1 Introduction
Endogenous metabolites are the intermediates and end products of life metabolic activities under the joint action of the body and the external environment. Their types and content changes can reflect the metabolic state of the body in various life activities, and provide important clues for the study of life processes such as diseases. There are many kinds of metabolites in organisms, and their isomers are diverse, and their content ranges span nine orders of magnitude, which poses a great challenge to the sensitivity and selectivity of analytical techniques.
Mass spectrometry (MS) has become a common technique for the analysis of endogenous metabolites because of its fast analysis speed, high throughput and wide dynamic range, which can provide qualitative and quantitative information of compounds at the same time. In recent years, a series of research achievements have been made in the analysis and application of endogenous metabolites by chromatography-Mass spectrometry, open ionization and Mass spectrometry imaging (MSI)
[1,2][3⇓~5][6,7]. However, the application of mass spectrometry is limited to some extent because it requires that the compounds to be detected have easily ionized groups. On the other hand, although primary mass spectrometry can provide information about the mass to charge ratio of the analyte ion, it cannot distinguish between isomers. Although tandem mass spectrometry can reflect the structural information of the analyte to some extent, it is difficult to identify the isomers with the same cleavage pathway by single mass spectrometry, even with a high-resolution mass analyzer. Therefore, how to improve the application scope of mass spectrometry for metabolite isomer analysis is a technical bottleneck that needs to be solved urgently.
The introduction of chemical derivatization reaction in mass spectrometry is an effective means to improve the application range of mass spectrometry. The detection sensitivity can be effectively improved by labeling specific compounds through derivatization reaction to enhance their ionization efficiency in mass spectrometry detection. In addition, chemical derivatization can also improve the specificity of tandem mass spectrometry, that is, the difference of physical and chemical properties between isomers can be amplified by derivatization reaction, and converted into the difference of abundance of corresponding product ions in the collision induced fragmentation (CID) process, so as to achieve the distinction of isomers. By selecting appropriate derivatization reagents for labeling, it can accurately distinguish the isomers of lipids, sugars, amino acids and other compounds in vivo. In this review, we will focus on the analysis of endogenous metabolite isomers, and review the methods of chemical derivatization mass spectrometry of metabolites and the application of chemical derivatization mass spectrometry of different types of endogenous metabolites.This review focuses on the derivatization methods of metabolites based on independent mass spectrometry techniques, such as online chemical derivatization mass spectrometry, and the research methods and progress of chemical derivatization for mass spectrometry analysis of lipids, sugars and chiral amino acid isomers.
2 Metabolite chemical derivatization mass spectrum analysis method
With the wide application of mass spectrometry in the field of biological analysis, researchers at home and abroad began to introduce chemical derivatization reactions into the analytical methods based on chromatography-mass spectrometry, open ionization and mass spectrometry imaging
[8,9][10][11,12]. The early chemical derivatization-chromatography-mass spectrometry techniques focused on improving the volatility, thermal instability and chromatographic retention behavior of samples to improve the chromatographic separation ability. The chemical derivatization research for mass spectrometry detection mainly focuses on the development of derivatization reaction mass spectrometry labeling reagents with high proton affinity. Most of these labeling reagents react with the analyte in solution, and the conversion products are purified and used for mass spectrometry analysis, which can significantly improve the ionization efficiency of the analyte. With a series of studies such as the phenomenon of accelerated chemical reaction under confined conditions such as microdroplets reported by Cooks et al., Department of Chemistry, Purdue University, the method of applying chemical derivatization reaction to on-line mass spectrometry has been developed and applied, which provides a new option for chemical derivatization mass spectrometry
[13,14].
Chemical derivatization mass spectrometry can be divided into online chemical derivatization and offline chemical derivatization according to whether the derivatization reaction occurs simultaneously with mass spectrometry detection. Off-line chemical derivatization mass spectrometry usually carries out the derivatization reaction in the solution system, and introduces the protophilic group into the sample to derivatize the analyte, and its detection efficiency is closely related to the mechanism of the derivatization reaction. Online chemical derivatization mass spectrometry introduces reagents in the ionization process or mass analyzer (such as ion trap), and the chemical derivatization and product detection of the analyte are carried out simultaneously, which can detect the analyte quickly and sensitively.
