1 Introduction
mass spectrometry is used to deduce the molecular structure by accurately determining the mass-to-charge (m/Z) ratio of the ions. Because of its high sensitivity, high resolution and high specificity, it is widely used to identify the structure of small molecular drugs, proteins, nucleic acids, lipids and carbohydrates
[1⇓⇓~4]. Because mass spectrometry is the detection of charged particles, the development of new Ionization/Dissociation techniques is the key to the development of mass spectrometry instruments and analytical methods. In view of the close relationship between Dissociation and Ionization, in this paper, the properties, characteristics and further dissociation ways of molecular ions produced by various ionization techniques are analyzed on the basis of reviewing various ionization techniques, and then the Photo Dissociation and its combination with various Soft Ionization techniques are emphatically described.
2 Summary of Mass Spectrometric Ionization Methods
At present, ionization techniques widely used in mass spectrometry are mostly based on the principles of electron gain/loss, protonation/deprotonation or formation of metal ion adducts. Table 1 summarizes the characteristics and applications of major ionization techniques such as electron impact/capture ionization, electrospray ionization, matrix-assisted ionization, surface ionization and atomic/ion beam ionization.
2.1 Electron impact/capture ionization
Electron Impact Ionization (EI), established by Dempster in 1918, is the most classical Ionization method, which is widely used in gas chromatography. In this method, the hot electrons emitted from the filament are accelerated, and the high-energy electron beam collides with the neutral molecules, causing them to lose their low ionization potential electrons and produce cationic radicals with high reactivity, which are further subjected to homolytic bond cleavage or heterolytic bond cleavage initiated by the free radical centers or positive charge centers, or accompanied by molecular rearrangement. The high-energy electron bombardment method (generally using 70 eV electron beam) is usually called "hard ionization", because the de Broglie wavelength of 70 eV electrons is about 0.14 nm, which is equivalent to the bond length of general chemical bonds. The strong interaction between high-energy electrons and molecules often causes non-specific chemical bond breaking activated by vibration, which affects the fragment ions in the analysis spectrum
[5]. In 2012, Zhong Hongying et al. Reported that low-energy Electron trapping is a new soft ionization method, namely Laser Activated Electron Tunneling (LAET), which uses photoelectrons generated by Laser irradiation on the semiconductor surface to achieve ionization and dissociation of neutral molecules
[6⇓⇓⇓~10]. In the electrostatic field, the photoexcited electrons jump from the valence band to the conduction band of the semiconductor, and the interface-transferred photoelectrons are captured by the electron-deficient atoms adsorbed in the sample molecules on the surface of the semiconductor, resulting in anion radicals with one unpaired electron, and further dissociation initiated by radical centers or negative charge centers. In contrast to high-energy electron bombardment, electron capture is an exothermic process that can proceed spontaneously and therefore does not require high energy, and low-energy electron (< 20 eV) capture effectively reduces vibrationally activated nonspecific chemical bond cleavage. Both ionization modes initiated by electron gain and loss produce radical cations or radical anions with unpaired electrons, and these types of molecular ions are unstable.The radical or charge center initiated chemical bond cleavage can further occur without other additional devices, thereby obtaining the characteristic fragment ions required for structure identification.
2.2 Electrospray ionization and matrix-assisted ionization
Electrospray Ionization (ESI) reported by Fenn et al in 1984 and Matrix Assisted Laser Desorption Ionization (MALDI) reported by Tanaka et al in 1988 can ionize neutral molecules by protonation/deprotonation or adduct formation with cations such as metal ions Matrix Assisted Laser Desorption Ionization, K
+ and Li
+[11][12]. Compared with the hard ionization technique, this soft ionization method has little damage to the sample and is widely used in the analysis of biological macromolecules, especially in the analysis of amino acid sequences and post-translational modifications of proteins
[13⇓⇓~16]. These two soft ionization techniques have greatly advanced biomedical research, and Tanaka was awarded the Nobel Prize in Chemistry in 2002. Similar to electrospray ionization/matrix-assisted ionization, proton transfer/charge reduction (PTR) technique was established by Lindinger et al. In 1995, which is mainly used to analyze volatile gaseous organic molecules with proton affinity higher than that of water
[17]. According to the technology, the hollow cathode is used for discharging water vapor, and the generated high-purity H
3O
+ is subjected to a molecular-ion reaction in a drift tube, so that the technology can be used for real-time on-line detection of atmospheric trace organic pollutants, and can also be used for auxiliary diagnosis of diseases such as lung cancer and the like
[18]. The quasi-molecular ions produced by these three methods are even-electron ions, which are more stable than cation/anion radicals with odd electrons, and are generally difficult to dissociate spontaneously, so there are fewer fragment ions in the spectrum. In order to obtain more structural information, it is necessary to combine with other dissociation techniques to further decompose these quasi-molecular ions into fragment ions.
2.3 Surface ionization
Surface Enhanced Laser Desorption Ionization (SELDI), proposed by Bill Hutchens and Tai Ying Yip in 1993, is a Surface Ionization method developed on the basis of matrix assisted ionization.Combine MALDI with protein chip, using chemical (ion exchange, reverse/forward separation or metal ion coordination, etc.) Or biochemical (antibody or receptor, etc.) Technology to modify that target surface of the sample, selectively binding the target molecules in the sample, and using appropriate solvent to remove other coexisting components
[19][20]. Proteins retained on the chip are co-crystallized with organic small molecule matrices that can absorb laser energy. Under the irradiation of specific wavelength laser, the target molecules to be detected are usually ionized from the protonation pathway. The protein chip SELDI technology has the characteristics of high integration, miniaturization, automation, high throughput and the like, and can be widely applied to the fields of disease marker screening, early diagnosis, new drug research and development and the like when combined with a time-of-flight mass analyzer
[21,22].
