Advances in capillary electrophoresis-mass spectrometry for cell analysis

Trends in Analytical Chemistry xxx (xxxx) xxx

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Advances in capillary electrophoresis-mass spectrometry for cell analysis Wei Zhou a, b, Baofang Zhang a, Yikun Liu a, Chenlu Wang a, Wenqi Sun a, Wen Li a, Zilin Chen a, b, * a

Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Beijing, 10080, China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

Mass spectrometry has become a powerful tool in the field of bioanalysis. The combination of capillary electrophoresis with mass spectrometry (CE-MS) offers fast and high-resolution separation, sensitive and selective detection with small sample volumes, which is suitable for cell analysis. Here, we review the recent technical development of CE, MS and hyphenated strategies as well as their potential in cell analysis. In particular, we discuss the multiple MS compatible CE modes including capillary zone electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic capillary chromatography and capillary electrochromatography for achieving better separation efficiency and selectivity before MS detection. For enhancing the sensitivity of CE-MS, several online preconcentration methods including dynamic pH junction, transient isotachophoresis and solid phase extraction are also reviewed and discussed. We also summarize the existing applications of CE-MS in cell analysis including cell biomacromolecule analysis, cell metabolism and single cell analysis. © 2019 Elsevier B.V. All rights reserved.

Keywords: Capillary electrophoresis Mass spectrometry Cell analysis Interface Biomacromolecule analysis Cell metabolism Single cell analysis

1. Introduction As the basic structural and functional units in biology, cells play vital roles in the functioning of living organisms and have been widely researched for their constituents and biological processes [1,2]. The analysis of cell is an essential and important part of disease diagnosis, treatment and drug discovery. Various analytical techniques like nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and ambient mass spectrometry have been applied for the study of their biological process, and the research targets are various from organs, tissues, single cell and even subcell [3e11]. CE emerged as an exciting and promising micro electric separation method since the late 1980s due to its advantages like low sample consumption, high resolution, short separation time and low cost. Various separation modes including capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary

* Corresponding author. School of Pharmaceutical Sciences, Wuhan University Wuhan, 430071, China. Fax: þ86 27-68759850. E-mail address: [email protected] (Z. Chen).

isotachophoresis (CITP), micellar electrokinetic capillary chromatography (MEKC) and capillary electrochromatography (CEC) have been applied in pharmacology, food analysis, biomarker research and biomolecular analysis [12,13]. Meanwhile, multiple detectors including ultraviolet (UV), fluorescence (FL), laser-induced fluorescence (LIF), electrochemical detector (ECD) and mass spectrometry (MS) have been equipped with CE instrument. Among them, MS serves as one of the most effective tools, especially for the analysis of biological samples due to its high sensitivity, high accuracy and powerful molecular structure elucidation [14,15]. However, the hyphenation of CE with MS is not straightforward, much emphasis of CE-MS methods for cell analysis has been placed on the development of coupling interface with high stability, good ionization efficiency and sensitivity. In recent years, for solving the dilution effect and section effect of commonly used coaxial sheathflow interface, a variety of novel nanospray interfaces were developed for better sensitivity and elimiating section effect [16]. Beside, investigation of MS compatible CE separation methods with good separation efficiency and selectivity is also important. The improvement of normal CZE methods, utilization of other separation methods including MEKC, CIEF, CITP, CEC and two-dimensional capillary electrophoresis (2D CE) were also investigated for

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coupling with MS [15,17]. In addition, in consideration of the complexity, low concentration and high throughput requirement for the analysis of biological samples, many other technologies such as online solid phase extraction (SPE), dynamic pH junction and sequential sample injection were also developed [14]. In this review, we provide an overview of the recent advances (mainly between 2014 and 2019) in CE-MS for cell analysis. The improvements and relevant innovations in CE-MS interfaces including CE-ESI-MS, CE-ICP-MS and CE-MALDI-MS, separation modes including CZE, MEKC, CITP, CIEF and CEC, some other related strategies like online preconcentration and improving of the column stability are summarized. Some innovative strategies that have not been applied in cell analysis until now but have the potential to be used are also included and discussed in detail. The applications including biomacromolecule analysis, cell metabolism analysis and single cell analysis are separately summarized. Microfluidics based CE-MS for cell analysis that have been reviewed in the recent two literature are not included in this paper [3,18]. 2. Development of instrumentation and methodology 2.1. CE-ESI-MS interface For online coupling CE with MS, ESI is the most frequently used ionization technique because it is relatively easy to be combined with CE, meanwhile, ESI is the most common source which enables the ionization of a wide variety of analytes into MS [16,19]. There are two major challenges in hyphenation of CE with ESI-MS. Firstly, the ESI source has strict requirements to the volatility of running buffer, it is common to use aqueous buffer with low concentration of formic acid or acetic acid as background electrolytes (BGEs) for online CE-ESI-MS. Most additives with special functions like sodium dodecyl sulfate, cyclodextrin are not allowed in normal CEESI-MS. So the selection of suitable CE separation method is important for CE-ESI-MS analysis. Secondly, in CE-ESI-MS system, the end of separation capillary in CE is not in the outlet vial, but connected with the ESI chamber of MS. It is necessary to keep a closed electric circuit for CE separation and ESI processes. So it is important but difficult to ensure stable current in the interface where multiple electrochemical reactions occur and the flow rate of running buffer is relatively low [20]. For solving this problem, a variety of interface designs have been proposed in order to maximize the potential of this hyphenated technique during these years. The most commonly used CE-ESI-MS interface is the pneumatically-assisted co-axial sheath liquid interface which is stable for using in various conditions and this interface was commercialized by Agilent Technologies. However, the substantial dilution of the sheath liquid (2e10 mL/min) leads to the significant decrease of sensitivity. In addition, the nebulizing gas that assists the ESI spray and the sheath liquid are known to cause a hydrodynamic flow inside the separation capillary, which is called suction effects. The suction effects have been reported to cause peak broadening and decrease the migration time [21,22]. In recent years, many techniques have been developed for improving of the sensitivity of the CE-MS while keeping flexibility and robustness. Among them, nanoflow sheath liquid interface and sheathless interface are two attractive kinds of interfaces [23]. Electrokinetically pumped sheath flow ESI interface was commercialized by CMP scientific, and porous tip-based sheathless interface was commercialized by Sciex. The commercialization of CE-MS interfaces promoted the development of CE-MS in various fields. 2.1.1. Nanoflow sheath liquid interfaces According to the flow rate, the CE-MS interfaces can be classified as two different working ranges including ion spray and nanospray.

