Introduction
Using a laser energy-absorbing matrix, Matrix-Assisted Laser Desorption Ionization (MALDI), an ionization process used in mass spectrometry, produces ions from larger molecules with minimum fragmentation. It examines a wide range of organic compounds, such as dendrimers, polymers, and other macromolecules; also, it examines biomolecules, which are biopolymers that include proteins, peptides, polysaccharides, and DNA, which break down rapidly when subjected to conventional ionization methods. Like Electrospray Ionization (ESI), MALDI generally produces a substantially lower number of multi-charged ions.
History of MALDI
In 1985, Franz Hillenkamp, Michael Karas, and others used the term Matrix-Assisted Laser Desorption Ionization (MALDI) (Karas et al., 1985). These scientists discovered that when tryptophan and alanine were combined and exposed to a pulsed 266 nm laser, the amino acids were ionized more readily. Tryptophan helps ionize the non-absorbing alanine by absorbing the laser energy. This type of “matrix” can potentially ionize peptides up to the 2843 Da peptide melittin (Karas et al., 1987).
In 1987, Koichi Tanaka of Shimadzu Corporation and his colleagues used the “ultra-fine metal plus liquid matrix method” by combining 30 nm cobalt particles in glycerol with a 337 nm nitrogen laser for ionization (Tanaka et al., 1988). They made significant progress toward large-molecule laser desorption ionization. Tanaka ionized biomolecules as large as the 34,472 Da protein carboxypeptidase-A using this laser and matrix combination. Tanaka got one-quarter of the Nobel Prize in Chemistry in 2002 for demonstrating that a protein can ionize by properly mixing laser wavelength and matrix .
Karas and Hillenkamp were then able to ionize the 67-kDa protein albumin with a nicotinic acid matrix and a 266 nm laser (Karas and Hillenkamp, 1988). Further improvements were made using a 355 nm laser and the cinnamic acid derivatives ferulic acid, caffeic acid, and sinapinic acid as the matrix (Beavis et al., 1989). The availability of small and relatively inexpensive nitrogen lasers with a wavelength of 337 nm, and the first commercial equipment developed in the early 1990s, made MALDI more accessible to researchers (Karas and Bahr, 1990). MALDI mass spectrometry is currently performed mostly on organic matrices.
Steps of MALDI
The three steps of the MALDI approach are as follows:
The sample is mixed with a suitable matrix material before being applied to a metal plate. The sample and matrix material experience ablation and desorption following exposure to a pulsed laser. The analyte molecules are ultimately ionized by protonating or deprotonating in the heated plume of ablated gases. Then they are propelled into the preferred mass spectrometer for examination afterward.
MALDI Matrix
The matrix is made up of crystallized molecules, including sinapinic acid, α-cyano-4-hydroxycinnamic acid (α-CHCA, also known as alpha-cyano or alpha-matrix), and 2,5-dihydroxybenzoic acid (DHB). One of these molecules is converted into a solution, often a mixture of highly purified water and an organic solvent like acetonitrile (ACN) or ethanol. To generate [M+H] ions, a counter ion source, such as trifluoroacetic acid (TFA), is typically used. A good example of a matrix solution is 20 mg/mL of sinapinic acid in ACN: water: TFA (50:50:0.1).
The identification of suitable matrix compounds is partly determined by trial and error, but it is also influenced by some specific molecular design criteria. They have a relatively low molecular weight (allowing for easy vaporization), but are large enough (with a low vapor pressure) to not evaporate during sample preparation or while standing in the mass spectrometer. They commonly become acidic and serve as a proton source to promote analyte ionization. Basic matrices were also reported. They have excellent optical absorption in the UV or IR ranges, allowing them to absorb laser irradiation quickly and efficiently. This efficiency is usually associated with chemical compounds that contain multiple conjugated double bonds, such as cinnamic acid. They are functionalized with polar groups, making them suitable for usage in aqueous liquids. They typically have a chromophore.