Because the organic small molecule matrix used in MALDI produces large background interference at the low mass end, and the laser thermal effect makes the actual dissociation area larger than the laser spot area, which affects the spatial resolution of mass spectrometry analysis. DIOS (Desorption Ionization on Porous Silicon), reported by Buriak et al in 1999, is a new matrix-free surface Ionization technique. This technique utilizes nanostructured Porous Silicon to absorb laser energy and ionize the molecules to be detected, which effectively overcomes the matrix interference problem of MALDI and can be used for mass spectrometry of small molecular metabolites
[23][24⇓~26]. In 2007, Siuzdak et al reported another matrix-free surface ionization technique, which is called Nanostructure-Initiator Mass Spectrometry (Nanostructure-Initiator).NIMS) aggregates initiator molecules such as bis (tridecafluoro-1,1,2,2-tetrahydrooctyl) tetramethyldisiloxane in the "clathrate" nanostructures on the porous silicon surface. When irradiated by ion beams or light, the initiator molecules are thermally volatilized and the sample molecules adsorbed on the surface are ionized
[27]. Because the starting molecule is very stable, it does not produce background ion interference. This soft ionization technique can be used not only for the analysis of small molecule metabolites, drugs, proteins and peptides, but also for cell and tissue imaging. Blood and urine samples can be analyzed directly because of the simple pretreatment of samples
[28⇓⇓~31]. These surface ionization methods are all soft ionization techniques, and the produced quasi-molecular ions are even-electron ions with high stability, which generally can not spontaneously break chemical bonds, and need other dissociation techniques to further produce fragment ions.
2.4 Atomic/ion beam ionization
The atom/ion beam ionization method uses the interaction between the atom/ion beam moving at a high speed and the molecule. Fast Atom Bombardment (FAB), reported by Barber et al in 1981, uses an atomic beam (Ar or He) with an energy of 3 ~ 10 keV to bombard liquid or solid sample molecules mixed with the matrix. Similar to MALDI, FAB is a soft ionization technique, which also produces even electron ions with high stability and is difficult to form fragment ions
[32]. Both use atomic beam and laser bombardment of sample molecules, respectively, but FAB is less sensitive than MALDI and cannot analyze proteins with molecular weights greater than 10 kDa
[33].
Ion beam ionization technology includes two types, one belongs to hard ionization technology, and the other belongs to soft ionization technology. Secondary Ion Mass Spectrometry (SIMS) is a hard ionization technique in which the Ion beam (Secondary Ion Mass Spectrometry, a
, B
, Cs
+, etc.) With energy of several kilo electron volts bombards the sample surface. There are a series of physical and chemical changes including the scattering of the ion beam on the sample surface, the sputtering of the sample particles and the chemical reaction on the surface of the adsorption layer
[34]. The primary ions bombarding the sample undergo elastic or inelastic collision with lattice atoms in the process of penetrating the surface of the solid sample, and after the lattice atoms acquire energy,Some of them move to the surface and transfer energy to the surface sample to sputter positive and negative ions such as atoms, atomic groups, elemental ions, fragment ions or molecular ions, or produce secondary ions through surface chemical reactions. SIMS can be used to identify chemical elements and compounds on the surface or inside of samples, and to image the surface of solid materials or biological tissues and cells
[35]. By alternately sputter and stripping that surface of the sample and acquire a sputter area map, depth profiling of different components can also be performed to obtain a three-dimensional image of the micro-area distribution of chemical component
[36⇓~38]. Secondary ion mass spectrometry (SIMS) has a high sensitivity down to ppm or even ppb, and a spatial resolution as low as 1 nm
[39]. However, the energy of ion beam is as high as several thousands of electron volts, which causes the non-specific chemical bond cleavage activated by vibration and produces a large number of fragment ions, so it is difficult to obtain quasi-molecular ions of intact biomolecules. According to the ion beam current density, SIMS is divided into static SIMS(Static-SIMS,>10
13 (ion /cm
2) and Dynamic-SIMS (> 10 ion /cm
2), which are respectively used with Time-of-Flight (TOF) and double-focusing mass analyzers. Since the first TOF-SIMS was developed by Benninghoven, a German scientist, in 1979, the fifth generation of TOF-SIMS has been developed
[40]. During this period, different ion sources have been developed for different analytical purposes, including gas discharge sources, surface ionization sources, thermal gap sources and liquid metal and cluster sources, which are widely used in the fields of materials, energy and biomedicine
[41]. Using an electron neutralization gun based on the charge compensation principle, SIMS can even analyze insulating samples
[42].
Desorption Electrospray Ionization (DESI) is a liquid ion beam Ionization technique developed by Cooks et al. In 2004. This normal-pressure soft ionization technique integrates ESI and Desorption
[43]. The organic solvent is sprayed with high-speed nitrogen at a certain voltage, the liquid ion beam composed of protonated cations or metal ion adduct cations impacts the surface of the sample, sputtering occurs, and the droplets carrying sample information enter the gas phase. Under the action of nitrogen purging and drying, the droplet containing the analyte is desolvated and enters the mass analyzer along the ion transfer tube. DESI can be used to analyze complex sample structures, chemical imaging and real-time dynamic detection of chemical reactions at atmospheric pressure, but its spatial resolution is limited by droplet size, and sample ionization efficiency is inhibited by solvents and high salts
[44⇓~46].
Direct Analysis Real Time (DART), invented by Laramee and Cody in 2005, is a surface non-contact atmospheric pressure gaseous atom/ion beam hybrid mass spectrometry ionization technique
[47]. Under atmospheric pressure, a neutral or inert carrier gas (such as nitrogen or helium) is discharged to generate ions, electrons and long-lived electronically excited atoms or molecules, and most of the ions and electrons are removed when passing through an ionization zone, leaving long-lived electronically excited atoms or molecules. When the carrier gas is nitrogen, these excited atoms or molecular beams will undergo Penning ionization with water molecules in the air when they impact the sample, resulting in hydrate ion clusters and electrons. In the positive ion mode, the hydrate ion clusters undergo an instantaneous proton exchange reaction with the molecules of the sample analyte to produce protonated cations. In the negative ion mode, the hot electrons produced by Penning ionization react with oxygen and water molecules in the atmosphere, and the resulting ionized clusters of oxygen and water molecules react with the molecules of the analyte and produce deprotonated negative ions. Both DART and DESI are soft ionization techniques at atmospheric pressure. Solvents and high salt samples have little effect on DART, especially they do not produce multi-charged ions and adducts of metal ions (such as Na
+, K
+ and Li
+). The spectra are simple and easy to identify, which can be used for qualitative structure identification and quantitative analysis
[48⇓⇓~51].
To sum up, ionization methods based on different physical and chemical principles have their own characteristics and are suitable for different analytical purposes. In addition to laser-induced tunneling electron capture soft ionization (LAET), electron impact hard ionization (EI) and secondary ion hard ionization (SIMS), several other common soft ionization techniques (such as ESI and MALDI) produce stable even-electron ions, which need to be combined with other dissociation techniques to achieve further dissociation of molecular ions.