The former one is operated at flow rates between 1 and 1000 mL/ min, produces micrometer diameter droplets and is often pneumatically assisted by a nebulizing gas. The later one is operated at flow rates below 1000 nL/min and allows obtaining smaller droplets (under 200 nm) without the assist of nebulizing gas. Compared with ion spray, nanospray is more compatible with CE with higher ionization efficiency and matrix effects tend to be diminished [16]. In the sheath flow interface, the sheath liquid is pumped into the outlet of the separation capillary for establishing the electrical contact. Besides, it increases the flexibility of the interface because changing the composition of CE running buffer and postmodification of the analytes can be achieved by specific design of the sheath liquid [24]. Although the existence of sheath liquid causes the dilution effect, decreasing of the flow rate of sheath liquid to nL/min can produce nanospray, which significantly reduces the dilution effect while keeping the inherent advantages of sheath flow interface unchanged [25]. Several innovative strategies developed for the construction of nanoflow sheath liquid interface which have been or have the potential to be used in cell analysis are reviewed below. The concept of “junction-at-the-tip”, that is, an outer tube surrounding the separation capillary used for mixing the CE running buffer and sheath liquid at the tip of electrospray emitter. It was utilized for design of low sheath flow interfaces [26,27]. As shown in Fig. 1a, by modifying junction-at-the-tip interface with a beveled metal emitter tip, a nanoflow sheath liquid interface that the flow rate of sheath liquid can be as low as 0.1 mL/min was developed. The volume of the micro-vial was only approximately 12 nL and the Taylor cone was located at the sharpest point of the tip rather than the orifice, which can eliminate the disrupt of small bubbles to the stability of Taylor cone. This interface showed better sensitivity than normal sheath-flow interface for the analysis of various amino acids and was also used for the cationic metabolites analysis of zebrafish embryos. Another simple nanoflow sheath liquid interface operating in the submicroliter nanospray regime without the assistance of nebulizing gas was developed with different design concept [28]. As shown in Fig. 1b, the separation capillary (50 mm i.d., 363 mm o.d.) was passed through a blunt metal needle (420 mm i.d., 720 mm o.d.) for construction of a coaxial spray tip. By adjusting the key parameters including the working distance from the MS inlet, ESI voltage, sheath liquid composition and flow rate, the Taylor cone with small diameter shown in Fig. 1b was obtained, which resulted in better performance. The second generation of this kind of interface was developed by reducing the diameter of the separation capillary and the emitter, which led to less peak broadening associated to the ESI process [24]. The novel interface was applied to the analysis of neural cell culture samples with good migration time repeatability and sensitivity. Similar interface was also developed by Peter Nemes et al. [29]. Besides, as shown in Fig. 1c, by threading the chemical etching separation capillary with thin wall (20 mm i.d., 30 mm o.d.) into a glass electrospray tip (200 mm i.d., 375 mm o.d.) with tapered tip (40 mm o.d.), an extendable sheath flow interface with only 4 pL of interface dead volume was developed for improving the CE separation efficiency and detection sensitivity [30]. Dovichi et al. exploited a series of electrokinetically pumped nanoflow sheath liquid interface for coupling CE with MS [25,31,32]. As shown in Fig. 1d, in general, the separation capillary was threaded into the glass emitter, and electrospray voltage also drove the EOF in the glass emitter as sheath liquid at a rate of nanoliter per minute. Three generations of this kind of interfaces were reported which were different in electrospray tip. The thirdgeneration interface employed a large diameter emitter orifice with only 20 mm distance between the separation capillary tip and emitter orifice. This interface reduced the possibility of plugging

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Fig. 1. Nanoflow sheath liquid CE-MS interfaces. (a) Junction at-the-tip interface with a beveled metal emitter tip. Reprinted with permission from Ref. [26]. Copyright© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (b) Technical diagram of the low sheath-flow interface assembly and detailed geometry of the fluid domain. Reprinted with permission from Ref. [28]. Copyright© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (c) Schematic diagram of the structure of the extendable sheath-flow interface. Reprinted with permission from Ref. [30]. Copyright© 2017 Elsevier. (d) Comparison of three generation of electrokinetically pumped nanoflow sheath liquid interface. Reprinted with permission from Ref. [32]. Copyright© 2015 American Chemical Society.

and was much more robust with no loss in sensitivity. For applying in negative mode electrospray MS, the normal glass emitter was no longer suitable because the EOF was from emitter orifice to the cross [33]. So 3-aminopropyltrimethoxysilane with positive charge was modified onto the inner wall of glass emitter for reversing the EOF. After that, negative mode operation was achieved with good reproducibility. By employing electrokinetically pumped nanoflow sheath liquid interface, plenty of works in bottom-up proteomics and metabolite analysis have been reported by Dovichi's group [34]. 2.1.2. Sheathless interfaces Directly coupling CE with MS without additional liquid is a good choice since no sample dilution occurs, but establishing a long term stable electrical contact is challenging. The most commonly used methods were coating the capillary tip with conductive materials like graphite, polyaniline, gold, copper, or silver to make the electrical contact for CE and ESI. However, the main problem comes from the rapid deterioration of the coating material under high voltage condition, which is detrimental to durable operations [14]. In recent years, using cracks in the capillary wall and sheathless porous tip are two main strategies for achieving electrical contact with long lifetime and stability. As shown in Fig. 2a, a sub-micrometer fracture was fabricated on the fused silica capillary after being stripped of the polyimide coating and protected by a piece of PTFE heat shrink tubing for electrical contact [35]. The fracture had a dimension of less than 2 mm in width, and high backpressure prevented leaking of the bulk flow. A metal electrode was inserted into the buffer reservoir for providing outlet voltage of CE and ESI voltage for spraying (the voltage was in a range of 1.0e2.2 kV). The simple interface allowed stable CE separation and ESI spray at nanoliter flow rates between 45 and 90 nL/min. The novel sheathless CE-MS interface was successfully applied for the analysis of peptide and protein samples with high sensitivity. Another crack based sheathless interface was fabricated by a programmable CO2 laser ablation and then coated with electrically conductive semi-permeable membrane [36]. The existing of semi-permeable cellulose acetate cast can not only prevent the leaking in the crack, but also make the electrical