The matrix solution is combined with the analyte (for example, a protein sample). A solution containing water and an organic solvent permits both hydrophobic and water-soluble (hydrophilic) molecules to dissolve. This solution is placed on a MALDI plate (often a metal plate). The solvents evaporate, leaving only the recrystallized matrix, which now contains analyte molecules embedded in MALDI crystals. The matrix and analyte are said to co-crystallize. Co-crystallization is an essential factor in selecting a suitable matrix for obtaining a high-quality mass spectrum of the analyte of interest.
In the 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 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 adjust the device to ionize samples in various ways. As previously mentioned, acid-base processes are frequently used to ionize samples; however, molecules with conjugated pi systems, such as naphthalene-like compounds, can also used as electron acceptors and serve as a matrix for MALDI/TOF. This is especially useful in analyzing compounds with conjugated pi systems. The most common application for these matrices is the research of porphyrin-like substances such as chlorophyll. These matrices demonstrated to have improved ionization patterns that do not cause unusual fragmentation patterns or full loss of side chains. It is also proposed that conjugated porphyrin-like molecules can act as a matrix and cleave themselves, omitting the requirement for a separate matrix compound.
MALDI Instrumentation
MALDI technology has many variations, and comparable equipment is now built for many applications, from academic and analytical to industrial and high throughput. The mass spectrometry field has grown to include ultrahigh-resolution mass spectrometry, FT-ICR equipment, and more high-throughput instruments. Because many MALDI MS instruments come with interchangeable ionization sources (electrospray ionization, MALDI, atmospheric pressure ionization, and so on), the technologies frequently overlap, and any soft ionization method can be utilized.
Laser
MALDI procedures commonly use UV lasers like nitrogen lasers (337 nm), and frequency-tripled and quadrupled Nd: YAG lasers (355 nm and 266 nm, respectively). Infrared MALDI uses lasers with wavelengths of 2.94 μm Er: YAG, a mid-IR optical parametric oscillator, and 10.6μm carbon dioxide. Infrared lasers, however less common, are utilized because of their milder way of ionization. IR-MALDI also offers the advantage of removing more material (suitable for biological samples), reducing low-mass interference, and being compatible with other matrix-free laser desorption mass spectrometry techniques.
Time of flight
The most common type of mass spectrometer used with MALDI is the time-of-flight mass spectrometer (TOF), because of its wide mass range. The TOF measuring approach is also well adapted to the MALDI ionization process, as the pulsed laser operates in individual shots rather than continuously. MALDI-TOF equipment frequently includes a reflectron (an “ion mirror”) that reflects ions with an electric field. This increases the ion flight path, increasing the time between ions with differing m/z values and improving resolution.
Modern commercial reflectron TOF instruments have a resolving power m/Δm of 50,000 FWHM (full-width half-maximum, defined as peak width at 50% of peak height) or higher.
MALDI was used with IMS-TOF MS to determine phosphorylated and non-phosphorylated peptides. MALDI-FT-ICR MS is an effective approach for obtaining high-resolution MALDI-MS observations.
Atmospheric pressure
Atmospheric pressure (AP) MALDI is an ionization technology (ion source) that, unlike vacuum, functions in a normal atmospheric environment. The fundamental difference between vacuum MALDI and AP-MALDI is the pressure at which the ions are formed. In vacuum MALDI, ions typically form at 10 mTorr or less, whereas AP-MALDI ions are produced under atmospheric pressure. Formerly, the fundamental disadvantage of the AP-MALDI technology compared to standard vacuum MALDI was its less sensitivity; however, ions may be transported into the mass spectrometer with high efficacy, and attomole detection limits have been observed.