3 Overview of Mass Spectrometric Dissociation Techniqu
Collision activation, electron gain and loss, and optical radiation are three common dissociation methods in mass spectrometry, which use different principles to produce different fragment ions.
Collision Activated Dissociation (CAD) or Collision Induced Dissociation (CID) is the most classical ion Dissociation technique in Tandem MS/MS
[52⇓⇓⇓⇓⇓~58]. Ions with a certain energy collide with inert gas molecules or atoms in the collision chamber, causing the chemical bonds activated by the collision to break and producing fragment ions. The kinetic energy of ions directly affects the efficiency of collision-activated dissociation. Magnetic mass spectrometers generally use high-energy CAD, and the ion acceleration voltage is more than 1000 V
[59]. Low-energy CID is generally used in quadrupole and ion trap, and the acceleration voltage is not more than 100 V
[60]. For example, in the positive ion mode, high-energy CAD cleaves three different chemical bonds in the polypeptide backbone, C-C, C-N and N—C
α, and the resulting spectrum contains all X, y, Z, a, B and C fragment ions, while low-energy CID generally forms mainly B and y ions (Fig. 1). CAD or CID is an ergodic process of vibrational energy levels near thermodynamic equilibrium conditions, that is, the absorbed energy is distributed to all vibrational energy levels according to thermodynamic statistical laws.Due to the lack of selectivity of chemical bond cleavage, the dissociation of glycosyl, phosphate and lipid groups covalently bound to proteins occurs simultaneously with the cleavage of chemical bonds in the backbone, resulting in the loss of important fragment ion information related to post-translational modifications. Moreover, for cations such as non-trypsin-cleaved peptides, high-quality long peptides and highly basic peptides, it is difficult for collision activation/induction to effectively break chemical bonds and produce fragment ions.
It is an important way to improve the dissociation efficiency by introducing electrons into the cations to transform the stable even-electron ions into unstable odd-electron ions. Electron Capture Dissociation (ECD) proposed by Zubarev and McLafferty in 1998 and Electron Transfer Dissociation (ETD) established by Hunt and Syka in 2004 are both non-ergodic Dissociation techniques far from thermodynamic equilibrium
[61]. In the ECD technique, a low-energy free electron (usually less than 1 eV) interacts with a protonated protein or peptide ion. When the electron is close to a positive ion, the electrostatic Coulomb force causes the electron to be captured by the protein or peptide cation, converting it from an even-electron ion to an odd-electron ion, and further causing radical-initiated N — Cα bond cleavage to form C and Z type ions (Fig. 1). This approach enhances the ability of non-tryptic cleavage of peptides, long peptides, and highly basic peptides to generate fragment ions, and preserves weaker post-translational modification groups. However, the coexistence of cations and free electrons is facing great difficulties, which limits the wide application of ECD in proteomics and can only be used in FTICR-MS
[62⇓⇓~65]. In order to solve this problem, Chemical Ionization (CI) is used to produce anions carrying electrons, which interact with protein or peptide cations and initiate Chemical bond cleavage similar to ECD
[66⇓~68]. ETD technology has been widely used in linear ion trap mass spectrometers, which can produce fragmented ions in 100 ~ 300 ms and is suitable for high-throughput proteomics analysis
[69]. Like ECD, ETD can selectively cleave the main chain, not only to obtain abundant C, Z sequence ions, but also to retain post-translational modification groups, including phosphorylation modification, N- and O-glycosylation modification, sulfonation modification and so on, which provides an effective means for the analysis of protein amino acid sequence and post-translational modification
[70][71][72]. ETD technology has significantly improved the ability to analyze large peptides or intact proteins with multiple charges, highly basic proteins such as histones, and has great application prospects in top-down proteome analysis and de novo sequencing
[73][74][75].
Compared with the above ionization/dissociation methods, the photoionization/dissociation technology uses light radiation with adjustable wavelength/energy to ionize sample molecules and activate the breakage of specific chemical bonds.This review focuses on the basic principles, instrumental features and applications in the structure determination of biomolecules (including small organic molecules, proteins, nucleic acids and sugars).
4 Basic principle of photoionization/dissociation
Under the irradiation of photons of different wavelengths (X-ray, ultraviolet light, infrared light and microwave), sample molecules or atoms (gas, liquid or solid) can be ionized through a variety of channels. When the photon energy is greater than the ionization energy, the direct ionization of molecules or atoms by absorption of photons is the main photoionization channel. Among them, infrared and microwave need to be excited step by step, so that atoms and molecules gradually enter the highly excited state and ionize. The final product ions of photoionization/dissociation depend on the competition between the multiphoton absorption process and the ionization, energy relaxation and dissociation processes, and the fragment ion species depend on the laser intensity, wavelength and pulse width. When the pulse width and the excitation wavelength are fixed, it is very convenient to control the degree of dissociation by adjusting the laser intensity. In addition, excited atoms and molecules can also be ionized by collisions with electrons, ions, neutral particles and the same kind of particles.
4.1 Direct photoionization and dissociation
Laser desorption Ionization is an early application example of photoionization. When a laser with a certain wavelength and pulse width directly irradiates a sample, neutral molecules absorb photons and undergo Photo ionization (PI), losing electrons with low ionization potential and producing cation radicals. If more than one photon is absorbed and enough energy is obtained, further photoionization/Dissociation (Photo Ionization Dissociation, PID) can occur, that is, the homolysis of chemical bonds initiated by free radical centers, the heterolysis of chemical bonds initiated by charges or the non-specific cleavage of chemical bonds activated by vibration can occur to produce positive ions and negative ions. If the sample itself is a positive ion such as methyl violet hydrochloride, as shown in fig. 2 (A), the thermal effect of the laser vaporizes it, Photo Desorption (PD) occurs, and it enters the mass analyzer. Laser Desorption Ionization (LDI) has no matrix interference and can be used for the analysis of small organic molecules
[76⇓⇓⇓~80]. Fig. 2 (B) shows the structural formula and photoionization mechanism of Methyl Violet, the main dye of ballpoint pen. This molecule has a group that can absorb laser (337 nm). The electrons in the bonding orbital absorb photons and jump to the anti-bonding excited state orbital. The excited state molecule loses electrons and generates m/Z 372 cation radical ions. Because the charge can be dispersed and delocalized in the macrocyclic conjugated system, the quasi-molecular ion has higher stability, is a base peak in the mass spectrum, and generates less fragment ions. The LDI technique requires the sample molecule to have a group that can absorb the laser energy. Because the laser directly irradiates the sample molecule, it is only suitable for photostable samples. In contrast, matrix-assisted laser desorption ionization (MALDI) is more common, which uses small organic molecules as matrix to co-crystallize with sample molecules, not only to absorb laser energy and protect the sample, but also to provide protons to protonate the sample molecules and convert the neutral molecules of the analyte into cations.