contact. Hirayama et al. developed a sheathless interface by making a small crack at the end of the fused silica capillary (Fig. 2b) [37]. The crack was carefully created by fixing the capillary on a plastic plate. An electrodialysis membrane made with cellulose acetate covered the crack to minimize the leaking of liquid while keeping the electrical contact. With the similar strategy, a thin conducting liquid film based sheathless CE-MS interface was designed [38]. As shown in Fig. 2c, a poly(dimethylsiloxane) (PDMS)-based microdevice was fabricated to facilitate alignment of the capillary and construction of microchannel. The microchannel (50 mm i.d.) in the microdevice was used for the connection of the CE separation capillary and ESI spray capillary. The polyimide coating at the left end of ESI sprayer was stripped, so the size of hole in the PDMS plate was slightly larger than the outer diameter of the ESI sprayer, and the electrical conduction was formed by the thin conducting liquid film between the sprayer and microchannel. The transparent PDMS material with channels allowed the facile alignment of the two columns and the separation capillary or spray tip can be easily changed. For further improving this interface, the same group used a triangular PDMS emitter with a microchannel instead of tapered fused silica capillary as spray emitter [39]. The PDMS emitter was suitably robust and the novel interface provided stable electrospray with flow rates ranging from 30 to 350 nL/min. For applying counterflow-assisted preconcentration technique in CE-MS, a twoleveled cross-type microchannel was integrated into the above thin conductive liquid film based interface for facilitating an additional flow into the CE inlet [40]. Significant improvement of the sensitivity for detection of five peptide standard samples was obtained by using this technique. In general, the key point of this kind of interface is making small crack for achieving electrical contact while preventing the leaking of buffer solution. Moini has developed a porous tip based sheathless interface in 2007 which significantly promoted the development of sheathless interface [41]. In this design, a porous capillary etched with hydrofluoric acid was used as spray tip, and the emitter was surrounded by conductive liquid for providing electrical connection. The interface showed stable spray and good ionization efficiency even with low flow rates. In recent years, several designs were introduced for improving this interface to some extent. By using a

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Fig. 2. Sheathless CE-MS interfaces. (a) A sheathless interface with a sub-micrometer electrical contact fracture. Reprinted with permission from Ref. [35]. Copyright© 2016 Elsevier. (b) Schematic of the sheathless CE-ESI-MS interface: (1) plastic plate (2 mm thick); (2) electrodialysis membrane; (3) separation capillary; (4) platinum electrode; and (5) buffer reservoir. Reprinted with permission from Ref. [37]. Copyright© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (c) Schematic of thin conducting liquid film based two capillary sheathless interface. Reprinted with permission from Ref. [38]. Copyright© 2012 Elsevier. (d) Schematic of the porous tip based sheathless interface. Reprinted with permission from Ref. [41]. Copyright© 2007 American Chemical Society.

short (<20 cm) and narrow (5 mm) capillary, the sheathless interface based CE-MS method achieved the analysis of peptide mixture and protein digest in ~1 min [42]. A dopant enriched nitrogen gas was combined with porous tip based CE-MS interface for enhancing the spray efficiency [43]. Using acetonitrile as dopant, up to 25-fold higher sensitivity was obtained for analysis of model glycopeptides with good precision. Besides, a commercially available capillary with metal-coating instead of chemical etched porous tip was used for electrical connection and ESI spray [44]. The metal-coated capillary overcame the reproducibility and ruggedness problems of etched porous tip and also possessed good stability and sensitivity. In conclusion, for porous tip based sheathless interface, the advantage of high sensitivity is apparent, however, the separation and ESI spray are integrated into one capillary, so if the clogging or contaminating events occur, the whole capillary needs to be changed, and chemical etching of the porous tip is relatively difficult with low reproducibility when operated in the laboratory. Besides the two main sheathless interface designs, a concept of “interface-free” CE-MS was also proposed in recent years. In this design, the electrical connection of the electrospray was completely omitted, and only one power supply was used for both CE separation and ESI spray. This hyphenation method was simple without the additional equipment [45]. However, strict requirements for small capillary diameter (<20 mm i.d.), limited range of applied voltages made it difficult for routine analysis. This configuration was further improved by coating the emitter tip with hydrophobic material for improving the nanospray ionization [46]. During the CE-MS analysis process, high voltage of 30 kV was firstly applied with no liquid flow. Fast separation of sample ions occurred in the capillary and corona discharge took place at the ESI tip and they were unsuitable for MS analysis. After several minutes, the voltage was decreased to 5 kV for ESI spray and additional pressure was added in the inlet of the capillary to generate a flow of 30 nL/min. The operation was relatively complex. 2.2. CE-MS with other ionization methods Although ESI is the predominant ionization source for coupling CE with MS, other sources including matrix assisted laser desorption/ionization (MALDI), inductively coupled plasma (ICP), and

ambient ionization are also utilized in CE-MS for special using. Especially for CE-MALDI-MS, it has good potential for the application in cell analysis. In the following section of this review, CE-MS with MALDI, ICP and ambient ionization method are included, while CE-MALDI-MS is detailedly summarized. MALDI is an attractive ionization technique due to the ability for dealing with non-volatile compounds, and it is suitable for the analysis of biomacromolecules like proteins, DNA and sugars. CE hyphenated to MALDI-MS is mostly achieved by offline mode. So non-volatile additives in running buffer or offline sample pretreatment can be integrated into CE-MALDI-MS. The first totally automated off-line CE-UV/MALDI-MS/MS instrument was developed based on the modification of commercial CE instrument, spotting device and adding an integrated delivery matrix system [47]. As shown in Fig. 3a, a co-axial sheath liquid deliver device was used as outlet of CE which was positioned by a robotic x-y-z axis motion system. The matrix was supplied by other capillaries connected to the CE instrument which can add pressure. The automated CE-MALDI-MS system was successfully applied for the analysis of intact proteins, protein digestion samples and monoclonal antibodies (mAbs). Based on this automated system, the same group added a fraction collection enrichment procedure to enhance the sensitivity of the top-down and middle-down analysis of mAbs [48]. As shown in Fig. 3b, a novel off-line approach was developed for coupling of a single run of CE separation to both MALDI-MS and substrate-assisted laser desorption (SALD) ICP-MS [49]. The CE fractions of 20-nL droplets were removed from the capillary outlet to 10-nm gold layer coated polyethylene terephthalate glycol target plate. The gold layer guaranteed the compatibility with both MALDI and SALD ICP ionization method. The combined method allowed workers to obtain both proteomic and metallothionein isoforms. MALDI-MS imaging (MALDI-MSI) has also been hyphenated with CE separation for metabolites study [50]. By coupling with in vivo microdialysis sampling, the method was used for studying neurotransmitters and other metabolites in the crustacean model Cancer borealis. ICP-MS offers several advantages including high elementspecificity, direct detection without derivatization, high sensitivity and simultaneous multi-element detection. After combining with CE separation, CE-ICP-MS has great potential for applying in elemental analysis of biological samples which always have limited

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Fig. 3. CE-MALDI-MS. (a) Schematic representation of the experimental setup of the CE-UV/MALDI-MS interface. Reprinted with permission from Ref. [47]. Copyright© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (b) Experiment workflow of the offline coupling of CE(UV)-MALDI MS/SALD ICP-MS. Reprinted with permission from Ref. [49]. Copyright© 2013 American Chemical Society.

