI ion source is easily coupled to an ion trap mass spectrometer or any other MS system equipped with
electrospray ionization Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules becaus ...
(ESI) or nanoESI source. MALDI with ionization at reduced pressure is known to produce mainly singly-charged ions (see "Ionization mechanism" below). In contrast, ionization at atmopsheric pressure can generate highly-charged analytes as was first shown for infrared and later also for nitrogen lasers. Multiple charging of analytes is of great importance, because it allows to measure high-molecular-weight compounds like proteins in instruments, which provide only smaller ''m/z'' detection ranges such as quadrupoles. Besides the pressure, the composition of the matrix is important to achive this effect.


Aerosol

In
aerosol mass spectrometry Aerosol mass spectrometry is the application of mass spectrometry to the analysis of the composition of aerosol particles. Aerosol particles are defined as solid and liquid particles suspended in a gas (air), with size range of 3 nm to 100 ...
, one of the ionization techniques consists in firing a laser to individual droplets. These systems are called single particle mass spectrometers (SPMS). The sample may optionally be mixed with a MALDI matrix prior to aerosolization.


Ionization mechanism

The
laser A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The fir ...
is fired at the matrix crystals in the dried-droplet spot. The matrix absorbs the laser energy and it is thought that primarily the matrix is desorbed and ionized (by addition of a
proton A proton is a stable subatomic particle, symbol , H+, or 1H+ with a positive electric charge of +1 ''e'' elementary charge. Its mass is slightly less than that of a neutron and 1,836 times the mass of an electron (the proton–electron mass ...
) by this event. The hot plume produced during ablation contains many species: neutral and ionized matrix molecules, protonated and deprotonated matrix molecules, matrix clusters and nanodroplets. Ablated species may participate in the ionization of analyte, though the mechanism of MALDI is still debated. The matrix is then thought to transfer protons to the analyte molecules (e.g., protein molecules), thus charging the analyte. An ion observed after this process will consist of the initial neutral molecule with ions added or removed. This is called a quasimolecular ion, for example +Hsup>+ in the case of an added proton, +Nasup>+ in the case of an added
sodium Sodium is a chemical element with the symbol Na (from Latin ''natrium'') and atomic number 11. It is a soft, silvery-white, highly reactive metal. Sodium is an alkali metal, being in group 1 of the periodic table. Its only stable iso ...
ion, or -Hsup>− in the case of a removed proton. MALDI is capable of creating singly charged ions or multiply charged ions ( +nHsup>n+) depending on the nature of the matrix, the laser intensity, and/or the voltage used. Note that these are all even-electron species. Ion signals of radical cations (photoionized molecules) can be observed, e.g., in the case of matrix molecules and other organic molecules. The gas phase proton transfer model, implemented as the coupled physical and chemical dynamics (CPCD) model, of UV laser MALDI postulates primary and secondary processes leading to ionization. Primary processes involve initial charge separation through absorption of photons by the matrix and pooling of the energy to form matrix ion pairs. Primary ion formation occurs through absorption of a UV photon to create excited state molecules by :S0 + hν → S1 :S1 + S1 → S0 + Sn :S1 + Sn → M+ + M where S0 is the ground electronic state, S1 the first electronic excited state, and Sn is a higher electronic excited state. The product ions can be proton transfer or electron transfer ion pairs, indicated by M+ and M above. Secondary processes involve ion-molecule reactions to form analyte ions. The lucky survivor model (cluster ionization mechanism) postulates that analyte molecules are incorporated in the matrix maintaining the charge state from solution. Ion formation occurs through charge separation upon fragmentation of laser ablated clusters. Ions that are not neutralized by recombination with
photoelectron The photoelectric effect is the emission of electrons when electromagnetic radiation, such as light, hits a material. Electrons emitted in this manner are called photoelectrons. The phenomenon is studied in condensed matter physics, and solid sta ...
s or
counter ion 160px, Polystyrene sulfonate, a cation-exchange resin, is typically supplied with as the counterion.">cation-exchange_resin.html" ;"title="Polystyrene sulfonate, a cation-exchange resin">Polystyrene sulfonate, a cation-exchange resin, is typical ...
s are the so-called lucky survivors. The thermal model postulates that the high temperature facilitates the proton transfer between matrix and analyte in melted matrix liquid. Ion-to-neutral ratio is an important parameter to justify the theoretical model, and the mistaken citation of ion-to-neutral ratio could result in an erroneous determination of the ionization mechanism. The model quantitatively predicts the increase in total ion intensity as a function of the concentration and proton affinity of the analytes, and the ion-to-neutral ratio as a function of the laser fluences. This model also suggests that metal ion adducts (e.g., +Nasup>+ or +Ksup>+) are mainly generated from the thermally induced dissolution of salt. The
matrix-assisted ionization In mass spectrometry, matrix-assisted ionization (also inlet ionization) is a low fragmentation (soft) ionization technique which involves the transfer of particles of the analyte and Matrix (mass spectrometry), matrix sample from atmospheric pres ...
(MAI) method uses matrix preparation similar to MALDI but does not require laser ablation to produce analyte ions of volatile or nonvolatile compounds. Simply exposing the matrix with analyte to the vacuum of the mass spectrometer creates ions with nearly identical charge states to electrospray ionization. It is suggested that there are likely mechanistic commonality between this process and MALDI. Ion yield is typically estimated to range from 10−4 to 10−7, with some experiments hinting to even lower yields of 10−9. The issue of low ion yields had been addressed, already shortly after introduction of MALDI by various attempts, including post-ionization utilizing a second laser. Most of these attempts showed only limited success, with low signal increases. This might be attributed to the fact, that axial time-of-flight instruments were used, which operate at pressures in the source region of 10−5 to 10−6, which results in rapid plume expansion with particle velocities of up to 1000 m/s. In 2015 successful laser post-ionization was reported, using a modified MALDI source operated at an elevated pressure of ~3 mbar coupled to an orthogonal time-of-flight mass analyzer, and employing a wavelength-tunable post-ionization laser, operated at wavelength from 260 nm to 280 nm, below the two-photon ionization threshold of the matrices used, which elevated ion yields of several lipids and small molecules by up to three orders of magnitude. This approach, called MALDI-2, due to the second laser, and the second MALDI-like ionization process, was afterwards adopted for other mass spectrometers, all equipped with sources operating in the low mbar range.