4.2 Combination of Photodissociation and Other Ionization Techniqu
For stable even-electron ions produced by other ionization techniques, such as protonated cation and metal ion adducts, if the ergodic high-energy collision activated dissociation (CAD) technique is used, the vibrationally activated non-specific chemical bond rupture is often caused. In recent years, photodissociation technology has been developed, which can improve the selectivity of chemical bond cleavage by using the selective light absorption of characteristic functional groups. Depending on the wavelength used, the photodissociation technique is mainly applied in the infrared and ultraviolet regions
[81].
Infrared Multiphoton Dissociation (IRMPD) is a typical photodissociation technique, which can be used to detect post-translational modifications of proteins, especially Phosphorylation and Sulfation, which are difficult to be detected by CAD.IRMPD absorbs multiple photons continuously, so that the even-electron quasi-molecular ions gradually enter the highly excited state, and the chemical bond cleavage activated by the vibration of infrared light with specific wavelength occurs, which is a "slow heating" fragmentation principle
[82,83][84][85]. Using the protonated diglyme cation as an example, fig. 3 compares the electron impact ionization and the photodissociation that occurs under 944 cm
-1 infrared radiation
[86]. Fig. 3 (A) shows that under electron bombardment, the neutral molecule loses an electron with a low ionization potential to become a cation radical m/Z 134. Due to the instability of this ion, this molecular ion is difficult to detect. The homolysis and heterolysis of the chemical bond are initiated by the radical center and the positive charge center, resulting in m/Z 59 and 89 fragment ions, respectively. Due to the strong absorption of C — O — C stretching vibration in the 1150~900 cm
-1 of the infrared fingerprint region, the adjacent C — O bonds of the C positive charge center undergo vibrationally activated heterolysis under 944 cm
-1 infrared radiation, as shown in fig. 3 (B), and the two positive charge centers produce m/Z 59 and 103 positive ions, respectively. Moreover, the positive ion of m/Z 103 can continue to absorb infrared light and break similar chemical bonds to obtain the positive ion of m/Z 59. It was also found that in the range of 933~953 cm
-1, the excitation wavelength had no effect on the fragmentation pathway, but affected the dissociation efficiency. The intensity of m/Z 59 fragment ion is directly affected by the laser irradiation time at the 944 cm
-1. The longer the irradiation time, the stronger the m/Z 59 fragment ion peak. It can be seen that the infrared multi-photon dissociation technology selectively enhances the chemical bond cleavage initiated by the positive charge of cations based on the absorption of specific functional groups to specific wavelength radiation.
The energy of each Ultraviolet photon is much larger than that of infrared photons. After absorbing ultraviolet photons, molecules can accumulate energy to reach the ionization threshold in nanoseconds, and Ultraviolet Photo Dissociation (UVPD) initiated by single photon occurs. This dissociation greatly reduces the ion collision inactivation, and the dissociation effect is similar to HCD (high energy collision induced dissociation). Ultraviolet photodissociation is also divided into Vacuum Ultraviolet Photo Dissociation (VUVPD) and near Ultraviolet photodissociation according to the wavelength of ultraviolet light. The most commonly used ultraviolet wavelengths for near ultraviolet photodissociation are 266 nm (4.61 eV) and 355 nm (3.49 eV). Ultraviolet light near 260 nm can be absorbed by aromatic amino acids, including tryptophan, tyrosine and phenylalanine. As shown in fig. 4, a polypeptide contain a tyrosine residue undergoes a specific homolytic cleavage of that C
α—C
β side chain, result in a neutral loss of 107 Da
[87]. Near-UV photodissociation is rarely used because many biomolecules cannot directly absorb near-UV light and need to introduce chromophores. Vacuum ultraviolet photodissociation has higher photon energy and can be absorbed by most biomolecules, so it has been widely used
[88⇓~90]. Commonly used 157 nm and 193 nm lasers can provide photon energies up to 7.9 eV or 6.4 eV, respectively, but need to be used under vacuum conditions to avoid absorption of light by air
[91,92][93].
5 Structure and Characteristics of Photodissociation Mass Spectrometer
Photodissociation techniques are often combined with electrospray ionization and matrix-assisted laser ionization techniques to enhance their ability to produce characteristic fragment ions. As previously describe, neutral sample molecules are convert to even-electron ions by different ionization modes, either by protonation/deprotonation or by forming metal ion adduct, with further dissociation depending on that wavelength (photon energy) and intensity (number of photons) of the absorbed light. Increasing the optical path length and the effective collision cross section to make the photons fully interact with the even-electron ions is the key to the reasonable design of the optical path, which directly affects the dissociation efficiency of the quasi-molecular ions in the sample.
5.1 Infrared multiphoton dissociation mass spectrometer
Infrared multiphoton dissociation technology is mainly used in ion traps and Fourier transform ion cyclotron resonance mass analyzers. CO
2 laser is often used as the light source of infrared multiphoton dissociation, and its 10.6 μm wavelength infrared laser can trigger the vibration activation of C — C, C — N, C — O, P — O and other chemical bonds widely existing in biomolecules
[94]. ZnSe is not only the best optical material for high power CO
2 lasers, but also an optical component of multiphoton infrared dissociation mass spectrometers because of its low absorption of 10.6 μm infrared light and high thermal shock resistance.