sample amount [51]. However, due to the specialty of ICP-MS, the CE-ICP-MS is rarely used in cell analysis. The CE-ICP-MS interfaces are similar with CE-ESI-MS to some extent. In early years, commercial pneumatically-assisted co-axial sheath liquid ESI-MS sprayer was fabricated for using in CE-ICP-MS with the mixture of water and methanol containing 100 ng/mL rhodium as sheath liquid [52]. The interface also showed good performance in CE-ICPMS. Recently, the same research group developed a novel directinjection high-efficiency nebulizer chamber to couple with this interface for improving the sensitivity of CE-ICP-MS [53]. As shown in Fig. 4a, a miniaturized direct-injection high-efficiency nebulizer chamber was designed instead of regular Scott double pass spray chamber for minimizing the gas phase dead volume and maintaining the high resolution of CE. It was found that the peak intensity of arsenic increased apparently when compared with the regular chamber. As shown in Fig. 4b, a microflow polyfluoroalkoxy nebulizer with lower flow rate at 10e20 mL/min was used for decreasing the dilution and improving the nebulization [54]. The sheath flow was introduced by a peristaltic pump for electrical connection and carried the analytes to the ICP-MS. This design of the interface can decrease the suction effect caused by nebulization gas. The concept of ambient ionization MS emerged in 2004 for direct ionization of analytes under ambient pressure and it has developed quickly in the last decade [55]. Various ambient ionization methods were developed but they were rarely used in CEMS. Bai's group developed an interface for combining CE with dielectric barrier discharge ionization (DBDI) MS [56]. As shown in Fig. 4c, the spray tip of co-axial sheath liquid interface was put between the DBDI outlet and MS inlet. The nebulized sample and sheath liquid were ionized by DBDI before being analyzed by MS. It was interesting that this kind of interface showed high tolerance of non-volatile salts and surfactants, which was beneficial for CE with special additives. 2.3. Capillary electrophoresis Introducing high efficiency separation technique before MS detection is essential especially for complex biological samples

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because it can reduce the interference of complex matrix, enhance the sensitivity and provide the possibility for analyzing of chiral compounds. As an alternative separation method, which possesses advantages like low sample consumption, high separation efficiency and multiple separation modes, various strategies have been reported for improving the CE process. In recent years, 2D CE, different capillary coating and multiple separation modes including CEC, CITP, MEKC and NACE were reported for enhancing the separation efficiency of CE. Online preconcentration methods like solid phase extraction (SPE), dynamic pH junction and field-enhanced sample injection were integrated before CE separation for enhancing the sensitivity of CE-MS. For improving the stability of commonly used polyimide coated fused silica capillary, two strategies including adjusting the buffer constituents and changing the separation capillary material were proposed. The recent progresses about the above topics were reviewed below. The development of 2D separation improves the separation ability of CE and enables the use of CE-ESI-MS incompatible additives in the first separation dimension [17]. Christian Neusüß’s group developed a 4-port-nL-valve based CE-CE-MS approach for achieving online interface-free 2D CE-MS [57,58]. As shown in Fig. 5a, the ESI incompatible electrolytes and additives were used in the first dimensional separation with a UV detector, while ESI compatible electrolytes were used in the second dimensional for CE-ESI-MS analysis of the selected fractions in the first dimension. Utilizing this strategy, successful analysis of ascorbic acid, acetylsalicylic acid and their related degradation products were achieved with non-volatile tricine of 100 mM as BGE for the first dimension CZE and ammonium acetate of 25 mM as BGE for the second dimension CZE [59]. The same valve interface was also applied in the CIEF-CZE-MS for analysis of proteins and mAbs with CIEF as the first dimension separation [60,61]. These applications demonstrated that the four-port valve based 2D CE-MS approach was a good way for online hyphenation of CE and MS with incompatible mode and additives. CZE is the simplest and most common separation mode in CE. When bare fused-silica capillary with silanol groups in the inner wall is directly used as separation capillary, the poor EOF adjustment ability and adsorption of positively charged samples may cause low resolution and poor reproducibility. So various coating materials were proposed for adjusting the EOF and reducing adsorption. After covalent bonding of cyanomethyl [3-(trimethoxysilyl)propyl]-trithiocarbonate, acrylamide and 4,40 -azobis(4cyanovaleric acid) were introduced into the capillary for forming linear poly(acrylamide) coated capillary [62]. The poly(acrylamide) coating can also be prepared by introducing the mixture of acrylamide and ammonium persulfate solution into the capillary and reacted in a water bath [63]. The coating not only reduced the adsorption of proteins and peptides, but also provided a very low EOF for increasing the separation window. Three kinds of capillaries including bare fused silica capillary, neutral capillary (with two coating layers including one hydrophobic coating and one hydrophilic polyacrylamide coating) and positive polyethyleneimine coated capillary were used for studying a set of 70 synthetic posttranslationally modified peptides [64]. The neutrally coated capillary showed the highest overall signal intensity of singly modified peptides, while bare capillary was better in identification of multiphosphorylated peptides. The results suggested that the coating was essential for adjusting the separation efficiency and selectivity of CZE-MS. Nonaqueous CE-MS is a kind of CZE-MS which uses pure organic solvents instead of normal aqueous solvents. It is suitable for analysis of hydrophobic ionic compounds that are more soluble in organic solvents than in water. In addition, most organic solvents are more volatile and have lower surface tension than water, making NACE more suitable for online coupling with MS [65,66].

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Fig. 4. CE-ICP-MS and ambient ionization based CE-MS. (a) Schematic diagram of the interface for coupling CE with ICP-MS. Reprinted with permission from Ref. [53]. Copyright© 2014 American Chemical Society. (b) Schematic diagram for CE-ICP-MS. Reprinted with permission from Ref. [51]. Copyright© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (c) Schematic diagram of the CE-DBDI-MS interface. Reprinted with permission from Ref. [56]. Copyright© 2016 Springer.

Fig. 5. Different CE methods for CE-MS. (a) General arrangement of the 2D CIEF-CZE-MS setup. Reprinted with permission from Ref. [61]. Copyright© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (b) Schematic of the new sheathless CITP/CZE-nanoESI-MS setup. Reprinted with permission from Ref. [44]. Copyright© 2016 American Chemical Society. (c) CE-MS analysis of herb sample using robust PEEK capillary. Reprinted with permission from Ref. [21]. Copyright© 2019 Elsevier.

Completely nonaqueous solvents in both BGE and sheath liquid based NACE-MS method was developed for determining active ingredients in plant extracts [67]. The results showed the sensitivity was 1e2 orders of magnitude higher than previous NACE-MS methods. CEC is a hybrid technique which combines the virtues of CE and HPLC. Compared with CZE, the existence of stationary phase can improve the separation efficiency and selectivity [68e70]. Although there are various kinds of CEC columns like open tubular CEC, monolithic column and packed column, only a few of them were used for CEC-MS analysis. A poly(stearyl methacrylate-divinylbenzene) monolithic column was coupled

with atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) for analysis of 16 polycyclic aromatic hydrocarbons (PAHs) [71]. In this method, the utilization of hydrophobic monolithic stationary phase successfully separated the neutral PAHs, which can't be separated by charge difference, and the APCI achieved the ionization of PAHs which can't be ionized by ESI. Pressurized CEC coupled with ESI-MS was used for metabolomics study [72]. The additional pressure on the inlet of C18 packed CEC column can ensure a stable flow of the running buffer. Selecting of proper stationary phase and MS compatible running buffer for different analytes are important in CEC-MS analysis.