2.1 Metabolite on-line chemical derivatization mass spectrometry
Because of the fast scanning speed of mass spectrometry, the time consumption of derivatization reaction is the restriction of online mass spectrometry when chemical derivatization is carried out online with mass spectrometry. Gas-phase ion-molecule reactions based on free radicals are often used in online chemical derivatization mass spectrometry because of their fast reaction rate. Ozone-induced dissociation (OzID) technique introduces oxygen radical ions into the ion trap mass analyzer, which react with the C-C double bonds in the skeleton structure of unsaturated lipid ions and induce dissociation, and the abundance ratio of the related fragment ions can be used to identify the position of unsaturated double bonds in the precursor ion structure
[15]. Nie Zongxiu et al. Developed a semiconductor assisted laser desorption/ionization mass spectrometry (SA-LDI MS) platform for online monitoring of photocatalytic reactions, which can be used for ultra-fast reaction screening
[16]. Free radical reactions can also be introduced into the atmospheric pressure ionization process for on-line derivatization mass spectrometry. Cooks et al. Found that the C — C/C — H bonds of saturated alkanes can be activated by nitrogen atoms in nitrogen under the action of an electric field at atmospheric pressure
[17]. On this basis, the atmospheric pressure ionization technology of nitrogen insertion reaction was developed, and the method of functionalization of saturated alkanes by nitrogen insertion reaction was established by integrating the process of in situ derivatization and desorption ionization, which solved the problem that saturated alkanes were difficult to ionize at atmospheric pressure
[18]. Although chemical derivatization based on free radical reaction has the advantage of fast reaction speed, it still faces the limitation that there are few reaction types to choose from.
There are many types of chemical derivatization reactions that can be carried out in solution system, and how to accelerate the derivatization reaction is a new direction of online mass spectrometry
[19]. The results show that the chemical reaction process will be accelerated under the confined conditions formed by heterogeneous reaction interfaces (such as condensed phase, liquid-gas interface, micro-solvation environment)
[20]. In 2011, Cooks et al. Reported the phenomenon of accelerated chemical reaction rate in the process of spray ionization, and proposed that the chemical reaction in the micro-droplet system is suitable for the theory of chemical reaction acceleration under confined conditions.That is to say, the reactant droplets are produced by spraying, and the droplets are continuously desolvated to form tiny droplets with a volume of several microliters, which provides a limited and high surface area place for chemical reaction and accelerates the derivative reaction process at the gas-liquid interface
[21]. The results of this study provide a new theoretical basis for on-line chemical derivatization mass spectrometry. They systematically studied the reaction acceleration phenomenon under the condition of micro-droplets, and found that the reaction rate of Hantzsch reaction under the condition of micro-droplets was UNK 1 times faster than that of solution system, and the required time was shortened to milliseconds
[22]. Similar accelerated reaction phenomena were found in various spray ionization modes such as electrospray ionization (ESI), electrospray extraction ionization (EESI), desorption electrospray ionization (DESI) and paper spray ionization (PS) (Fig. 1). Among them, reactive-Desorption Electrospray Ionization (rDESI) uses a droplet beam containing a reactive reagent to extract the analyte on the surface of a solid sample and react with it, and the secondary droplets formed can rapidly generate reaction products in milliseconds during the desolvation process. This method of accelerated chemical reaction mass spectrometry based on electrospray and other methods has been used in the following three aspects: (1) the principle and mechanism of on-line reaction monitoring for solution reactions
[23]; (2) preparing a mass spectrum method, and collecting a product generated by a liquid-phase droplet ion reaction; And (3) carrying out derivative reaction on the substance to be detected during spray ionization to generate a product which is easy to detect by mass spectrometry.
Reaction desorption electrospray ionization mass spectrometry (RD-ESI-MS) has been widely used in the detection of metabolites in biological samples. Ouyang Zheng et al. Combined rDESI with tandem mass spectrometry to achieve rapid screening of anabolic steroids in whole urine
[24]. Hydroxylamine was added to the spray solution as a derivatization reagent and reacted with the carbonyl group of the steroid in the process of ionization.The reaction product and the oxime formed by its dehydration were observed. Seven steroids, including glucocorticoids, were detected by this method. The method was also applied to the detection of acetone in human plasma. The linear response range of steroid and acetone was about 2 orders of magnitude, the detection limit was less than 1 ng, and the analysis time was only a few seconds. Ifa et al. Used the reaction desorption electrospray mass spectrometry imaging (rDESI-MSI) method to derivatize the positive ion derivatization candidate DC9 for the derivation of lipids in tissues, and simultaneously detected phosphatidylethanolamine (PE) and small molecular lipids in rat brain tissue and zebrafish in positive ion mode
[25]. The results showed that the response intensity of DC9 derivatization products was significantly improved compared with the conventional DESI-MSI detection, and the data acquisition time and sample consumption were saved. In polyhydroxy compounds, the cis-diol with close space position can react with phenylboronic acid to form a negatively charged cyclic borate, which can be used for on-line mass spectrometry to detect sugars in organisms and distinguish cis-estriol from trans-estriol
[26,27]. Recent studies have shown that microdroplet accelerated reactions are also suitable for the derivatization of metabolites in single biological cells, and the concentration gradient of derivatization reagents formed by the gradual evaporation of derivatization solvents during on-line reactions is similar to the gradient concentration process of substances transported in cells
[28]. Therefore, this method is also expected to be a simple and controllable method for simulating intracellular reactions, which will help to further understand the process of intracellular biochemical reactions
[29,30].