Infrared multiphoton dissociation (IRMPD) can be used with three-dimensional and two-dimensional ion traps (linear ion traps), and it has three significant advantages over collisionally activated dissociation (CAD)
[95]. First, in conventional CAD analysis, a higher RF voltage is usually required to ensure the energy required for the collision activation of the parent ions, but this reduces the ability to detect ions with a low mass-to-charge ratio. Although the RF voltage can be used as low as possible to capture ions, it is still unavoidable to sacrifice these fragment ions with a low mass-to-charge ratio (m/Z). However, IRMPD is independent of RF voltage, and the photodissociation technique can produce fragment ions that cannot be observed in CAD. Second, when the CAD technique is used in an ion trap, only one auxiliary voltage excites the parent ion resonance, resulting in that fragment ions having different resonance frequencies from the parent ion cannot be activated. IRMPD is a dissociation mode independent of ion resonance. Its parent ions and fragment ions will absorb photons and initiate the dissociation of secondary ions, which can obtain more abundant fragment ions than CAD. Thirdly, IRMPD is not a collision-activation-initiated dissociation mode and does not initiate translational activation, which effectively reduces the loss of ions caused by particle scattering or unstable ion trajectories when using CAD. After passing through the small window of ZnSe, the infrared laser enters the ion trapping chamber through the small hole of the ring electrode or the end electrode. The radiation time is usually between 2 ms and several seconds, which is mainly determined by the laser power, beam diameter, dissociation degree, ion trap pressure and the infrared absorption ability of the target ions
[96,97]. In the IRMPD technique, due to the low energy of infrared photons, sample ions need to absorb multiple photons over a period of time (ms) before they can be activated, and an environment without ion collisions is needed to avoid ion deactivation during light irradiation. However, there is often 10
-3Torr helium in the ion trap, and the collision between helium and ions consumes ion energy to achieve the purpose of damping ion motion, which is a common strategy of ion trap technology to trap ions in the center of the trap. In this case, how to improve the photodissociation efficiency becomes the key problem to be solved in IRMPD. The existing instruments mainly improve the photodissociation efficiency by increasing the optical path, increasing the laser power, thermal assistance or reducing the pressure to reduce the collision deactivation
[86][98][99][100]. In addition, the fragment ions are too small, which is another problem faced by IRMPD. Because infrared multi-photon dissociation has no ion-selective function, the product ions of molecular ion dissociation will continue to absorb photons, and the fragmentation reaction will continue, resulting in some fragment ions too small, which makes it difficult to analyze the spectrum and molecular structure. The axial expansion method developed by Payne et al. Uses auxiliary RF resonance to excite the product ions, increase their amplitude in the z-axis direction, and guide these product ions to move to the edge of the ion trap, so that the parent ions remain in the laser path to absorb laser energy and undergo fragmentation reactions
[99].
Infrared multiphoton dissociation technique can be efficiently applied to Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometry
[100]. FTICR mass spectrometry was first reported by Marshall and Comisarow at the University of British Columbia in 1974 in Chemical Physics Letters
[101]. Ion cyclotron motion is an important experimental basis for FTICR mass spectrometric analysis. The equation of motion ω
c=qB/m (where ω
c is the angular frequency of ion cyclotron motion, B is the magnetic field strength, Q is the ion charge, and m is the ion mass) indicates that the angular frequency of ion cyclotron motion is only related to the ion charge-to-mass ratio Q/m and the applied magnetic field strength B. If a linear accelerator is used, the particles are accelerated step by step along a nearly linear trajectory, and the longer the linear distance, the higher the kinetic energy gained by the accelerated ions. In 1930, Ernest Lawrence, a professor of physics at the University of California, Berkeley, put forward the cyclotron theory, which greatly improved the efficiency of the accelerator. The ion kinetic energy up to 1 MeV can be obtained by using a small electric field. Lawrence also won the Nobel Prize in Physics in 1939 for his outstanding achievements in the cyclotron
[102][103]. Based on the cyclotron principle, ions can be excited to a larger orbit radius when the frequency of the applied transverse alternating electric field is equal to the frequency of the ion cyclotron. A Penning Trap for trapping ions in FTICR mass spectrometry has excellent performance of ion manipulation. It uses a uniform axial magnetic field and a non-uniform quadrupole electric field to Trap ions, that is, a broadband radio-frequency signal (chirp) including all ion cyclotron frequencies is introduced into the excitation plate.All ions are resonantly excited to a larger radius to do rotary motion, the image current generated by the ions on the receiving plate comprises all ion information, the time domain signal can be converted into a frequency domain signal through Fourier transform, and a mass spectrogram with mass-to-charge ratio (m/Z) as an abscissa and signal intensity as an ordinate is obtained. Compare with a linear ion trap, that Penning trap has high vacuum degree and long ion trapping time, can effectively reduce collision cooling, and can realize continuous multi-photon absorption and step-by-step excitation, thereby enhancing the efficiency of the IRMPD and improve the detection sensitivity of a sample
[104⇓⇓~107]. Moreover, in the infrared multi-photon dissociation technology, the sample molecular ions continuously absorb infrared photons, and the continuous fragmentation reaction produces a large number of fragment ions. Other mass analyzers are limited by mass resolution and are difficult to resolve the structure of similar or too small fragment ions, but FTICR has high mass resolution and accuracy, which makes it the best partner of multi-photon dissociation technology. FTICR is a very unique mass analyzer. Its signal detection is not to use ions to collide with the multichannel plate (MCP) integrated by a miniature electron multiplier, but to detect the image current generated by induction and containing all the ion information. The mass-to-charge ratio is determined by frequency, which is conducive to the analysis of multiphoton dissociation products.
In recent years, Chinese scientist Zhou Mingfei and other scientists have also used infrared multiphoton dissociation and time-of-flight mass spectrometry to discover rare alkaline earth metal -(CO)
n complexes and (N
2)
n complexes, and put forward a new concept that alkaline earth metals are also applicable to the 18-electron rule
[108,109]. Alkaline earth metal complex cations were obtained in the gas phase using a pulsed laser evaporation-ultrasonic expansion ion source. Through the red shift of the C — O stretching vibration of these compounds, they demonstrated that the largest contribution to the formation of the M — CO bond comes from the feedback of π electrons on the d orbitals of alkaline earth metals to the ligands.
5.2 Ultraviolet photodissociation mass spectrometer
Ultraviolet photodissociation can be carried out in the near and far ultraviolet regions with wavelengths ranging from 200 to 400 nm and 10 to 200 nm, respectively. Compared with the infrared multiphoton dissociation, the higher energy ultraviolet photons (3 ~ 8 eV) can initiate the photodissociation of molecules by single photon. Near-ultraviolet photodissociation is more selective than infrared multiphoton photodissociation and requires specific chromophores. If the sample lacks chromophores, the selectivity of detection can be improved by introducing specific groups through chemical derivatization. VUV photodissociation is less selective because of its higher photon energy, which widely induces electronic energy level transitions of biomolecules. There are two main ways of UV photodissociation. The first possible way is that molecular ions absorb UV photons and then transition to high-energy electronic excited States, triggering a unique UVPD fragmentation pathway completely different from low-energy collision activation
[110]. It is also possible to relax to a low electronic energy state or ground state, where chemical bond breaking similar to conventional collisional activation occurs
[111]. The second possible way is that electrons are detached from multi-charged anions after absorbing UV photons, and then become anionic radicals, which are then used with CAD to initiate the fragmentation reaction. This method is also called Activated-Electron-Photo Detachment Dissociation (A-EPD)
[112].
