Matrix-assisted laser desorption/ionization comprises photochemical (ionization and fragmentation) and mechanical (desorption) events. In MALDI, analytes are incorporated into organic matrices in a solid or viscous phase (ionic liquid matrices). A pulsed laser irradiates the sample to facilitate the ionization/desorption reactions. During laser excitation, matrices take up and redistribute the laser energy for desorption and ionization of analytes. In this section, we first review critical factors determining efficiencies of ionization and desorption. Models proposed for ionization and desorption processes from the perspectives of both experimental observation and theory are discussed. After the review of general MALDI mechanisms, the specific aspects of ionization mechanisms on carbohydrates and methods that improve ion abundances of carbohydrates are reviewed.
Ion production in MALDI
Owing to the high complexity of the MALDI process, a single ionization model that could elucidate phenomenon universally to every MALDI experiment is highly unlikely. Ionization environment varies among different matrix systems as well as excitation conditions.22) For investigating mechanisms in MALDI, the ion-to-neutral ratio (ionization efficiency) is normally discussed.23–26) Typically, ionization efficiency differs across types of analytes. The ionization efficiency for molecules with higher proton affinity than matrices, such as peptides and proteins, is 10−3–10−4.27–32) For analytes with lower proton affinity, such as carbohydrates, the ionization efficiency drops by a few orders of magnitude to 10−7–10−8.23,33,34) A better understanding of mechanisms leads to improvements in the ionization efficiency and increases its effectiveness toward more fields. Over the last three decades, fundamental ionization mechanisms have been systematically studied by manipulating one or more parameters among different matrix systems.22,35) Effects of the incorporation of analytes into matrix solid,36–40) matrix-to-analyte ratio,41,42) solvent constituents,43–47) laser excitation conditions,22,48,49) and the crystal morphology of samples47,50–55) on ion production were investigated.
A prerequisite of successful MALDI processes is absorption of laser energy by matrices. The optimal MALDI-MS sensitivity is generally achieved when the utilized laser wavelength corresponds to the high optical absorption band of the solid-phase matrices.56) After photo-absorption, a two-step ionization process suggests that ionization of matrices is as an initial ionization step, followed by ion–molecule reactions to produce charged analyte species.35,57) The second step is less controversial, and ion yields of analytes can be estimated by relative thermodynamic properties between matrices and analytes.58–61) However, how matrices are initially ionized remains open to debate. Two major arguments of postulated initial ionizations include 1) photoionization in the gas phase (Eq. (1)),35,62–65) and 2) thermal energy promoted ionizations in the condensed phase (Eq. (2)).34,66–74)
in which M and M* represent the matrix in the electronic ground and excited states, respectively. In Eq. (1), the positive matrix radical ion and photoelectrons are generated by multi-photon ionization via
energy pooling/annihilation processes. It should be noted that, although the impact of photoelectrons on the generation of MALDI ion and trigger of fragmentation has been extensively discussed,75,76)
the major source of electrons is still in debate.75,77)
In Eq. (2), absorbed photon energy is converted to thermal energy via
ultrafast non-radiative relaxation. In this equation, an increase in the system temperature facilitates both ionization and desorption. Proton disproportionation of matrices is thermally induced to create protonated/deprotonated matrices. Our group proposed a quantitative thermal model and successfully predicted the laser energy dependence of the ion yield.68,69)
Both thermal and chemical equilibria can presumably be established by collisions on the solid surface. In our thermal model, ion abundance depended on the concentration on the surface and desorption rate constant.