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The main problem for coupling other CE modes like MEKC and CIEF with MS is the incompatible BGE and additives. Besides 2D CEMS, other strategies have also been proposed for coupling these CE modes with MS. In MEKC, electrokinetic migration and partitioning of analytes between bulk solution and micellar phases are combined for separation. Polymeric surfactants are suitable for MEKCESI-MS because molecular micelles are covalently stabilized micellar aggregates, which remain intact in solution phase in the capillary. By using polysodium N-undecenoyl-L,L-leucylvalinate as chiral pseudophase in MEKC-MS, baseline separation of warfarin and its five metabolites was completed in 45 min [73]. Ammonium perfluorooctanoate is a volatile surfactant, which presents less effect on analyte response and ion suppression. Several MEKC-ESI-MS methods based on volatile ammonium perfluorooctanoate were reported for multiple analysis with good performance [74e77]. Using ICP-MS instead of ESI-MS for tolerance of non-volatile surfactants is also a good way. Carsten Engelhard's group developed a MEKC-ICP-MS method using normal sodium dodecyl sulfate as pseudophase for fast separation, characterization and speciation of gold and silver nanoparticles and their ionic counterparts [78]. CIEF is a separation mode which is suitable for analysis of amphoteric compounds like amino acids, peptides, proteins that based on the pI difference of analytes. A pH gradient is firstly formed in the capillary under the influence of electric field. Then the sample molecules migrate through the electric field and focus in the region where environment pH value is equal to their pI. Directly coupling CIEF with ESI-MS suffers from the interference caused by commercial ampholytes. These ampholytes have similar molecular weight as peptides, which generated large background signal and competed with peptides in ESI process. Six amino acids with different pIs ranging from 3.2 to 9.7 were employed as ampholytes for CIEF-ESIMS. The background signal in m/z between 300 and 1800 of amino acids as ampholytes was significantly lower than that of commercial ampholytes [79]. This method was successfully used for protein digests analysis with high resolution [80]. A glycerol/water medium in bare fused silica capillary was used to replace normal aqueous gel medium in coated capillary [81]. The glycerol/water medium was anti-convective and minimized protein adsorption, which was suitable for online CIEF-ESI-MS analysis. Another alternative method for coupling CIEF with MS was using MALDI-MS in off-line mode [82]. So the ESI-MS incompatible reagents like sodium hydroxide, orthophosphoric acid and poly(ethylene glycol) can all be used in CIEF separation. CZE employs an open-tube capillary for separation and has no stationary phase, in order to keep high separation efficiency, the sample injection volume usually needs to be less than 1% of the total capillary volume. Therefore, the sample injection amount in CZE is limited. In order to increase the sample loading capacity of CE-MS, many sample preconcentration methods including SPE, dynamic pH injection, field-enhanced sample injection and CITP were online coupled with CE-MS. Solid phase microextraction (SPME) allows for both preconcentration of peptides and sequential elution of peptide fractions for improved proteomic coverage. Yates's group developed a SPME method with multistep elution and coupling with transient isotachophoresis CE-MS/MS for bottom-up proteomic analysis using a high sensitivity porous ESI sprayer [83]. The results demonstrated that this method was 3 times more effective than nanoflow liquid chromatography at identifying proteins when dealing with mass-limited amounts (5 ng) of Pyrococcus furiosus tryptic digest. A microcartridge (7 mm  250 mm i.d.  360 mm o.d.) filled with C18 particles was integrated into the fused silica capillary by two plastic sleeves at 7.5 cm from the inlet [84]. Two frits were used to retain the particles. The existence of C18 particles in the capillary extracted the hydrophobic target compounds in the large injection volume. After washing with

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buffer solution, the target compounds were eluted by less volume (about 50 nL) elution buffer and then separated by applying a voltage. The online SPE microcartridge can not only enhance the sensitivity of CE-MS, but also clean the complex matrix. A 1.5 cm length of sulfonated-silica hybrid strong cation exchange (SCX) monolith was covalently immobilized into the inlet of separation capillary for online SPE-CE-MS [85]. The protein digest samples dissolved in 1 M acetic acid were loaded by pressure and then eluted by 200 mM ammonium bicarbonate (pH 8). The SCX monolith based online SPE-CE-MS method showed good performance for the analysis of Xenopus laevis eggs protein digest. Besides SPE, electrophoretic preconcentration methods were also applied for CE-MS. Dynamic pH junction preconcentration method was integrated with CZE-ESI-MS for bottom-up proteomics [86,87]. Compared with normal injection method, this method performed large injection volume and a significantly larger identified number of proteins and peptides. Sun group reported a dynamic pH junction method and linear polyacrylamide coated capillary for CE-MS. They achieved a microliter-scale capacity, a 140 min separation window and a high peak capacity (~380) for bottom-up proteomics [88]. The similar dynamic pH junction method was also used for single-shot top-down proteomics after coupling with CZE-ESI-MS [89]. In addition, the capture efficiency of target analytes was defined and studied by Chen group, they demonstrated that dynamic pH junction could reproducibly focus more than 95% of the target molecule injected into the capillary [90]. CITP was also coupled with CZE-MS for enhancing the sample injection amount. As shown in Fig. 5b, the inner wall of separation capillary was coated by hydroxypropyl cellulose for suppressing the EOF [44]. 25 mM ammonium acetate in deionized water (pH 4) was used as leading electrolyte solution and 0.1 M acetic acid in 9:1 vol ratio of deionized water to methanol was used as BGE. The peptide samples were dissolved in leading electrolyte solution with lower electrophoretic mobility than BGE. By using CITP/CZE method, the sample loading capacity can reach over 30% of the total separation capillary volume. After loading of large sample volume, the metal coated emitter was protected by a movable Teflon tube and high voltage was applied for 15 min to make CITP/CZE separation. Then the protective Teflon tubing was moved back and small nitrogen backpressure was applied in the inlet for eluting the samples to MS inlet for detection. Transient isotachophoresis was introduced for the full antibody primary structure and microvariant characterization, allowing injection of up to 25% of the total capillary volume for CE-MS analysis [91]. In addition, field-enhanced sample injection micelle-to-solvent stacking method was also applied for CZEMS analysis of eight penicillins and sulfonamides [92]. Sensitivity enhancement factors (peak height) were 1623e3328 compared to typical injection. In summary, dynamic pH junction is well-suited for preconcentration of amphiprotic analytes, especially for peptides and proteins. ITP can achieve concentrate trace of components in high concentration of matrix ions and is efficient for analysis of low conductivity sample. Compared with electric stacking preconcentration method, online SPE method is more difficult to operate because much effort needs to be applied on the preparation of SPE column and carefully connecting it with separation capillary. But the selectivity of SPE is better because various materials with different extraction functions can be used in the SPE column [93]. The long-term stability of CE-MS remains a major obstacle hampering its widespread application. For commonly used polyimide coated fused silica capillary, the polyimide coating is prone to aminolysis in alkaline buffer and swell in organic solvents. Especially in CE-MS system, when the polyimide coating is exposed in the spray chamber with high temperature and high humidity [94]. For avoiding current drop, irregular electrospray and clogging events, several strategies were proposed. Using weak alkaline