UVPD technology generally uses high-energy short-pulse laser (about several nanoseconds), whose pulse number, energy density and wavelength are the key technical parameters to control the energy transfer and affect the efficiency of photoactivation and lysis. UVPD techniques generally employ excimer laser sources, including F
2(157 nm,7.9 eV), ArF (193 nm, 6.4 eV), XeF (351 nm, 3.5 eV), and Nd
3+:YAG solid-state lasers (213 nm, 5.8 eV; 266 nm, 4.7 eV; 355 nm, 3.5 eV)
[113]. In addition, gas discharge lamps, synchrotron radiation and light-emitting diodes can also be used for photodissociation. The combination of photodissociation and mass analyzer requires two modifications to the instrument. The first is to add an optical system, using a CaF
2 or a quartz window to introduce ultraviolet light into the instrument. The second is to add a trigger system to synchronize light radiation with ion detection and control the degree of ion dissociation.
Ion traps are the most common mass analyzers used in conjunction with UV photodissociation techniques, including 3D quadrupole ion traps, 2D linear ion traps, and orbital ion traps
[114⇓⇓~117]. All ion traps are very suitable for photodissociation because ions can be well accumulated and stored during photoactivation, which facilitates the control of energy transfer and the degree of dissociation, and enhances the signal-to-noise ratio of spectra. However, the photodissociation is generally not carried out in the electrostatic field orbital ion trap, but is carried out in a C-shaped trap or a linear ion trap in front of the orbital trap, and the product fragment ions of the photodissociation enter the orbital ion trap at a certain initial velocity and angle,While moving radially around the central spindle-shaped electrode, it moves axially synchronously along the central electrode to realize the measurement of the mass-charge ratio of various ions.
Photodissociation techniques can also be used in conjunction with time-of-flight mass analyzers. Ions with different mass-to-charge ratios take different times to move a certain distance in the vacuum field-free region, and the time-of-flight mass analyzer measures the ion velocity by time and converts it into mass-to-charge ratio. Compared with ion trap mass analyzers, time-of-flight mass analyzers face more technical challenges when combined with photodissociation technology, mainly because the laser pulse is difficult to synchronize with the flight time. The measurement of time often needs to determine a starting point, but such a time point is difficult to determine, and the initial velocity and direction of movement of ions are different. One of the effective solutions is to change the continuous ion source to the pulsed ion source. Quadrupole-Time of Flight (Q-TOF) tandem mass analyzer is further improved to be combined with the photodissociation technology. The molecular ions produced by the ion source are first focused in the quadrupole and then enter the orthogonal acceleration region. Under the action of the pulse voltage perpendicular to the incident direction, they obtain a constant velocity in the orthogonal direction, which improves the disadvantage of the linear flying mass analyzer that the initial velocity and direction are uncontrollable. Photodissociation generally takes place in the Trap Cell before entering the TOFMA. In recent years, the combination of ion mobility analyzers with time-of-flight and quadrupole mass analyzers has been reported, which enables photodissociation technology to be further used to resolve biomolecular isomers
[118].
6 Photodissociation and Structural Identification of Biomolecules
Molecular structure identification of complex samples is an urgent and important problem in chemistry and related fields, including biomedicine, pharmacy, agriculture and environmental science. The development of new Ion Activation and Dissociation (Ion Activation and Dissociation) techniques has been the most active research field of mass spectrometry. Photodissociation technology can make atoms/molecules in excited state lose electrons through photostimulation, and chemical bond cleavage initiated by charges or free radicals occurs. Or through excited-state energy relaxation, triggering vibrationally activated chemical bond breaking. Photodissociation technology can provide different fragment ions from conventional CAD, which is of great significance for the analysis of the primary structure of complex biological macromolecules (proteins and nucleic acids), the types and sites of protein post-translational modifications, and small organic molecules (such as drugs and metabolites).
6.1 Structural identification of small organic molecules
Compared with proteins and nucleic acids, small organic molecules do not have linear structures and are not limited repetitive sequences of specific structural units. The identification of their diverse structures requires not only highly accurate resolution and determination of the mass-to-charge ratio (m/Z) of fragment ions,It is also necessary to be able to trace the cleavage pathways of different chemical bonds with a high degree of selectivity, and ultimately to accurately deduce the molecular structure from the mass spectra obtained. The selective absorption of molecular ions to specific wavelengths of light can provide an important basis for the traceability and identification of specific functional groups and their related chemical bonds.
C — C, C — N, O — H, N — H, C — O and C — O bonds in common small organic molecules can be activated by infrared laser induced chemical bond vibration. Figs. 5 (A) and 5 (B) show the infrared multiphoton dissociation mechanism of cephalosporins and penicillins, respectively. Since the N atom has a lone pair of electrons, the positive charge of the protonated cephalosporin is located on the N atom as shown in fig. 5 (A)
[117]. The molecule absorbs multiple photon energies, activates the C — N bond directly adjacent to the positive charge, and initiates the homolytic cleavage of the C — N bond, resulting in the m/Z 158 fragment ion via lactam ring opening. This fragment ion is a base peak in the mass spectrum, because the four-membered ring has a large ring tension, which is conducive to the homolysis of the two C-N bonds and the cleavage of the lactam ring. And that positive charge carry by the generated product ion is delocalize in a p-π conjugated system, thereby having higher stability. It was found that β-lactam drugs could undergo this kind of cleavage, and the fragment ions produced by β-lactam drugs became the characteristic ions for identifying these drugs. Fig. 5 (B) is the infrared multiphoton dissociation mechanism of the adduct of penicillin with K
+ cation, whose base peak m/Z 160 fragment ion also arises from the cleavage of the lactam ring.