Contrary to the two equations, the lucky survivor model suggests that matrices and analytes are pre-charged in solids.78,79) In this model, the laser energy overcomes ion-pair interactions to free analyte ions from their respective counter ions. It should be emphasized that a single ionization mechanism that is applied universally to elucidate all MALDI observations is highly unlikely owing to different photochemical properties of matrices as well as excitation conditions. Contributions of different ionization channels need to be considered even for a single matrix system in Fig. 1.22)
|Fig. 1. Initial ionization channels, including photoionization and thermally induced ionization, as well as radiative and radiationless relaxation processes after the absorption of laser photons by matrices. Reprinted with permission from ref. 22. Copyright (2012) American Chemical Society.|
Desorption (phase transition)
Desorption in MALDI is the phase transition of samples from the condensed phase to the gas phase. It is a prerequisite for molecules to be detected by MALDI mass spectrometry. Desorption processes, including thermal desorption occurring at low and moderate laser energy density to phase explosion happening at high laser energy density, were studied quantitatively on both experimental and theoretical bases.22,80) Results of these studies suggest that factors affecting the desorption efficiency can be classified into two categories: excitation parameters36,81–86) and intrinsic properties of samples.51,87–92) Generally, desorption efficiency increases when MALDI crystal size decreases51) and the system temperature increases.87–92)
On the basis of factors influencing desorption efficiency, mathematical equations as well as computational methods, such as molecular dynamics (MD) simulations, have been used to quantitatively describe the desorption process.86,93,94) As shown in Fig. 2, Zhigilei et al. utilized MD simulations to describe desorption of MALDI after laser irradiation.95) However, most of the aforementioned desorption models simplified the impact of ionization, which deviates from the fact that MALDI relies on the complex interplay of both desorption and ionization. By utilizing transition state theory to derive desorption rate, our group developed a comprehensive model that both considered desorption and ionization to predict ion abundance as a function of laser fluence,69)
in which, A
, represent the pre-exponential factor, Boltzmann constant, Planck constant, vibrational frequency of the intermolecular bond, and activation energy for desorption, respectively. [ion]s
stands for the concentration of surface ions. T0
is the initial system temperature, and r
is the conversion factor between system temperature and laser energy density. Linear relations between temperature and laser energy density were obtained within typical MALDI conditions.69,86,91)
|Fig. 2. Snapshots from MD simulations of laser ablation of a molecular solid illustrating different mechanisms of material ejection: (a) desorption of monomers; (b) phase explosion of the overheated material; (c) hydrodynamic sputtering due to the fast melting and motion of liquid in the surface region; (d) photomechanical spallation of the surface layer caused by the relaxation of laser-induced thermoelastic stresses. The laser pulse durations are 150 ps (a, b) and 15 ps (c, d), fluences are 34 J/m2 (a), 61 J/m2 (b), 40 J/m2 (c), and 31 J/m2 (d). The laser penetration depth is 50 nm in all simulations. The irradiation parameters correspond to the regime of thermal confinement in (a) and (b) and to the regime of stress confinement in (c) and (d). Reprinted with permission from ref. 95. Copyright (2003) American Chemical Society.|
System temperature is a particularly important factor because it not only affects desorption efficiency but also ionization/fragmentation processes. The predicted system temperature varies with respect to experimental conditions, such as the type of matrices, energy densities, and irradiation wavelengths. Many efforts have been made to evaluate system temperatures of MALDI, such as predictions by theoretical models,62,69,70,86,96) MD simulations,32) the measurement of infrared emission of the system based on the theory of black body radiation,22,91) and the use of thermometer molecules as probes of internal energy.87,88,90,92) Although evaluations by different methods have certain inconsistencies, a consensus of transient MALDI temperature might reach ∼1000 K, which is detrimental to thermally labile carbohydrates.
Mechanistic studies of carbohydrate ionization in MALDI
Unlike proteins or nucleic acids that are protonated or deprotonated, carbohydrates are predominately ionized by forming adducts with alkali metal ions (mainly sodium and potassium ions) in MALDI.97,98) Several groups have attempted both experimentally and theoretically to understand ionization mechanisms of carbohydrates in MALDI. Mohr et al. found that the binding affinities of proton and alkali metal ions to carbohydrates followed the order: H≪Li<Na<K<Cs.99) Since carbohydrates are incorporated into the matrix solid, the relative binding competition between carbohydrates and matrices needs to be considered. The binding affinity of protons to carbohydrates is lower than that to commonly used matrices such as 2,5-dihydroxybenzoic acid (DHB) and 2,4,6-trihydroxyacetophenol (THAP).30,100,101) In contrast to the proton affinity, the sodium affinity of carbohydrates is higher than that of matrices.98,101,102) Thus, it is energetically favorable for carbohydrates to form the adduct with alkali metal ions rather than with protons. A reaction model proposed by Lee et al. suggests that the increase in thermal energy can effectively induce the production of alkali ion adducts of carbohydrates.103) They suggested alkali metal ions were generated from the dissolution of salts at high-temperature MALDI condition. Those alkali metal ions attached to nearby matrices and analytes during desorption. However, mechanisms about how carbohydrates are alkalized are still unclear.104)
Ion yields of carbohydrates are related to multiple parameters, such as reaction enthalpies for forming adducts with alkali metal ions, volatility, and structural stabilities.101,105) Owing to the thermally labile nature of carbohydrates, Chen et al. reported that low ion yield might be the result of rates of fragmentation and/or desodiation (loss of sodium ions back to its neutral form) being faster than sodiation in the high-temperature ionization environment.101) Chen et al. also suggested that protonated carbohydrates were easy to decompose through the dehydration reaction. Cancilla et al. reported that the fragmentation behavior of carbohydrates was also related to their degree of branching.106) A branched glycan can establish multiple bindings to an alkali metal ion, resulting in lesser fragments. Computational methods, including MD simulation, semi-empirical and ab initio calculations, were utilized by Fukui et al. to investigate the relationship between the structure and reactivity (fragmentation) of sodiated oligosaccharides.105) Fukui et al. found that the coordination of metal ions to oligosaccharides affected the propensity for fragmentation. Based on the calculation result, increasing the number of oxygen atoms interacting with the sodium ion stabilized the carbohydrate structure (less fragmentation) and increased the ion yield.