AP-MALDI is utilized in mass spectrometry (MS) in many applications, including proteomics and drug discovery. Popular themes addressed by AP-MALDI mass spectrometry include proteomics; mass analysis of DNA, RNA, PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small compounds, and synthetic polymers; similar uses are also accessible for vacuum MALDI equipment. The AP-MALDI ion source can be connected to an ion trap mass spectrometer or a different MS system equipped with electrospray ionization (ESI) or nanoESI source.
MALDI ionization at low pressure produces primarily singly charged ions (see “Ionization mechanism” below). In contrast, ionization at atmospheric pressure can produce highly charged analytes, as demonstrated first with infrared and then with nitrogen lasers. Multiple charging of analytes is critical because it enables the measurement of high-molecular-weight molecules such as proteins in equipment with limited m/z detection ranges, such as quadrupoles. Aside from pressure, the composition of the matrix is essential to achieving this effect.
Aerosol
In aerosol mass spectrometry, one of the ionization methods is to fire a laser at individual droplets. These systems are known as Single-Particle Mass Spectrometers (SPMS). Before aerosolization, the sample can be combined with a MALDI matrix.
MALDI Ionization mechanism
In the dried droplet spot, the laser is fired at the matrix crystals. The matrix absorbs the energy from the laser, and it is assumed that this event predominantly desorbed and ionizes the matrix (by adding a proton). The hot plume formed during ablation contains a variety of species, including neutral and ionized matrix molecules, protonated and deprotonated matrix molecules, matrix clusters, and nanodroplets. Ablated species may participate in analyte ionization, although MALDI’s process is still controversial. The matrix is then assumed to transmit protons to analyte molecules (e.g., protein molecules), hence charging them.
After this process, an ion will be composed of the initial neutral molecule [M] and ions that were added or removed. These are quasimolecular ions i.e. [M+H]+ for added proton, [M+Na]+ for added sodium ion, and [M-H]− for removed proton. MALDI can produce singly charged ions or multiply charged ions ([M+nH]n+), depending on the matrix, laser intensity, and voltage utilized. It is worth noting that all of these species have even numbers of electrons. Ion signals from radical cations (photoionized molecules) can be seen in matrix molecules and other organic compounds.
The gas phase proton transfer model of UV laser MALDI’s, coupled physical and chemical dynamics (CPCD) model, proposes primary and secondary processes that lead to ionization. Primary processes require initial charge separation via photon absorption by the matrix and energy pooling to produce matrix ion pairs. Primary ion production involves the absorption of a UV photon to form excited state molecules:
S0 + hν → S1
S1 + S1 → S0 + Sn
S1 + Sn → M+ + M−
where S0 represents the ground electronic state, S1 is the first electronic excited state, and Sn is the higher electronic excited state. The product ions can be proton or electron transfer ion pairs, represented by M+ and M− respectively. Secondary processes include ion-molecule reactions that produce analyte ions.
The lucky survivor concept (cluster ionization mechanism) proposes incorporating analyte molecules into the matrix while keeping the charge state from the solution. Charge separation happens during the fragmentation of laser-ablated clusters, resulting in the formation of ions. Lucky survivors are those ions that are neutralized by recombination with photoelectrons or counter ions.
The thermal model proposes that in melted matrix liquid, high temperature supports proton transport between matrix and analyte. The ion-to-neutral ratio is an essential factor used to explain the theoretical model, and its incorrect citation may result in a wrong interpretation of the ionization mechanism. The model quantitatively estimates the rise in total intensity of ions as a function of the analytes’ concentration and proton affinity, as well as the ion-to-neutral ratio as a function of laser fluence. This model also proposes that metal ion adducts (e.g., [M+Na]+ or [M+K]+) are mostly formed by the thermally induced dissolution of salt.
The matrix-assisted ionization (MAI) approach produces analyte ions of volatile or nonvolatile molecules by using matrix preparation identical to MALDI but without the use of laser ablation. Simply exposing the matrix containing the analyte to the vacuum of the mass spectrometer produces ions with almost identical charge states as electrospray ionization. It is believed that this mechanism and MALDI share mechanistic similarities.