Fig. 5 (C) shows the infrared multiphoton dissociation pathway of chlorophyll A. Since the stretching vibration of the C — O bond and the bending vibration energy level of the vinyl C — H are both close to the photon energy, two types of dissociation can occur
[118]. After McLafferty rearrangement, part of the side chain was lost, resulting in m/Z 593 ion. When the side chain with the ester group is lost, the m/Z 533 ion is produced. The other is the cleavage of the pyrrole ring. Through multi-photon activation, the conjugated pyrrole ring loses electrons and becomes a cation radical, which is a very active odd-electron ion, prone to electron-induced chemical bond homolysis, and obtains a series of fragment ions at the low mass end. The above experimental facts show that the vibrational activation of chemical bonds excited by infrared photons with specific wavelengths determines the selectivity of chemical bond cleavage, making infrared multiphoton dissociation an important means for structural analysis of small molecules.
Although most of the chemical bonds of organic small molecules can be activated by the vibration of infrared photons, it takes a long time to accumulate multiple photon energies due to the low energy of each photon. Ultraviolet photoelectrons have higher energy, and ultraviolet light of different wavelengths can also be selectively absorbed by different chemical bonds, and further chemical bond rupture initiated by excited state electrons occurs, or chemical bond rupture initiated by vibration after relaxation to the ground state occurs. The higher the photon energy is, the more fragment ions are generated, and the richer the molecular structure information is
[119].
6.2 Structural identification of monosaccharides and polysaccharides
Sugars are one of the largest families of natural products. The complexity of their structures lies not only in the diversity of monosaccharides and their intricate connections and branching structures, but also in the fact that they are often linked to other molecules through glycosidic bonds. There are mainly four kinds of glycosides in nature, most of which are O-glycosides, N-glycosides (such as nucleoside compounds), C-glycosides when the sugar C
1 is directly bound to the carbon atom of the aglycone, and S-glycosides when thiols are condensed with the sugar. Due to the lack of chromophores, saccharides generally have no characteristic absorption in the near-UV and visible regions, and if near-UV photodissociation is used, the sample needs to be derivatized. IRMPD is a common technique for structural analysis of monosaccharides and polysaccharides. 10.6μm(943 cm
-1) infrared photons from a CO
2 laser can activate C — O — C vibrations, but the transannular structure of most O-glycosides is preserved
[120]. Figs. 6 (A) and (B) show the C-O-C vibration-activated C-O bond cleavage of erythromycin and neomycin, respectively, by IRMPD. Both molecules undergo a dissociation dominated by the cleavage of the glycoside chain, removing the aminoglycoside ring
[96]. The macrocyclic structure of erythromycin was hardly cleaved, and the aminoglycosides of neomycin were lost in turn, which could be used to identify the glycosides conveniently. Infrared multiphoton dissociation generally leads to not only the cleavage of glycosidic bonds, but also the cleavage of N- and S-glycosides across the ring structure, resulting in abundant fragment ions, while CID requires several stages of tandem mass spectrometry to achieve the same results
[121,122][123].
VUV can be absorbed by most sugar molecules, because the photons provided by VUV have high energy, which can not only stimulate the cleavage of C-O bonds outside the ring, but also cause the cleavage of C-O bonds inside the ring. Fig. 7 shows the two dissociation pathways of an oligosaccharide. One is the dissociation of the glycoside C — O single bond connecting the two sugar rings to form y and B ions, which provide sequence information
[124,125]. The other is the homolysis of the C — O bond in the sugar ring, which initiates the homolysis of the C — C bond in the adjacent ring, and finally breaks the ring structure to produce X and a ions, which can be used to identify the connection mode. The a, B, y and X ions produced by VUV photodissociation of oligosaccharides are similar to the fragment ions obtained by HCD. In addition to glycosidic bond and ring structure cleavage, neutral loss fragments are also produced. For example, deprotonated sialic acid oligosaccharides were dissociated by vacuum ultraviolet light, and 62 Da of ethylene glycol was lost, which provided characteristic information for their identification
[126].
6.3 Identification of amino acid sequences and post-translational modifications in peptides/proteins
IRMPD mainly initiates the cleavage of C-N peptide bonds of peptides/proteins, and the resulting B and y ions are the main fragment ions, which is very similar to CAD. In order to ensure the accurate identification of amino acid sequences and post-translational modifications, it is necessary to retain the information of post-translational modifications, including phosphorylation, sulfonation, glycosylation and disulfide bonds, when cleaving peptide bonds. Because the IR absorption frequencies of C — N peptide bond and post-translational modification are quite different, it is possible to achieve selective peptide bond cleavage by controlling the excitation wavelength. For example, the infrared absorption frequency of the P-OH group is very close to the 943 cm
-1 photon energy provided by the CO
2 laser, so the phosphorylated modified peptide will be selectively cleaved
[127⇓⇓⇓⇓~132]. The infrared multiphoton dissociation mechanism of sulfonated peptides is very similar to that of phosphorylated peptides, because the infrared absorption frequency of the S — OH group is also close to the 943 cm
-1 photon energy
[133]. IRMPD has a high ability to break peptide bonds and reduce the ion discrimination effect of CAD technology, which has been used in protein "Top-Down" amino acid sequence analysis
[134⇓~136].
Near-UV light dissociation is less applicable to amino acid sequence and post-translational modification analysis because near-UV light can only be absorbed by peptides/proteins containing tryptophan, tyrosine, and phenylalanine residues
[137⇓⇓⇓⇓⇓~143]. A unique feature of the near UV photodissociation is that it can selectively cleave the disulfide bond. Near UV photons at 266 nm wavelength can effectively stimulate the S — S bond homolysis to produce product ions corresponding to the two constituent peptides. The near UV photodissociation of S — S bonds is highly selective, and the disulfide bond cleavage is still the dominant pathway even in the presence of weaker phosphate groups
[144].