Chemical reaction acceleration in that microdroplet system has high applicability, and obvious acceleration is realize for carbon-carbon and carbon-nitrogen bond formation, cyclization, oxidation-reduction and multi-component multi-step reactions
[31]. The on-line chemical derivatization mass spectrometry technology based on this method can realize the instantaneous derivatization of the analyte and the on-line detection of the products, and has been applied to the detection of metabolites such as aldehydes, ketones, alcohols, esters, mercaptans and alkanes, which will have a wide application prospect in the detection of metabolites and their isomers
[23].
2.2 Metabolite off-line chemical derivatization mass spectrometry
Off-line chemical derivatization mass spectrometry means that the substance to be detected is derivatized and labeled in the conventional reaction system, and then the product is detected by mass spectrometry. Off-line chemical derivatization in solution has been widely reported, and the suitable ionization methods are not limited to ESI, but also suitable for matrix-assisted laser desorption ionization (MALDI), such as alkylation and introduction of protophilic groups
[32,33][19]. Researchers have developed a series of endogenous metabolite profiling methods for off-line chemical derivatization of metabolites containing different functional groups: amino acids can be derivatized by reagents such as acyl chloride, dansyl chloride, anhydride, N-hydroxysuccinimide, etc. By nucleophilic reactions initiated by primary amines at pH > 8; Chemical derivatization of hydroxyl groups by mass spectrometry usually uses derivatization reagents containing basic groups, such as methylation of polyhydroxy substances or conversion of hydroxyl groups to esters using methyl halides. Off-line chemical derivatization can also be used for isotope labeling, which uses multiple isotopes to label different substances to be detected at the same time, thus realizing multi-channel analysis and improving the throughput of mass spectrometry detection
[32]. For example, Feng Yuzhen et al. Developed a parallel isotope derivatization method for metabolites containing carboxyl, carbonyl, amino and thiol functional groups, and identified more than 1400 metabolites in rat feces samples
[9].
2.3 Metabolite chemical derivatization mass spectrometry imaging analysis
In recent years, mass spectrometry imaging technology has developed rapidly, and has become one of the hot spots in the field of mass spectrometry. Mass spectrometry imaging technology has the advantages of broad spectrum, high sensitivity and good specificity, and can obtain the dynamic information of the spatial and temporal distribution of metabolites in biological tissues. In addition, mass spectrometry imaging analysis of structural isomers of metabolites to characterize their spatial distribution is conducive to the excavation and exploration of their biological significance. However, compared with liquid chromatography-mass spectrometry (LC-MS) method, it is difficult to obtain highly sensitive detection results similar to LC-MS because of the lack of hierarchical dimensionality reduction. Therefore, the development of mass spectrometry imaging analysis methods for low abundance, low ion response metabolites and isomers is an urgent problem to be solved.
At present, a small number of studies have reported in situ chemical derivatization mass spectrometry imaging analysis methods for metabolites, including off-line chemical derivatization methods, such as derivatization reagent spraying method, automated spotting method, slice pretreatment method, and on-line chemical derivation methods, such as on-line ozone-induced dissociation reaction mass spectrometry imaging method. The emerging chemical derivatization mass spectrometry imaging technology can be used to visualize the low abundance, low ion response metabolites and structural isomers in complex systems of biological tissues. For example, Guo Yinlong et al. Reported that N, N-dimethylpiperazine iodide (DMPI) was sprayed on thyroid cancer tissue slices as a derivatization reagent using a self-developed electrospray spraying device, and reacted with free fatty acid carboxyl groups in the tissue
[11]. The results of MALDI-MSI analysis of fatty acid derivatives and phospholipids in tissues under positive ion mode showed that the expression of saturated free fatty acids (such as FFA C16: 0, FFA C18: 0) was positively correlated with phospholipids in thyroid cancer tissues. The method effectively improves the detection sensitivity of the low-abundance fatty acid in vivo and increases the number of fatty acids detected by the mass spectrometry imaging method. At present, although some progress has been made in the study of chemical derivatization mass spectrometry imaging methods, there are still some problems, such as complex operation, poor repeatability and limited reaction types.