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ammonia containing buffer (pH < 9) or non-ammonium buffer was demonstrated as an effective way for enhancing the capillary stability [95]. In addition, Chen group directly used poly(ether ether ketone) (PEEK) capillary instead of polyimide coated fused silica capillary as separation column in CE-MS [21,94]. Although the bad optical permeability restricts the application of PEEK capillary in CE analysis with optical detector, the column-end detection character of MS solves this problem. As shown in Fig. 5c, the PEEK capillary possessed excellent properties like high chemical stability, high strength, good thermal stability and easy to cut. The experimental results suggested that PEEK capillary was robust enough in CE-MS system for tolerance to high content of organic solvents and alkaline buffer. However, the high chemical resistance of PEEK material make the modification of capillary wall become much more difficult than fused silica capillary. For solving the poor EOF adjustment ability and high chemical resistant problems of PEEK capillary, bioinspired polydopamine method was used to functionalize the inner wall of PEEK capillary. The zwitterionic polydopamine layer can generate cathodic EOF at high pH value (pH  5) and anodic EOF at low pH value (pH  4), which was beneficial for CE separation. 3. Application 3.1. Biomacromolecule analysis Biomacromolecules like proteins, peptides, nucleic acids and glycans, have significant biological functions in cell, such as signal transduction, cell division, cell-cell interaction and making up cellular structures. Due to the low abundance of target biomacromolecule and the complex composition of biological matrix, higher demands are put forward to the separation capability and the sensitivity of instrument. Reverse phase liquid chromatography coupled with mass spectrometry (RPLC-MS) has been routinely used for the separation and detection of biomacromolecules, and the separation mechanism is based on their hydrophobicity. However, sample loss of hydrophilic molecules during sample loading and separation is significant when using RPLC-MS. CE can provide separation mechanism based on size-to-charge ratios, which can be a complement to LC. Meanwhile, CE-MS has high separation efficiency and high detection sensitivity, which can be an efficient analytical method for biomacromolecule analysis [88,96,97]. It has been demonstrated that CE-MS method showed higher sensitivity than nanoLC-MS or ultra-performance liquid chromatography (UPLC)-MS methods in bottom-up and top-down proteomics when dealing with mass-limited samples like 5 ng [83,98e100]. In addition, the sensitivity can be improved when coupling with online preconcentration methods like transient isotachophoresis or SPE. In recent years, CE-MS has been effectively used for proteomics, protein post-translational modifications analysis, antibody and microRNA detection, and these applications are reviewed here. Some specific experiment conditions employed in the relevant application of CE-MS in cell analysis were summarized in Table 1. Proteomics has been a significant tool in molecular biology and medical science, and bottom-up proteomic analysis is an important part of proteomics [62,97]. In bottom-up proteomic, the protein is digested by enzyme into peptides, then the peptides are identified and quantified after separation. Better separation strategy can improve the proteome coverage [32,101,102]. Because of the large number of peptides cleaved by enzyme, routinely used HPLC cannot meet demands for peptides separation effectively. Lindner's group combined CE-MS with HPLC and applied it for quantitative proteomics [97]. As shown in Fig. 6a and 1371 phosphopeptides were successfully quantified from yeast strain lysates by RP-HPLC followed by CE-MS detection. For the aspect of identification of

peptides, 33854 unique peptide sequences were found and 8106 modified peptides including acetylation, phosphorylation, deamidation, and oxidized forms were identified. Sun group used nanoflow RPLC/CZE-MS/MS method to produce over 7500 protein identifications and nearly 60000 peptide identifications from 5 mg of MCF7 breast cancer cell proteome digest [87]. However, inherent problems also exist in CE-MS analysis, CE employs small sample injection volumes which result in low loading capacity and poor concentration detection limits. To solve these problems, different strategies were developed. Dynamic pH junction is a simple and intriguing method to improve the loading capacity of CE [86]. As shown in Fig. 6b, through dynamic pH junction, a larger volume of sample can be introduced with little loss of separation efficiency. The proposed method was used for the analysis of E. coli digests and intact protein, and compared to conventional injection, the peak strength can be increased by 10 times. Besides dynamic pH junction, Dovichi and his co-workers have combined solid-phase extraction (SPE) with CE-MS for bottom-up proteomics [85]. A polyacrylamide coating and a sulfonate-silica hybrid strong cation exchange (SCX) monolith were prepared sequentially in a single fused silica capillary and used for SPE to preconcentrate the protein digest from Xenopus laevis eggs. 330 protein groups and 872 peptides were identified through this method. Sun group established a strong cation exchange-RPLC-CZE-MS/MS platform for bottom-up proteomics. They identified around 8200 protein groups and 65000 unique peptides from a mouse brain proteome digest in 70 h, which are comparable to the performance of 2D-LC-MS/MS method [103]. Although bottom-up proteomics by MS is high-throughput, the proteoform information pertaining to variants, splice isoforms, and combinatorial post-translational modifications are often lost after proteolysis, intact protein analysis by top-down proteomics can better collect these information. Sun and his co-workers using dynamic pH junction-based CZE-MS/MS to achieve top-down analysis of an Escherichia coli proteome with high loading and peak capacity. About 2800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins were identified [89]. They also coupled size exclusion chromatography and reversed phase LC based protein prefractionation to CZE-MS/MS for deep top-down proteomics of Escherichia coli. 5700 proteoforms were identified [104]. Post-translational modification (PTM) of proteins refers to chemical changes that proteins may undergo after translation, and it plays a fundamental role in protein folding, stability and conformation so that is important to cellular functions. More than 40 PTMs have been identified that related to diseases such as cancer and neurological disorders [105]. Therefore it is of great significance to identify the PTMs of protein. Due to the high separation capability of CE and the high sensitivity of MS, CE-MS can preferably perform to identify the peptides or proteins that carry modification. A series of related researches has been carried out by Lindner's group [64,106,107]. Three differently coated separation capillaries have been applied for analysis of post-translationally modified peptides by CE-MS, including phosphorylation, acetylation, methylation, and nitration [64]. And through this method, 5685 phosphopeptides were identified and 4088 phosphopeptides were quantified from PC-12 pheochromocytoma cells. Besides, CEMS was also used for identification of myelin basic peptides from mouse brain which incorporate the same PTM at different sites [106]. In this work, 40 modifications at 33 different sites of myelin basic protein from brain of mice of different ages were identified, including Na-terminal acetylation, mono- and dimethylation, phosphorylation, oxidation, deamidation, and citrullination. CZEESI-MS/MS method was used for phosphorylated peptide identifications using an enriched phosphorproteome from the MCF-10A cell line. By coupling with dynamic pH junction method, 2313