Based on the understanding of mechanisms of MALDI and the nature of carbohydrates, three strategies are generally adopted to improve ion yield of carbohydrates: 1) thermalizing high-temperature ionization environments to reduce dealkalization and fragmentation; 2) doping ionization agents (permanent charges) to enhance ion yield; 3) changing the nature of glycans by derivatizations. We highlighted some of selected advancements in Table 1.
Table 1. Methods to improve ion abundances of carbohydrates in MALDI mass spectrometry.
|Mediating high-temperature ionization environment||Soft ionization by ionic liquid matrices (ILMs)|
|–1,1,3,3-Tetramethylguanidium (TMG) salt of p-coumaric acid (G3CA)||107|
|–3-Aminoquinoline (3-AQ) based ILM||108, 109|
|–2,4,6-Trihydroxyacetophene (THAP) based ILM||110|
|Ice as a soft matrix||111|
|Temperature-controlled sample plate allowing a frozen sample inside the vacuum||112|
|Trylayer sample preparation method with thermal energy-dissipating diamond nanoparticles||113|
|Doping ionization agent||Ammonium salts||114, 115|
|Introducing permanent charges with succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-AcOSu)||116|
|Nanoparticles containing ionization agents||117–123|
|Chemical derivatization||Permethylation||124, 127, 135|
|Amidation||126, 128, 132|
|Derivatization with hydrazines||125, 129|
The selection of matrices is a crucial step for the analysis of carbohydrates because every matrix produces unique ionization environment after the laser irradiation.22) Problems associated with decomposition of thermally labile moieties and low ionization efficiency could be overcome by a suitable selection of the matrix. As shown in Fig. 3, “cool” ionic liquid matrices: 1,1,3,3-tetramethylguanidium (TMG) salt of p-coumaric acid (G3CA),107) and 3-aminoquinoline/p-coumaric acid (3-AQ/CA)108,109) were developed by Tanaka and coworkers to yield a less locally heated ionization environment for enhancing the sensitivity and suppressing the losses of sialic acid moieties. Applying a similar concept, THAP-based ionic liquid matrices110) were also reported for automated identification and quantitative analysis of native oligosaccharides. Witt et al. utilized ice as an extremely soft matrix to prevent carbohydrates from decomposition and enhance the capability of MALDI mass spectrometry for the structural characterization of glycans.111) The use of ice as a matrix is advantageous because it provides an ideal “near-physiological” condition. Liang et al. modified the ion source of commercial MALDI time-of-flight mass spectrometer to enable the preparation of a frozen carbohydrate/DHB sample at 100 K. Enhancement of sensitivity (20–30 folds) and reduction of fragmentation were achieved by this method.112) Our group developed a trilayer sample preparation method, which changes sample morphology by consecutively depositing matrices, diamond nanoparticles, and carbohydrates. Owing to the high thermal conductivity, diamond nanoparticles thermalized the MALDI temperature. Diamond nanoparticles also adjusted the incorporation of matrices and carbohydrates. As shown in Fig. 4, we selectively enhanced the sensitivity of underivatized carbohydrates by more than an order of magnitude from protein/carbohydrate mixtures.113) This sample preparation method is potentially suitable for diagnosing carbohydrates in real samples because carbohydrates are often accompanied by proteins in real biological samples.