Applications


Biochemistry

In
proteomics Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...
, MALDI is used for the rapid identification of proteins isolated by using
gel electrophoresis Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
:
SDS-PAGE SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. T ...
,
size exclusion chromatography Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules ...
,
affinity chromatography Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the ...
, strong/weak ion exchange, isotope coded protein labeling (ICPL), and
two-dimensional gel electrophoresis Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first ...
.
Peptide mass fingerprinting Peptide mass fingerprinting (PMF) (also known as protein fingerprinting) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately ...
is the most popular analytical application of MALDI-TOF mass spectrometers. MALDI TOF/TOF mass spectrometers are used to reveal amino acid sequence of peptides using post-source decay or high energy
collision-induced dissociation Collision-induced dissociation (CID), also known as collisionally activated dissociation (CAD), is a mass spectrometry technique to induce fragmentation of selected ions in the gas phase. The selected ions (typically molecular ions or protonated ...
(further use see
mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
). MALDI-TOF have been used to characterise
post-translational modification Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
s. For example, it has been widely applied to study
protein methylation Protein methylation is a type of post-translational modification featuring the addition of methyl groups to proteins. It can occur on the nitrogen-containing side-chains of arginine and lysine, but also at the amino- and carboxy-termini of a num ...
and
demethylation Demethylation is the chemical process resulting in the removal of a methyl group (CH3) from a molecule. A common way of demethylation is the replacement of a methyl group by a hydrogen atom, resulting in a net loss of one carbon and two hydrogen ato ...
. However, care must be taken when studying post-translational modifications by MALDI-TOF. For example, it has been reported that loss of sialic acid has been identified in papers when
dihydroxybenzoic acid Dihydroxybenzoic acids (DHBA) are a type of phenolic acids. There are six main compounds, having all the same molecular formula C7H6O4. Those are: * 2,3-Dihydroxybenzoic acid (2-Pyrocatechuic acid or hypogallic acid) * 2,4-Dihydroxybenzoic acid ...
(DHB) has been used as a matrix for MALDI MS analysis of glycosylated peptides. Using sinapinic acid, 4-HCCA and DHB as matrices, S. Martin studied loss of sialic acid in glycosylated peptides by metastable decay in MALDI/TOF in linear mode and reflector mode. A group at Shimadzu Corporation derivatized the sialic acid by an
amidation In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a compound with the general formula , where R, R', and R″ represent organic groups or hydrogen atoms. The amide group is called a peptide bond when it is p ...
reaction as a way to improve detection sensitivity and also demonstrated that ionic liquid matrix reduces a loss of sialic acid during MALDI/TOF MS analysis of sialylated oligosaccharides. THAP, DHAP, and a mixture of 2-aza-2-thiothymine and phenylhydrazine have been identified as matrices that could be used to minimize loss of sialic acid during MALDI MS analysis of glycosylated peptides. It has been reported that a reduction in loss of some post-translational modifications can be accomplished if IR MALDI is used instead of UV MALDI Besides proteins, MALDI-TOF has also been applied to study
lipids Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
. For example, it has been applied to study the catalytic reactions of
phospholipase A phospholipase is an enzyme that hydrolyzes phospholipids into fatty acids and other lipophilic substances. Acids trigger the release of bound calcium from cellular stores and the consequent increase in free cytosolic Ca2+, an essential step in ...
s. In addition to lipids,
oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids c ...
s have also been characterised by MALDI-TOF. For example, in molecular biology, a mixture of 5-methoxysalicylic acid and
spermine Spermine is a polyamine involved in cellular metabolism that is found in all Eukaryote, eukaryotic cells. The precursor for synthesis of spermine is the amino acid ornithine. It is an essential growth factor in some Bacterium, bacteria as well. ...
can be used as a matrix for
oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids c ...
s analysis in MALDI mass spectrometry, for instance after
oligonucleotide synthesis Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure (sequence). The technique is extremely useful in current laboratory practice because it provides a rapid and inexpens ...
.