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Table 1 Summarization of some specific experiment conditions employed in the relevant applications of CE-MS in cell analysis. Interface/MS

Comment

Ref

Proteomics of K562-cell digest

Analyte/Sample

CZE/LPA

1 M HAc

Electrokinetically pumped sheath flow interface/Orbitrap Fusion Lumos Tribrid MS

[62]

Proteomics of human lymphoblastic T-cells

RPLC-CZE/DMA-GMA-MAPS

10% HAc

Porous tip based sheathless interface/Q Exactive MS

Glycoprofiling of recombinant human erythropoietin mAbs

CZE/Polyacrylamide

50 mM HAc

CIEF/Neutral coating

NH4OH with 15% glycerol and 1% FA with 15% glycerol as catholyte and anolyte respectively 10% HAc (pH 2.2)

Porous tip based sheathless interface/TOF MS Electrokinetically pumped sheath flow interface/TOF MS

Over 27 000 peptide and nearly 4400 protein-group identifications in a single 120 min run 62 000 peptides and 6100 proteins were identified within 12.5 h Revealing 74 glycoforms in a 60-min run Showing linear correlation (R2 ¼ 0.99) between pI values and migration time with good repeatability LODs between 10 and 200 nM were obtained with an injection volume of only circa 20 nL Successful quantitative analysis of endogenous anionic metabolites in Aplysia californica neurons ~230 Different molecular features including 70 known metabolites were detected with <0.02% of the single cell content Identified and quantified ~750 e800 protein groups by analyzing just ~5 ng of protein digest in 16-cell Xenopus laevis embryos

Anionic metabolites in glioblastoma cell line extracts Single cell anionic metabolites from individual Aplysia californica neurons

Mode/Coating

CZE/Bare

BGE

Porous tip based sheathless interface/TOF MS

Sample stacking-CZE/Bare

20 mM ammonium bicarbonate

Coaxial sheath flow nanospray interface/micrOTOF MS

Metabolic analysis in live Vertebrate (Xenopus laevis) Embryo

CZE/Bare

1% FA

co-axial sheath flow CE-ESI interface/qoaTOF MS

Proteomics in live Xenopus laevis and zebrafish embryos

CZE/Bare

25% (v/v) MeOH and 1 M formic acid

electrokinetically pumped interface/Orbitrap MS

[101]

[112] [114]

[127]

[151]

[155]

[157]

Background electrolyte (BGE), poly(acrylamide) (LPA), acetic acid (HAc), formic acid (FA), dimethylacrylamide (DMA), glycidyl methacrylate (GMA), 3-methacryloyloxy propyltrimethoxysilane (MAPS), limit of detection (LOD).

Fig. 6. CE-MS for biomacromolecule and metabolism analysis. (a) Proteomic workflow used to characterize stable isotope labeling by amino acids in cell culture labeled yeast strains. Reprinted with permission from Ref. [97]. Copyright© 2015 American Chemical Society. (b) Schematic of bottom-up proteomics using dynamic pH junction preconcentration and CZE-ESI-MS. Reprinted with permission from Ref. [86]. Copyright© 2014 American Chemical Society. (c) Schematic of CE-MS detection of circulating miRNAs. Reprinted with permission from Ref. [117]. Copyright© 2016 Springer. (d) The experiment protocol used in tracking biochemical changes correlated with ultra-weak photon emission using metabolomics. Reprinted with permission from Ref. [137]. Copyright© 2016 Elsevier.

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phosphorylated peptides were identified in 100 min [99]. As shown in Fig. 7, strong cation exchange-RPLC was coupled with CZE-MS/ MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. 11555 phosphopeptides were identified by this platform [108]. mAbs are one of the fastest growing biological therapeutic agents, they have strong therapeutic potency and specificity. Proteoform is used to designate all of the different molecular forms, which the protein product from a single gene can be found. This difference may be caused by genetic variations, alternatively spliced RNA transcripts and post-translational modifications [109]. mAbs consist of mixtures of proteoforms, due to the differences in glycosylation and other PTMs, sequence truncation, or other modifications occur at the process of production, purification, or storage. Different proteoforms may lead to different biological activity, stability and toxicity [110]. Therefore, comprehensive characterization of mAbs is necessary for drug quality control [110,111]. CE-TOF-MS using a porous tip based sheathless ESI interface was reported for characterization of intact pharmaceutical glycoproteins. The sheathless interface showed a considerable improvement for the analysis of intact proteins over normal sheath-liquid CE-MS [112]. Alexander R. Ivanov has developed a research that characterizes the proteoforms of mAbs by CZE-MS [110]. Dovichi's group has applied CZE-MS for comprehensive analysis of host cell impurities in monoclonal antibodies [113]. A novel online CIEF-MS method was developed for intact mAb charge variant analysis using an electrokinetically pumped sheath flow ESI interface and a TOF-MS with pressure-assisted chemical mobilization. The method showed linear correlation (R2 ¼ 0.99) between pI values and migration time with good repeatability using a mixture of pI markers [114]. CE-MS method has also been successfully applied to the characterization of mAbs charge variants and monoclonal antibodies biosimilarity assessment [48,58,111,115,116]. Besides the above applications, CE-MS has been also applied in direct analysis of microRNAs (miRNAs). miRNAs are a group of

Fig. 7. Strong cation exchange-RPLC-CZE-MS/MS for large-scale phosphoproteomics. (A) Experiment design of the work. (B) Base peak electropherogram of one RPLC fraction (fraction 8) after CZE-MS/MS analysis. (C) Mass-to-charge ratio vs migration time of peptides identified by CZE-MS/MS from the RPLC fraction 8. Reprinted with permission from Ref. [108]. Copyright© 2019 American Chemical Society.