Various derivatization reactions have been widely used in the analysis of metabolites, providing clues and basis for the exploration of various biological processes. Off-line chemical derivatization mass spectrometry has universal applicability to a variety of derivatization reactions, and effectively improves the mass spectral response of low-abundance and hard-to-ionize metabolites. On-line chemical derivatization mass spectrometry is simple and efficient, and has a wide application prospect in rapid mass spectrometry analysis. Table 1 lists the methods and applications of online and offline chemical derivatization in metabolic mass spectrometry, indicating that the existing derivatization mass spectrometry methods have been successfully applied to the detection of lipids, sugars, amino acids and other compounds in biological tissues, body fluids and single cells.
3 Analysis of Metabolite Isomers by Chemical Derivatization Mass Spectrometry
High resolution mass spectrometry alone is not enough to distinguish the isomers of metabolites due to their small differences in structure and physical and chemical properties. At present, more and more chemical derivatization mass spectrometry methods have been successfully applied to the detection of isomers of different compounds. Lipids, sugars and amino acids are important metabolites in organisms, and they are also common compounds with multiple isomers. Chemical derivatization mass spectrometry methods and applications for the analysis of lipids, sugars, and amino acid isomers are described below.
3.1 Chemical derivatization mass spectrometry method for lipid
Lipid is an important component of cell membrane, which is diverse in chemical structure, endogenous abundance and physical and chemical properties, and plays an important role in a variety of physiological and pathological processes. The functions of different lipids are closely related to their types and structures. Therefore, the identification of the fine structure of lipids is of great significance for the study of their biological functions.
The structure of lipid molecules is diverse, and there are many isomers such as head group type, acyl chain length, connection position, unsaturation and stereochemistry, which poses a great challenge to the identification of lipid structure. Accurate identification of various lipid isomers will become the focus of mass spectrometry analysis of lipids in the future. The identification of lipids can be divided into five levels: (1) the head group and the classification of lipids; (2) that total carbon numb and the number of double bond of the acyl chain; (3) that numb of carbons and the numb of double bonds per acyl chain; (4) where the acyl chains are attached to the lipid backbone (e.g., the sn position), and (5) where the double bonds are located and their stereochemical conformation. Collision-induced dissociation (CID) mass spectrometry can be used to identify the first three levels, but conventional tandem mass spectrometry still has some limitations, such as low sensitivity and insufficient structural resolution ability for the analysis of fine structures such as sn positional isomers, double bond positions and cis-trans isomers. Therefore, a number of chemical derivatization-based tandem mass spectrometric methods have been developed for the identification of lipid fine structure isomers, such as photochemically activated Patern Paterní-B Büchi reaction, ozone-induced dissociation, and ultraviolet light dissociation.
3.1.1 Patern Paterní-B Büchi (P-B) reaction
The Patern Paterní-B Büchi reaction is a class of reactions involving the photochemical cycloaddition of carbonyl groups to alkenes [2 + 2] to form oxetanes. The derivatization reagent solution containing ketone or aldehyde carbonyl was introduced online during ESI ionization.Under the catalysis of 254 nm ultraviolet light, the diradical intermediate was generated, which reacted with the lipid double bond to form a product containing an oxane ring, and the specific fragments related to the position of the double bond could be identified by tandem mass spectrometry
[37]. The method has that advantage of high efficiency, rapidness and simple operation, and is universally applicable to various lipids. Ouyang Zheng and Xu Yu cooperated to improve the P-B reaction, and established a large-scale and complete lipid deep structure analysis method through 2-acetylpyridine online photochemical P-B reaction-nanoESI-MS/MS analysis. This study also found that MS
3 analysis without photochemical reaction can be used directly to distinguish sn isomers: underivatized phosphocholine loses the head group (-183 Da) in MS/MS analysis, forming a dioxolane ring where it binds to the fatty acyl chain, and the sn-2 fatty acyl chain binds to the dioxolane ring through the newly formed C C. In further CID cleavage, the dioxolane ring is cleaved, producing a characteristic ion that characterizes the sn site. The use of 2-acetylpyridine as a derivatization reagent is helpful for the generation of characteristic product ions with high abundance at the C-C position and the sn position, resulting in a higher P-B reaction yield. The method is applied to the research of lipidomics, improves the structural resolution of lipid molecular analysis, and realizes the simultaneous resolution and quantitative analysis of phospholipid types, double bond positions and n-position isomers of fatty acid chains in lipids. The P-B reaction can also be carried out offline. For example, Dreisewerd et al. Used benzaldehyde as a derivatization reagent to carry out the off-line P-B reaction on the tissue slices, and analyzed the slices after the reaction by MALDI-2-MSI and MALDI-2-MS/MS, and found that phosphatidylcholine and phosphatidylserine double-bond positional isomers had different distribution trends in the white matter and gray matter of the mouse cerebellum (Fig. 2)
[46].