Organic chemistry

Some synthetic macromolecules, such as
catenane In macromolecular chemistry, a catenane () is a mechanically interlocked molecular architecture consisting of two or more interlocked macrocycles, i.e. a molecule containing two or more intertwined rings. The interlocked rings cannot be sepa ...
s and
rotaxane In chemistry, a rotaxane () is a mechanically interlocked molecular architecture consisting of a dumbbell-shaped molecule which is threaded through a macrocycle (see graphical representation). The two components of a rotaxane are kinetically t ...
s,
dendrimer Dendrimers are highly ordered, branched polymeric molecules. Synonymous terms for dendrimer include arborols and cascade molecules. Typically, dendrimers are symmetric about the core, and often adopt a spherical three-dimensional morphology. The ...
s and hyperbranched polymers, and other assemblies, have molecular weights extending into the thousands or tens of thousands, where most ionization techniques have difficulty producing molecular ions. MALDI is a simple and fast analytical method that can allow chemists to rapidly analyze the results of such syntheses and verify their results.


Polymers

In polymer chemistry MALDI can be used to determine the
molar mass distribution The molar mass distribution (or molecular weight distribution) describes the relationship between the number of moles of each polymer species (Ni) and the molar mass (Mi) of that species. In linear polymers, the individual polymer chains rarely have ...
. Polymers with
polydispersity In chemistry, the dispersity is a measure of the heterogeneity of sizes of molecules or particles in a mixture. A collection of objects is called uniform if the objects have the same size, shape, or mass. A sample of objects that have an incons ...
greater than 1.2 are difficult to characterize with MALDI due to the signal intensity discrimination against higher mass oligomers. A good matrix for polymers is
dithranol Dithranol ( INN) or anthralin (USAN and former BAN) is a hydroxyanthrone, anthracene derivative, medicine applied to the skin of people with psoriasis. It is available as creams, ointment or pastes in 0.1 to 2% strengths (Drithocreme, Dithrocream, ...
or AgTFA. The sample must first be mixed with dithranol and the AgTFA added afterwards; otherwise the sample will precipitate out of solution.