Ion yields are commonly estimated to be between 10−4 and 10−7, with some studies indicating even lower yields of 10−9. The issue of low ion yields was solved quickly after the launch of MALDI by several approaches, including post-ionization with a second laser. Most of these attempts were only partially successful, with minimal signal increases. This could be related to the working of axial time-of-flight instruments, which operate at pressures in the source region of 10−5 to 10−6, resulting in fast plume expansion with particle velocities up to 1000 m/s.
In 2015, successful laser post-ionization was observed. This used a modified MALDI source operated at a higher pressure of ~3 mbar coupled to an orthogonal time-of-flight mass analyzer. A wavelength-tunable post-ionization laser was also used, operated at a wavelength from 260 nm to 280 nm, below the two-photon ionization threshold of the matrices utilized, which elevated ion yields of many lipids and small molecules by up to three orders of magnitude. This method, known as MALDI-2 due to the second laser and the second MALDI-like ionization process, was later used for various mass spectrometers, all of which had sources operating in the low mbar range.
Applications of MALDI
MALDI has many applications in different fields:
Biochemistry
MALDI is used in proteomics to quickly identify proteins extracted by gel electrophoresis, including SDS-PAGE, size exclusion chromatography, affinity chromatography, strong/weak ion exchange, isotope-coded protein labeling (ICPL), and two-dimensional gel electrophoresis. Peptide mass fingerprinting is the most common analytical application for MALDI-TOF mass spectrometers. MALDI TOF/TOF mass spectrometers are used to discover peptide amino acid sequences via post-source decay or high-energy collision-induced dissociation.
MALDI-TOF is used to study post-translational changes. For example, it is used to investigate protein methylation and demethylation. However, caution must be carried out while investigating post-translational alterations with MALDI-TOF. For example, loss of sialic acid is observed in studies where dihydroxybenzoic acid (DHB) was utilized as a matrix for MALDI MS analysis of glycosylated peptides.
S. Martin used sinapinic acid, 4-HCCA, and DHB as matrices to study the loss of sialic acid in glycosylated peptides via metastable decay in MALDI/TOF in linear and reflector modes. Shimadzu Corporation experts derivatized sialic acid via an amidation reaction to enhance detection sensitivity. They also demonstrated that an ionic liquid matrix minimizes sialic acid loss during MALDI/TOF MS analysis of sialylated oligosaccharides. THAP, DHAP, and a combination of 2-aza-2-thiothymine and phenylhydrazine were found as matrices capable of reducing sialic acid loss during MALDI MS analysis of glycosylated peptides. It is observed that using IR MALDI instead of UV MALDI can reduce the loss of some post-translational modifications.
Aside from proteins, MALDI-TOF is used to analyze lipids. For example, it is used to investigate the catalytic processes of phospholipases. MALDI-TOF is used to characterize both lipids and oligonucleotides. For example, in molecular biology, a mixture of 5-methoxy salicylic acid and spermine can be utilized as a matrix for oligonucleotide analysis in MALDI mass spectrometry, after oligonucleotide synthesis.
Organic chemistry
Some synthetic macromolecules, such as catenanes and rotaxanes, dendrimers and hyperbranched polymers, and other assemblies, have molecular weights in the thousands or tens of thousands, where most ionization techniques have difficulty producing molecular ions. MALDI is a simple and fast analytical approach that chemists can use to quickly analyze and validate the outcomes of such syntheses.
Polymers
In polymer chemistry, MALDI is utilized to calculate molar mass distribution. Polymers with a polydispersity above 1.2 are challenging to characterize with MALDI due to signal intensity selectivity against higher mass oligomers. Dithranol or AgTFA make excellent polymer matrices. The sample must be combined with dithranol first, followed by the AgTFA; otherwise, it will precipitate out of the solution.