3.1.2 Ozone-induced dissociation technique
Ozonation is an important method to identify the structure of derivatized lipids. The reaction of olefins with ozone can produce primary ozonated products, which are further converted to two carbonyl compounds. The two carbonyl compounds correspond to the skeletons on both sides of the C-C double bond of the olefin, respectively, so the structure of the original olefin and the position of the double bond can be inferred from the product
[15]. In the initial study, Murphy et al. Exposed the dried phospholipid film to ozone for ozonation reaction offline, and then analyzed the product by ESI-MS/MS to determine the double bond position through the characteristic ion produced
[58]. This method can achieve a higher yield of ozonides, but it requires an additional sample preparation step before mass spectrometry analysis, which is difficult to be compatible with online analytical methods such as shotgun lipidomics analysis. Thomas et al. Developed Ozone electrospray mass spectrometry (OzESI-MS), which uses oxygen as the atomizing gas during spray ionization, increases the capillary voltage to corona discharge to generate Ozone, ozonizes the lipid to be detected online, and analyzes the product by tandem mass spectrometry to analyze its structure
[59]. The method is simple to operate and can be carried out directly on a commercial mass spectrometer for the identification of single lipids or simple lipid mixtures. However, for complex lipid mixtures, there are great challenges in peak assignment and spectrum analysis.
Ozone-induced dissociation (OID) is an online lipid derivatization mass spectrometry method developed based on ozonization, which overcomes the limitations of OzESI-MS for the structural identification of lipids in complex mixtures. OzID technology is to install an ozone generator at the mass spectrometer ion trap detector, inject low concentration (~ 1%) ozone into the buffer gas, first select the target lipid ions through the ion trap, the selected ions react with ozone, and identify the target lipid structure through the product fragment ions. OzID technology is limited by the need for instrument modifications and strict adherence to safety requirements for highly reactive ozone. Nevertheless, the OzID technique enables the identification of lipid structures in complex mixtures, producing characteristic ions that are easily recognized, and has been reported to have the ability to identify a variety of lipid isomers at double bond positions, sn positions, and so on. Blanksby et al. Combined OzID method with data-independent shotgun lipidomics method to analyze the isomers of unsaturated lipids in plasma, and found that most of the unsaturated lipids existed in the form of isomer mixtures
[60]. Ellis et al. combined MALDI-MSI with OzID-ion trap mass spectrometry to characterize the specific distribution of various types of lipid isomers in rat brain tissue
[35]. As shown in fig. 3, the expression of various isoforms of PC (36:1) was significantly different in the gray and white matter of the rat brain. OzID technology is compatible with a variety of lipidomics methods, and can provide comprehensive structural information of lipids in complex matrices of biological tissues, which has a wide application prospect.
3.1.3 Other online chemical derivatization mass spectrometry method
P-B reaction and OzID technology are commonly used in on-line lipid derivatization mass spectrometry. In addition, the multilevel mass spectrometric information of unsaturated lipid alkali metal adducts can also be used to identify the position of the double bond
[61]. UV photolysis can cleave the carbon-carbon bond in the acyl chain by 193 nm UV irradiation, and the characteristic ion pair with a difference of 24 Da can be used to identify the position of the double bond in the corresponding lipid. It is particularly suitable for on-line MSI and LC-MS/MS analysis due to the extremely short time it takes for UV light to be absorbed by the lipid and to activate the latter. Low temperature plasma (LTP) in situ ionization mass spectrometry has also been used to rapidly identify the location of lipid double bonds
[62]. Oxygen radicals generated during the ionization of LTP probes oxidize unsaturated lipids, and the oxidation products can be used to determine the location of double bonds.
3.2 Chemical derivatization mass spectrometry method for sugar
Carbohydrates exist in organisms in the form of monosaccharides and their derivatives, oligosaccharides, polysaccharides, complex polysaccharides and glycosides. The diversity of sugar species, sugar chains and abnormal structures makes the structural differentiation of sugar isomers a difficult problem. To fully elucidate the structure of sugars, the following information is needed: (1) molecular weight and the types and molar ratios of the constituent monosaccharides; (2) the conformation of each sugar ring and the configuration of the anomeric carbon; (3) the way in which the sugar residues are linked; (4) the order of attachment of the sugar residues; (5) Secondary structure and spatial conformation. Mass spectrometry has become an indispensable research tool for characterizing the structure of sugar groups because of its small sample size, fast analysis speed and high sensitivity. At present, there are two main methods to identify sugar isomers based on mass spectrometry: chemical derivatization mass spectrometry and Ion mobility mass spectrometry (IMS).