Microbiology

MALDI-TOF spectra are often used for the identification of microorganisms such as bacteria or fungi. A portion of a colony of the microbe in question is placed onto the sample target and overlaid with matrix. The mass spectra of expressed proteins generated are analyzed by dedicated software and compared with stored profiles for species determination in what is known as biotyping. It offers benefits to other immunological or biochemical procedures and has become a common method for species identification in clinical microbiological laboratories. Benefits of high resolution MALDI-MS performed on a Fourier transform ion cyclotron resonance mass spectrometry (also known as FT-MS) have been demonstrated for typing and subtyping viruses though single ion detection known as proteotyping, with a particular focus on influenza viruses. One main advantage over other microbiological identification methods is its ability to rapidly and reliably identify, at low cost, a wide variety of microorganisms directly from the selective medium used to isolate them. The absence of the need to purify the suspect or "presumptive" colony allows for a much faster turn-around times. For example, it has been demonstrated that MALDI-TOF can be used to detect bacteria directly from blood cultures. Another advantage is the potential to predict
antibiotic susceptibility Antibiotic sensitivity testing or antibiotic susceptibility testing is the measurement of the susceptibility of bacteria to antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important typ ...
of bacteria. A single mass spectral peak can predict methicillin resistance of ''Staphylococcus aureus'' MALDI can also detect
carbapenemase Beta-lactamases, (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems (ertapenem), although carbapenems ...
of
carbapenem-resistant enterobacteriaceae Carbapenem-resistant Enterobacteriaceae (CRE) or carbapenemase-producing Enterobacteriaceae (CPE) are Gram-negative bacteria that are resistant to the carbapenem class of antibiotics, considered the drugs of last resort for such infections. They ...
, including ''
Acinetobacter baumannii ''Acinetobacter baumannii'' is a typically short, almost round, rod-shaped ( coccobacillus) Gram-negative bacterium. It is named after the bacteriologist Paul Baumann. It can be an opportunistic pathogen in humans, affecting people with compromi ...
'' and ''
Klebsiella pneumoniae ''Klebsiella pneumoniae'' is a Gram-negative, non-motile, encapsulated, lactose-fermenting, facultative anaerobic, rod-shaped bacterium. It appears as a mucoid lactose fermenter on MacConkey agar. Although found in the normal flora of the mouth ...
''. However, most proteins that mediate antibiotic resistance are larger than MALDI-TOF's 2000–20,000 Da range for protein peak interpretation and only occasionally, as in the 2011''Klebsiella pneumoniae'' carbapenemase (KPC) outbreak at the NIH, a correlation between a peak and resistance conferring protein can be made.


Parasitology

MALDI-TOF spectra have been used for the detection and identification of various parasites such as
trypanosomatid Trypanosomatida is a group of kinetoplastid excavates distinguished by having only a single flagellum. The name is derived from the Greek ''trypano'' (borer) and ''soma'' (body) because of the corkscrew-like motion of some trypanosomatid species. ...
s, ''
Leishmania ''Leishmania'' is a parasitic protozoan, a single-celled organism of the genus '' Leishmania'' that are responsible for the disease leishmaniasis. They are spread by sandflies of the genus ''Phlebotomus'' in the Old World, and of the genus '' ...
'' and ''
Plasmodium ''Plasmodium'' is a genus of unicellular eukaryotes that are obligate parasites of vertebrates and insects. The life cycles of ''Plasmodium'' species involve development in a blood-feeding insect host which then injects parasites into a vert ...
''. In addition to these
unicellular A unicellular organism, also known as a single-celled organism, is an organism that consists of a single cell, unlike a multicellular organism that consists of multiple cells. Organisms fall into two general categories: prokaryotic organisms and ...
parasites, MALDI/TOF can be used for the identification of parasitic insects such as
lice Louse ( : lice) is the common name for any member of the clade Phthiraptera, which contains nearly 5,000 species of wingless parasitic insects. Phthiraptera has variously been recognized as an order, infraorder, or a parvorder, as a result o ...
or
cercariae Trematodes are parasitic flatworms of the class ''Trematoda'', specifically parasitic flukes with two suckers: one ventral and the other oral. Trematodes are covered by a tegument, that protects the organism from the environment by providing secr ...
, the free-swimming stage of
trematode Trematoda is a class of flatworms known as flukes. They are obligate internal parasites with a complex life cycle requiring at least two hosts. The intermediate host, in which asexual reproduction occurs, is usually a snail. The definitive host ...
s.