|Fig. 3. Positive-ion mass spectra of 100 fmol fetuin GP1, 100 fmol NA4 glycan, and 10 fmol β-casein 1–25 using (a, d, g) 3-AQ/CA, (b, e, h) 3-AQ/CHCA, and (c, f, i) 2,5-DHB. Reprinted with permission from ref. 109. Copyright (2014) American Chemical Society.|
|Fig. 4. Mass spectra of 5 pmol each of dextran and ACTH fragment 18–39 obtained from various samples, including (a) dried-droplet, (b) thin-layer, and (c) trilayer samples. The numbers indicate the degrees of polymerization (n) of sodiated dextran. Reprinted with permission from ref. 113. Copyright (2013) American Chemical Society.|
In the second category, Yamagaki et al. improved ionization efficiency of neutral glycans by adding ammonium salts in MALDI samples to form chloride-anionized molecules.114) In a similar fashion, doping salts containing anions, such as NO3− and Cl−, was conducted by Domann et al. to enhance the ionization efficiency of neutral N-linked glycans.115) Domann et al. reported the best matrix for ionizing glycans was mixing THAP with ammonium nitrate (1 M) to form [M+NO3]−. Gao et al. reported a derivatization method that enhanced 20- to 50-fold ion abundance of N-linked glycans by introducing permanent charges using nonspecific proteolysis with pronase E and co-derivatization with succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-AcOSu) and methylamidation.116) Nanoparticles (NPs), such as gold, silver, titanium dioxide, iron oxide (Fe3O4), zinc oxide, and manganese oxide, have been utilized for the analysis of carbohydrates because they enhance ionization efficiency by releasing ionization agents.117–121) After absorbing laser energy, NPs also convert photon energy to thermal energy to facilitate material desorption. Charge and energy transfer from NPs to analytes triggers ionization/fragmentation and desorption of carbohydrates.117,122) Furthermore, functionalization of NPs with conventional organic matrices have been reported to serve as new matrix materials to preserve the intact glycan as well as the generation of extensive cleavages in MALDI.123) More details about methods on the structural identification of carbohydrates are reviewed in the following section.
Chemical derivatization has been accepted as a well-established option for the analysis of carbohydrates in MALDI124) because chemical derivatization protects glycans from losing acetyl groups or sialic acid moieties to increase carbohydrate ionization efficiency.125,126) Hakomori first demonstrated permethylation of complex carbohydrates.127) Derivatization was also reported to be beneficial to compensate differences in ionization efficiency. A novel amidation using acetohydrazide was demonstrated by Toyoda et al. to eliminate the discrepancy of ionization efficiency between α2,3- and α2,6-linked sialic acids-containing oligosaccharides.128) Derivatization also improves chromatographic analysis by reducing the polarity of glycans. Various methods for derivatization have been developed, such as uses of hydrazines129) and other labeling techniques.130)
Although derivatization provides advantages, conventional derivatization typically requires harsh conditions (strong base) and long reaction times. Most derivatization methods require clean-up procedures for the removal of chemicals, resulting in a loss of samples, which especially leads to a serious problem for trace analysis.131) Thus, derivatization methods have been improved to meet the demand of low sample loss and high efficiency.132) Miura et al. reported a solid-phase methyl esterification of sialic acid residues of glycans for rapid and quantitative glycoform profiling by MALDI-TOF MS.133) As shown in Fig. 5, the workflow of their on-bead/on gold nanoparticle derivatization is potentially suitable for the large-scale glycomics study. Using a combination of carboxylic acid activators in ethanol, Reiding et al. achieved nearly complete ethyl esterification of α2,6-linked sialic acids and lactonization of α2,3-linked variants in a short time and mild conditions.134) A solid-phase miniaturized approach was demonstrated by Kang et al. for quantitative permethylation of oligosaccharides in less than a minute.135) By mixing analytes with methyl iodide in dimethyl sulfoxide solution, this method avoided excessive clean-up and yielded picomole-level sensitivity.
|Fig. 5. Workflow for “on-bead methyl esterification” of sialic acids for immobilization and recovery of oligosaccharides. Reprinted with permission from ref. 133, John Wiley & Sons, Inc. (2007).|