3.2.1 Chemical derivatization mass spectrometry
Most glycans have strong hydrophilicity and low proton affinity, so they are not easy to ionize when detected by mass spectrometry. By derivatization, that invention can realize the follow purposes: (1) the ionization efficiency is improve, and the mass spectrum detection sensitivity is enhanced; (2) stabilize that structure of sugar molecules to prevent rearrangement; (3) protect unstable glycan residues; (4) Distinguish isomers. Charles et al. Reported the introduction of serine and Cu
2+ into the kinetic method of mass spectrometry to distinguish monosaccharide isomers
[42]. The derivatization process of glycan is usually relatively complex and is mostly completed under off-line conditions. Common derivatization methods include reductive amination, hydrazine reaction, carboxyl binding reaction, and permethylation and methylamination of sugars. Pan yuanjiang et al. combined hydrazine derivatization reagents 3-hydrazinobenzoic acid (3HBA) and quinoline-3-carbohydrazide (Q3CH) with the commonly used matrix 2,5-dihydroxybenzoic acid (DHB) in MALDI-MS, and proposed the parallel target plate derivatization strategy (POTDS), which not only improved the ionization efficiency of N-glycans, but also used them as the internal standard for mass correction in MS and MS
2, thus realizing accurate correction and identification in positive and negative ion modes simultaneously
[47]. Specific chemical derivatization methods can also be developed for glycan isomers of specific biological interest, such as esterification and amidation reactions, which can be used to distinguish acidic N-glycan isomers. Alpha- (2,3) and alpha- (2,6) sialic acid are two common N-glycan linkage isomers, and alpha- (2,6) sialic acid is considered to be a biomarker for a variety of cancers. Carlstedt et al. First reported the identification of α- (2,3) and α- (2,6) sialic acids using 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM) as a derivatization reagent
[63]. The molecular weight of α- (2,6) sialic acid increases by 14 Da after methyl esterification, while the molecular weight of α- (2,3) sialic acid decreases by 18 Da after reaction to form lactone. Therefore, the two kinds of sialic acids can be distinguished by a characteristic ion peak with a difference of 32 Da in the mass spectrum. Wuhrer et al. Found that differentiation of α- (2,3) and α- (2,6) sialic acids could be achieved using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) -activated derivatization
[53]. In that presence of EDC, the. Alpha.- (2, 6) -linked sialic acid form an ethyl ester and the. Alpha.- (2, 3) -linked sialic acid forms a lactone. The derivatization method has simple steps, does not need a purification process, and improves the specificity of the sialic acid isomer. In order to further simplify the analysis process and stabilize the structure of sialic acid, Cipollo et al applied this method to hydrophilic interaction liquid chromatography-solid phase extraction-MALDI-TOF MS analysis, which can directly detect glycan mixture samples
[64].
3.2.2 Ion mobility mass spectrometry
Ion mobility spectrometry (IMS) is an analytical method for the separation of chemical substances based on the difference of migration rates of different gas phase ions in an electric field. The migration rate of ions in the drift tube is affected by the ion mass, size, collision cross section (CCS) and dipole moment. ion mobility spectrometry (IMS) can be divided into several types: Drift time ion mobility spectrometry (Drift tube ion mobility spectrometry, DTIMS), traveling wave ion mobility spectrometry (Travelling-wave ion mobility spectrometry, TWIMS), Field asymmetric waveform ion mobility spectroscopy (Field asymmetric waveform ion mobility spectrometry, FAIMS) and trapping ion mobility mass spectrometry (Trapped ion mobility spectrometry, TIMS). The main difference between them is to push ions into the drift tub. Ion mobility spectrometry (IMS) is fast and can separate the analyte in tens of milliseconds, which is especially suitable for on-line detection with mass spectrometry. Ion mobility mass spectrometry (IMS) is a new analytical technique combining ion mobility spectrometry with mass spectrometry. Usually, an ion transfer tube is added between the mass spectrometer ion source and the mass analyzer, and the ions to be detected are pre-separated by the ion transfer tube according to the mobility and then further detected by the mass spectrometer, so that three-dimensional data of migration time, mass to charge ratio and response intensity can be obtained, and the separation performance and application range of the mass spectrometry are expanded.