Microbiology
MALDI-TOF spectra are commonly used to identify microorganisms like bacteria or fungi. A portion of the microbe’s colony is placed on the sample target and overlaid with the matrix. In biotyping, the mass spectra of expressed proteins are analyzed by specialist software and compared to stored profiles to determine the species. It supports other immunological or biochemical processes and has become a standard method for identifying species in clinical microbiological laboratories. The advantages of high-resolution MALDI-MS performed on a Fourier transform ion cyclotron resonance mass spectrometry (FT-MS) are demonstrated for typing and subtyping viruses using single ion detection known as proteotyping, with a special emphasis on influenza viruses.
One major advantage over other microbiological identification methods is the ability to identify a wide range of microorganisms quickly and accurately from the selective medium used to isolate them at a low price. The lack of the requirement of purifying the suspect or “presumptive” colony allows for significantly quicker turnaround times. For example, MALDI-TOF detects bacteria directly from blood cultures.
Another advantage is the ability to predict antibiotic susceptibility in bacteria. A single mass spectral peak can indicate methicillin resistance in Staphylococcus aureus. MALDI can also identify carbapenemase from carbapenem-resistant Enterobacteriaceae, such as Acinetobacter baumannii and Klebsiella pneumoniae. However, the majority of proteins that regulate antibiotic resistance are larger than MALDI-TOF’s 2000-20,000 Da range for protein peak interpretation, and only in rare cases, such as the 2011 Klebsiella pneumoniae carbapenemase (KPC) outbreak at the NIH, can a correlation between a peak and a resistance-conferring protein be established.
Parasitology
MALDI-TOF spectra have been used to detect and identify several parasites, including trypanosomatids, Leishmania, and Plasmodium. Moreover, MALDI/TOF can be used to identify parasitic insects like lice or cercariae, which are free-swimming stages of trematodes.
Medicine
MALDI-TOF spectra are frequently used together with other analytical and spectroscopic techniques to diagnose diseases. MALDI/TOF is a promising diagnostic tool because it enables the quick identification of proteins and modifications to proteins without the cost or computational capacity of sequencing, nor the skill or time required to determine a crystal structure in X-ray crystallography.
One example is necrotizing enterocolitis (NEC), a fatal disease that affects premature newborns’ guts. The symptoms of NEC are quite similar to those of sepsis, and many newborns die while awaiting diagnosis and treatment. MALDI/TOF was utilized to detect bacteria found in the feces of NEC-positive newborns. The purpose of this study was to characterize the fecal microbiota associated with NEC rather than to investigate disease mechanisms. There is a belief that similar technology can be utilized as a rapid diagnostic tool that does not need sequencing.
Another application for MALDI/TOF is cancer diagnosis. Pancreatic cancer is still one of the most dangerous and difficult-to-identify cancers. Impaired cellular signaling caused by mutations in membrane proteins is recognized to contribute to pancreatic cancer. MALDI/TOF is utilized to identify a membrane protein linked to pancreatic cancer, and it may eventually function as an early detection method.
MALDI/TOF is utilized to direct treatment as well as diagnosis. MALDI/TOF can detect drug resistance in bacteria, particularly to β-lactams (Penicillin family). The MALDI/TOF detects carbapenemases, indicating drug resistance to standard antibiotics. It is believed that the technique might be utilized to identify drug-resistant bacteria in as little as three hours. This method could assist physicians in determining whether to prescribe stronger antibiotics initially.
Detection of protein complexes
Following initial findings that some peptide-peptide complexes could resist MALDI deposition and ionization, studies of large protein complexes utilizing MALDI-MS are reported.
Small molecules
While MALDI is commonly used for large macromolecules, it is also used to examine small molecules with masses less than 1000 Da. The issue with small molecules is matrix effects, where signal interference, detector saturation, or suppression of the analyte signal is possible because matrices are usually made up of small molecules. The choice of matrix is significantly dependent on the molecules to be analyzed.
References
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