Medicine

MALDI-TOF spectra are often utilized in tandem with other analysis and spectroscopy techniques in the diagnosis of diseases. MALDI/TOF is a diagnostic tool with much potential because it allows for the rapid identification of proteins and changes to proteins without the cost or computing power of
sequencing In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succ ...
nor the skill or time needed to solve a crystal structure in
X-ray crystallography X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
. One example of this is
necrotizing enterocolitis Necrotizing enterocolitis (NEC) is a devastating intestinal disease that affects premature or very low birth weight infants.Gephart S.M., Quinn M. A call to action to fight for equity and end necrotizing enterocolitis disparities. ''Adv. Neonata ...
(NEC), which is a devastating disease that affects the bowels of premature infants. The symptoms of NEC are very similar to those of
sepsis Sepsis, formerly known as septicemia (septicaemia in British English) or blood poisoning, is a life-threatening condition that arises when the body's response to infection causes injury to its own tissues and organs. This initial stage is follo ...
, and many infants die awaiting diagnosis and treatment. MALDI/TOF was used to identify bacteria present in the fecal matter of NEC positive infants. This study focused on characterization of the fecal microbiota associated with NEC and did not address the mechanism of disease. There is hope that a similar technique could be used as a quick, diagnostic tool that would not require sequencing. Another example of the diagnostic power of MALDI/TOF is in the area of
cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
.
Pancreatic cancer Pancreatic cancer arises when cell (biology), cells in the pancreas, a glandular organ behind the stomach, begin to multiply out of control and form a Neoplasm, mass. These cancerous cells have the malignant, ability to invade other parts of t ...
remains one of the most deadly and difficult to diagnose cancers. Impaired
cellular signaling In biology, cell signaling (cell signalling in British English) or cell communication is the ability of a cell to receive, process, and transmit signals with its environment and with itself. Cell signaling is a fundamental property of all cellula ...
due to mutations in membrane proteins has been long suspected to contribute to pancreatic cancer. MALDI/TOF has been used to identify a
membrane protein Membrane proteins are common proteins that are part of, or interact with, biological membranes. Membrane proteins fall into several broad categories depending on their location. Integral membrane proteins are a permanent part of a cell membrane ...
associated with pancreatic cancer and at one point may even serve as an early detection technique. MALDI/TOF can also potentially be used to dictate treatment as well as diagnosis. MALDI/TOF serves as a method for determining the
drug resistance Drug resistance is the reduction in effectiveness of a medication such as an antimicrobial or an antineoplastic in treating a disease or condition. The term is used in the context of resistance that pathogens or cancers have "acquired", that is, ...
of bacteria, especially to
β-lactams A beta-lactam (β-lactam) ring is a four-membered lactam. A ''lactam'' is a cyclic amide, and ''beta''-lactams are named so because the nitrogen atom is attached to the β-carbon atom relative to the carbonyl. The simplest β-lactam possible is ...
(Penicillin family). The MALDI/TOF detects the presence of carbapenemases, which indicates drug resistance to standard antibiotics. It is predicted that this could serve as a method for identifying a bacterium as drug resistant in as little as three hours. This technique could help physicians decide whether to prescribe more aggressive antibiotics initially.


Detection of protein complexes

Following initial observations that some peptide-peptide complexes could survive MALDI deposition and ionization, studies of large protein complexes using MALDI-MS have been reported.


See also

*
Peptide mass fingerprinting Peptide mass fingerprinting (PMF) (also known as protein fingerprinting) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately ...
*
PEGylation PEGylation (or pegylation) is the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG, in pharmacy called macrogol) polymer chains to molecules and macrostructures, such as a drug, therapeutic protein ...
*
MALDI imaging MALDI mass spectrometry imaging (MALDI-MSI) is the use of matrix-assisted laser desorption ionization as a mass spectrometry imaging technique in which the sample, often a thin tissue section, is moved in two dimensions while the mass spectrum is ...
*
Matrix (mass spectrometry) In mass spectrometry, a matrix is a compound that promotes the formation of ions. Matrix compounds are used in matrix-assisted laser desorption/ionization (MALDI), matrix-assisted ionization (MAI), and fast atom bombardment (FAB). Matrix-assisted ...


References


Bibliography

* * * *


External links


Primer on Matrix-Assisted Laser Desorption Ionization (MALDI)
National High Magnetic Field Laboratory {{DEFAULTSORT:Matrix-assisted laser desorption ionization Ion source Biochemistry methods