Ion mobility mass spectrometry has been successfully used for isomer analysis, but this method still has some limitations for the identification of macromolecular glycans. The study of sugar isomers by IMS began in the late 1990s. Clemmer et al. Used self-made DTIMS to analyze a series of oligosaccharides and first demonstrated that isomers have different drift times
[65]. In 2003, Froese et al. First separated the mixture of oligosaccharide isomers using FAIMS
[66]. Recent studies have found that by introducing metal ions to form non-covalent adduct ions, the conformational differences of sugar isomers can be amplified to facilitate their separation by IMS. Most of these studies are based on alkali metal ions, such as Pagel et al., who reported the use of DTIM-MS to detect the CCS values of [M+Na]
+, [M+K]
+, [M+H]
+ and [M+Cl]
-,[M+H
2PO
4]
- ions of high mannose N-glycan isomers
[39]; Dodds et al. studied the gas-phase ion-ion electron transfer reaction and found that although the CCSs spectra of the divalent adduct ions of sugar isomers [ [sugar, M: metal]] may be the same, the electron transfer products [C+M]
(N-1)+· of different isomer adduct ions can be separated by IMS, so this method can be used to identify sugar isomers
[67]. Leary et al found that transition metals such as Mn
2+, Co
2+, Fe
2+ and Ni
2+ could induce the conformational change of adduct ions of heparin oligosaccharide isomers by introducing exogenous ions, and then separated them by IMS
[43].
3.3 Chemical derivatization mass spectrometry method for amino acid
Amino acids are important organic molecules in organisms, which play an important role in protein synthesis, maintaining physiological functions and various metabolic activities. Amino acids are also an important class of chiral molecules, and most of them are chiral compounds. Chiral amino acids are closely related to their receptors, enzymes and other biological macromolecules in vivo, and different chiral enantiomers show unique biological functions. L-amino acids are the main active forms in mammals, but more and more reports show that D-amino acids play an important role in physiological and pathological processes such as diseases. For example, D-Ser, as an agonist of N-methyl-aspartate (NMDA) receptor, is closely related to stroke, Alzheimer's disease and schizophrenia. The commonly used chiral analysis methods, such as high performance liquid chromatography, electrophoresis, optical rotation spectrometry, can obtain good chiral discrimination results, but there are some shortcomings, such as complex operation, large amount of samples and narrow application range. In recent years, a variety of analytical methods based on mass spectrometry have been successfully applied to the identification and analysis of chiral substances, mainly including the following two ways: (1) Chiral resolution technology combined with mass spectrometry, which combines mass spectrometry with chiral separation technologies, such as high performance liquid chromatography, capillary electrochromatography and ion mobility spectrometry. This method enhances the sensitivity and accuracy of the analysis, but there is a problem that the hyphenated method is incompatible with mass spectrometry. (2) a chiral recognition mass spectrometry, in which a chiral compound to be detected is chemically derived into a diastereoisomer, and then a mass spectrometry is used for detecting the small difference between the kinetics and the thermokinetics of the chiral diastereoisomer in the reaction process for chiral recognition.
3.3.1 Chiral recognition mass spectrometry
According to the mechanism of chiral recognition, chiral recognition mass spectrometry can be divided into three types: (1) host-guest binding method; (2) guest exchange ion molecular reaction method; (3) Diastereomer dissociation method. Among them, the diastereoisomer dissociation method is one of the most widely used methods for chiral analysis by mass spectrometry, which can obtain the specific MS/MS fragmentation patterns of the derived diastereoisomers. For example, the kinetic method of mass spectrometry uses tandem mass spectrometry to convert the small free energy differences between enantiomers into large differences in the fragmentation mode of the diastereomeric complexes formed, thus achieving chiral discrimination. Cooks et al. introduce a transition state divalent metal cation (M
2+) to react with a chiral analyte enantiomer (A
RorS) and a chiral reference (ref
*The complex dissociates as a precursor ion via CID competitive loss of the reference or analyte to the dimer ion [M
2+(A
RorS)(ref
*)-H]
+ or [M
2+(ref
*)
2-H]
+[68]. The activation energy required for the dissociation of diastereomers is different, so the abundance of product ions produced by the fragmentation of diastereomers in the secondary mass spectrum is different, thus realizing chiral recognition (Fig. 4). Enantiomeric quantitation can also be performed by constructing a standard curve from the natural logarithm of the change in the product dimer to dimer abundance ratio and the mole fraction of one of the enantiomers
[36]. The kinetic method of mass spectrometry is widely used in the analysis of chiral amino acids, and chiral amino acids are often introduced as chiral reference substances. Cooks et al. Used Cu
2+ and Trp as ligand ions and chiral reference agents, respectively, to carry out chiral recognition of selective N-alkylation products, and realized on-line monitoring of ee values of products
[36]. Zeng Su et al reported a series of studies in this area, such as the establishment of a kinetic mass spectrometry method for the [Ni
II(PPIs)(tadalafil)
2]
2+ of doubly charged trimeric complexes for chiral recognition and quantitative analysis of four tadalafil enantiomers and three proton pump inhibitors
[69]. In addition, Pan yuanjiang et al. Developed a chiral probe of N-benzenesulfonyl-2-pyrrolidinyl chloride, which realized the simultaneous determination of enantiomeric excess of amino acids in organic solvent systems and animal plasma
[70]. They also recently reported a new brominated chiral aldehyde probe (D-BPBr), which has good recognition effect and detection sensitivity for D-amino acids, and can realize the labeling, separation and detection of trace chiral amino acids and amino-containing metabolites combined with reversed phase C18 chromatographic column HPLC-MS/MS analysis. Using the characteristic ion of bromine isotope 2 Da and the peak intensity ratio of 1 ∶ 1, false positive results were effectively eliminated, and 119 amino acid metabolites were rapidly detected in urine and plasma samples.This method has the potential to be used for the screening of D-amino acid biomarkers in complex biological samples
[54].