Vacuum ultraviolet photodissociation technique was first reported in Fourier transform ion cyclotron resonance mass spectrometer, which can be combined with soft ionization techniques such as MALDI and ESI
[145⇓⇓⇓⇓~150][151⇓⇓⇓~155][156⇓⇓⇓⇓⇓⇓⇓~164]. The dissociation of excimer ions produced by MALDI is the result of laser thermal effect and ultraviolet photons. The excimer ions produced by ESI are called "cold" ions, which have lower internal energy and are difficult to break the chemical bonds activated by vibration even if they collide with the buffer gas in the ion trap, thus ensuring that the specific chemical bond breaking initiated by photoelectrons occurs within a few milliseconds between the formation of ions and their entry into the mass analyzer
[165,166]. Fig. 8 shows the mechanism of ionization of different ions by 157 nm vacuum ultraviolet light. The C—C
α bond is homolyzed to produce a + 1 and X + 1 cation radicals, and then the ortho-position chemical bond initiated by the radical center is homolyzed and the H atom is eliminated to produce even-electron ions a and X, respectively, while the side chain is lost to produce d, V and w ions, respectively, and less B, y and C, z-type ions
[167]. The formation of an X ion or an a ion depends on the position of the protonated basic amino acid in the polypeptide. If the basic arginine appears at the N-terminus, the charge will remain at the N-terminus to form an a ion. If the basic arginine appears at the C terminus, the fragment ion charge remains at the C terminus, mainly producing X ions, a phenomenon known as the "arginine effect". On the contrary, lysine mainly produces y ion and B ion at the C terminal, and the aminoguanidylation of the side chain of lysine is similar to that of arginine, so ultraviolet ionization technology can provide relevant information for identifying the C terminal arginine peptide fragment
[168]. The formation of product ions is related to the energy required for chemical bond cleavage and the stability of the product. For example, the elimination of aspartic acid residues occurs in the side chain, while the elimination of glutamic acid residues occurs in the main chain. This is because the C
αC
β bond formed by aspartic acid residues can be conjugated with the amide bond, and the structure is more stable. In general, d ions do not appear in glycine and alanine residues without β-C substitution, and the elimination of aromatic rings requires higher energy, so aromatic amino acid residues do not produce d ions
[165]. When the gaseous ions produced by ECD and ETD are dissociated by ultraviolet light, if arginine is at the N-terminal, a ions are mainly produced, which is consistent with the arginine effect. However, if the arginine is at the C terminus, it will break the N-C
α bond to form C and Z ions
[169]. Ion dissociation is also related to charge localization, such as H
+ and Na
+, which are localized to basic amino acid residues and prone to charge-induced chemical bond cleavage
[170].
The post-translational modification sites can be well preserved by using vacuum ultraviolet photodissociation technology to identify the post-translational modification
[171]. It was found that the neutral loss of phosphoric acid molecules was closely related to the illumination time. When serine and threonine phosphorylated peptides were dissociated by 157 nm vacuum ultraviolet light, if the excitation time was controlled within 1 μs, the product ions almost did not lose 98 Da phosphate neutrality. Phosphate group loss began to occur after 1 μs, and the ions generally lost phosphate groups by 13 μs. In contrast, the phosphate group attached to tyrosine was more stable than that of serine and threonine, and few product ions lost the phosphate group, and no loss of phosphate group was observed even when the photoexcitation time was 13 μs
[172]. Because the phosphorylated peptide carries a negative charge, the dissociation efficiency is higher in the negative ion mode, a large number of a and X ions can be formed, and the phosphate group is well retained in the fragment ions, which is convenient for identifying the modification site
[173].
6.4 Nucleic acid sequence analysis
The use of mass spectrometry for nucleic acid analysis is challenging because the phosphate esters of nucleic acids are negatively charged, and the negative ion mode of mass spectrometry is generally less sensitive than the positive ion mode. Secondly, nucleic acid has only four bases, and a large number of isomeric fragment ions are produced after dissociation, which brings some difficulties to sequence analysis. In order to obtain more abundant fragment ions, various photodissociation techniques such as IRMPD and UVPD have been developed. As that phosphate group in the nucleotide has a lar absorption cross section for infrared photons (wavelength of 10.6 mu m) emit by a common CO
2 laser, a double-stranded structure of the nucleic acid can be dissociated into a single-stranded structure in the time as short as microsecond, and further chemical bond breakage can be further initiated by continuously absorbing a plurality of infrared photons, so that complete sequence information can be obtained for oligonucleotides, miRNA, siRNA and the like
[174][174,175]. As shown in fig. 9, IRMPD excites P — O and C — O bonds, accumulates multi-photon energy, and triggers vibrationally activated chemical bond cleavage to generate fragment ions containing sequence and modification group information. The degree of ion dissociation is related to the time of photon radiation, and the composition of fragment ions can be changed by changing the laser intensity and radiation time. However, if the irradiation time is too long, two 5'P-O bonds will be broken, resulting in internal ions that interfere with sequence analysis, as well as w ions and a ions that lose bases. In addition, small protonated base ions, including [GH
+], [AH
+], and [CH
+], which cannot be obtained by CAD techniques, can also be obtained by IRMPD dissociation techniques.
The principle of UVPD is completely different from that of IRMPD. It mainly dissociates nucleic acids through Electron Photodetachment (EPD), that is, deprotonated nucleic acids absorb high-energy ultraviolet photons and lose electrons to become odd-Electron ions. Since the internal energy of these odd-Electron ions is not high, they will not spontaneously dissociate into fragment ions, and they need to be activated by collision with CAD to obtain fragment ions, which is called Activation-electron Photodetachment (Activation-Electron Photo detachment, A-EPD). The odd-electron ion are subjected to collision activation and then are crack, so that complete a, B, C, d, X, y and Z series ions and low-mass w1, z1 and a1 ions can be obtain, that base loss is reduced, and the coverage of the nucleic acid sequence is improved.
7 Conclusion and prospect
The complex and diverse structures of biomolecules are the molecular basis for performing their unique functions, and the accurate identification of their complex structures requires the development of diverse analytical methods to reveal different structural characteristics. Ionization/dissociation is the key to mass spectrometry. Different neutral sample molecules are ionized into gaseous charged ions, in which even-electron ions have high stability and need to be further dissociated into fragment ions by other dissociation techniques. Photoionization/dissociation technology is based on the absorption of photons of specific wavelengths by different chemical bonds or functional groups. Compared with other dissociation technologies such as collision-activated dissociation, photoionization/dissociation technology improves the selectivity and dissociation efficiency. From small molecule structure to macromolecule sequence, covalent modification and binding site, from genetic information storage nucleic acid to function executor protein, drug and metabolite, photoionization/dissociation technology plays an important role (Table 2). At present, there are still some technical challenges in photoionization/dissociation technology, which limits its wider application in the field of life sciences. Because the existing photoionization/dissociation technology generally uses a single wavelength laser source, it can not carry out continuous wavelength scanning, so it can only obtain fragment ions formed by the breakage of specific chemical bonds. The structures of biomolecules are diverse, and there are a large number of isomers. Further, the combination of spectroscopy and mass spectrometry can not only obtain accurate mass-charge ratio, but also obtain functional group information, which is expected to further improve the ability of structural identification of unknown molecules.
the National Natural Science Foundation of China(21834002)
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