Chiral recognition mass spectrometry is expected to play an important role in high-throughput chiral analysis because of its high sensitivity, fast analysis speed, and no interference from solvents and impurities.
3.3.2 Ion mobility mass spectrometry chiral analysis method
Ion mobility mass spectrometry has also made great progress in the chemical derivatization analysis of chiral amino acids. However, the chiral enantiomers can not be separated directly by ion mobility mass spectrometry because their CCS values are exactly the same, and they need to be converted into diastereomers by chiral derivatization before analysis. Chiral derivatization reagents used in ion mobility mass spectrometry are mainly divided into the following categories: (1) gas phase chiral modifiers added to buffer gas; (2) a chiral selector containing metal ions; (3) a chiral selector that does not contain metal ions; (4) Other chemical derivatization reagents. Yao Zhongping et al. Found that better chiral discrimination results could be obtained by combining TWIMS analysis with tetra-ligand [(Cu
2+)
2XY
2-4H+Na]
+] ions formed by Cu
2+ in the kinetic method
[44]. Although the use of multi-ligand ions in kinetic mass spectrometry can achieve good chiral discrimination, it needs to optimize the selection of the best metal ion and chiral reference agent for each chiral amino acid, which is not universal. Recently, Dominguez-Vega et al. Used (+) -1- (9-fluorenyl) ethyl chloroformate (FLEC) to derivatize 21 amino acids, and combined with TIMS-TOF MS to achieve chiral resolution of 17 of them
[55].
Ion mobility mass spectrometry (IMS) has high resolution and high separation speed, and has great prospects in chiral analysis. However, up to now, mass spectrometry based on chemical derivatization is still difficult to distinguish chiral enantiomers in complex mixtures such as biological tissues.
4 Conclusion and prospect
The chemical derivatization mass spectrometry method converts the substance to be detected into a product ion which is easy to be detected by mass spectrometry by introducing a derivatization reaction, thereby improving the detection sensitivity and selectivity of mass spectrometry, expanding the application range of the method in metabolite analysis, and enabling the mass spectrometry technology to accurately distinguish and quantitatively analyze various isomers. The combination of derivatization with high performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS) has been widely used in the separation and detection of metabolite isomers. However, the application of chemical derivatization in metabolite mass spectrometry is still challenging: (1) the fine structure analysis of metabolites often requires MS/MS or even MSn analysis, which lacks high-throughput analysis methods; (2) The existing methods can accurately distinguish metabolites from standard samples, but the detection sensitivity needs to be further improved for complex biological tissue samples; (3) characterization of the spatial distribution of the isomers by mass spectrometric imaging techniques without pre-separation; (4) Quantitative analysis of metabolite isomers in biological samples.
On-line chemical derivatization mass spectrometry based on free radical reaction and microdroplet accelerated chemical reaction during electrospray ionization has the advantages of simple operation and time saving, which is expected to open a new chapter in the application of mass spectrometry
[30,31]. The newly developed chemical derivatization mass spectrometry (CDMS) imaging technique can be used to visualize the low abundance, low ion response metabolites and fine structure isomers in the complex system of biological tissues, so the exploration of chemical derivatization mass spectrometry imaging method will become one of the development frontiers of mass spectrometry imaging technology. Further development of chemical derivatization methods suitable for mass spectrometry of endogenous metabolite isomers will help to discover more metabolic markers, better understand the mechanisms of various life processes, and provide new evidence for early diagnosis of diseases and discovery of potential drug targets.
the National Natural Science Foundation of China(21927808)
the Medical Science and Health Technology Innovation Project of Chinese Academy of Medical Sciences(2017-I2M-3-010)
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