small, non-coding, single-stranded RNAs, which can regulate cellular messenger RNA and their corresponding proteins. The sequence and quantitative levels of miRNAs are closely related to cancer diagnosis and prognosis after anticancer treatment. As shown in Fig. 6c, Berezovski's group developed a CE-MS method for label-free quantitation and revealed modification of miRNAs [117]. In the research, two endogenous human circulating miRNAs isolated from B-cell chronic lymphocytic leukemia serum were detected. MicroRNA profiling with CE-MS has the potential to be a minimally invasive bioassay method for clinical diagnostics and disease treatment. 3.2. Cell metabolism analysis Cell metabolism analysis is a major aspect of cell analysis that analyzes numerous metabolites in cells simultaneously. It is important for that it can provide physiological status of organisms and describe the health status in principle. However, cell metabolism analysis is still a challenge due to dynamic change of metabolites, complexity of constituents, immense diversity of metabolite structures, and trace amounts of metabolites. CE-MS is now recognized as a strong analytical method for analysis of polar and charged metabolites in biological samples. Over the past few years, significant advancement has been made in CE-MS based analytical approaches for metabolites studies, and CE-MS has become a well complementary technique for LC-MS and NMR spectroscopy in metabolic profiling filed [118,119]. In the section, recent advances of CE-MS approach for cell metabolism studies are summarized. Our body is a sophisticated machine, many biomedical and clinical problems are complex and urgent to be solved. For biomedical and clinical applications, cell metabolism is a great angle to solve those problems because the alterations in the profiles of cell metabolites in our body may reflect changes or disorders in some physiological mechanisms. Through CE-MS approach, cell metabolism analysis was built and many biomedical and clinical problems were solved including uncovering the underlying physiological mechanisms [120,121], pathogenic mechanisms of disorders founding [122e126], analysis of complex biological samples [24,37,127,128], drug screening [129e132], finding diagnostic biomarkers [133,134], figuring out the mechanisms of drug action and resistance [135]. Koga et al. successfully employed a CE-ESI-TOF MS system to analyze T-cell metabolites with the effect of pharmacological inhibition of calcium/calmodulin-dependent protein kinase 4 (CaMK4) [136]. They found that CaMK4 may conduce to aberrant expression of glucose transporter 1 (GLUT1) in T cells from active systemic lupus erythematosus (SLE) patients. Through this method, the clinical significance of the expression of GLUT1 in patients with SLE was identified. As shown in Fig. 6d, Burgos et al. developed a CE-MS method to study the change of intracellular metabolites in HL-60 cells and investigated the biochemical changes based on the measured ultra-weak photon emission (UPE) profile [137]. They found that the levels of specific metabolites, including S-adenosylmethionine, creatine, putrescine, hydroxyproline, b-alanine, serine and methionine were significantly changed in HL-60 cells after inducing respiratory burst. Compared with the recorded UPE studies, they revealed that metabolic pathways in HL-60 cells especially the methionine pathway may play a role in UPE and the methionine pathway has potential for using UPE to monitor metabolic changes. Hepatocellular carcinoma (HCC) is one of the most prevalent human malignancies in the world. The lack of effective screening methods for early diagnosis has been a longterm problem to improve the survival rate. Therefore, the development of novel biomarkers for monitoring HCC would be

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imperative and of great clinical importance for diagnosis. Zeng et al. employed a CE-TOF MS-based metabolomics method to discover novel biomarkers of HCC [134]. In their research, a total of 183 human serum specimens were studied, a novel serum biomarker model including glutamine, tryptophan, and 2-hydroxybutyric acid was determined and evaluated as an effective tool for diagnosing HCC from non-HCC. Their CE-TOF/MS-based metabolomics study provided novel insights into the complicated biological process of tumor metabolism. Research found that methionine enkephalin (MENK) treatment in mouse model improved disease manifestations of multiple sclerosis to some extent experimentally. In order to explore the potential mechanisms of MENK in multiple sclerosis, CE-MS method was applied to profile intracellular metabolites fluctuation in 5 astrocytoma cell lines which were challenged by MENK [131]. This study exhibited the potential neuron protective effects of MENK. The study also demonstrated that CE-MS can be a promising tool for cell metabolomics. CE-MS based metabolism analysis has also been applied in plant and microbial analysis. Environmental conditions play an important role in the physiological metabolism of plants and microbes. However, relatively little is known about the effect of environmental factors on dynamic changes of metabolism. Recently many studies have focused on various environmental factors including cultivation characteristics, acid stress, dark incubation, nitrogen starvation, Rhizoctonia solani and water turbulence on metabolism of plants and microbes [138e144]. CE-MS method was employed to conduct transcript and metabolism analysis on Synechocystis sp. PCC 6803 under nitrogen starvation [142]. 161 metabolites in Synechocystis sp. PCC 6803 were analyzed simultaneously before and after nitrogen depletion. Their research found that under nitrogen starvation not only glycogen but also the metabolites downstream of sugar catabolism increased in Synechocystis sp. PCC 6803, which resolved the contradiction between sugar catabolic gene upregulation and glycogen accumulation in this unicellular cyanobacterium. Besides finding the influence of environmental conditions on the physiological metabolism of plants and microbes, CE-MS method can also be used for elucidating the comprehensive metabolic alterations and drug screening in plant samples [145,146]. A simple, rapid and effective affinity capillary electrophoresis (ACE) and CE-MS method was used for screening compounds with anti-HIV activity from the licorice extract [146]. In their study, glycyrrhizin and licorice saponin G2 were found to be effective by bioactive experiment of cellular level, the anti-HIV activity of glycyrrhizin was further confirmed. This strategy might be used for seeking HIV-1 inhibitors in natural products through the high throughput screening and identification platform. 3.3. Single cell analysis Cell is the basic unit of the organism's morphological structure and life activities. In the process of cell proliferation, differentiation and metabolism, internal and external factors work together to make the difference between cells. Even if two cells are homologous, the intracellular material composition and content could be very different [147]. However, the analysis of cell population can only give an average result of homeostasis and cannot characterize the differences between cells. Therefore, it is of great significance to study the substance composition and morphology of cells at the single cell level to reveal the differences between cells and explain some physiological behaviors. But the extremely small size makes it difficult to obtain single cell and perform sample pretreatment in it. The content of substances in single cells is extremely low and the concentration difference between components is large, spanning nine orders of magnitude [148], which further increases the

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difficulty of detection. Meanwhile, cells are constantly changing in the process of metabolism, thus requiring a high spatial and temporal resolution technology to reflect the real-time changes of cells. With minimum diameter of the capillary, CE requires very small sample consumption, that is, several nanoliters, in line with the characteristics of small size and less content of single cells. CE is an effective method that provides high resolving power and efficient separation in a short time [149]. The advantages of MS such as extensive molecular coverage, high sensitivity and specificity, relative structural and quantification identification make it address the requirement of understanding cell heterogeneity and single cell measurement [150]. Some research groups technologically bridge the gap by integrating capillary electrophoresis and mass spectrometry to enable analysis of identified single cells. Anionic metabolites from individual Aplysia californica neurons were analyzed by fabricating capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) platform. This system provided the ability to ensure a stable spray through the application of a customized coaxial sheath-flow nanospray interface. It showed good separation repeatability and credible detection sensitivity for 16 mono-, di- and triphosphate nucleosides [151]. Single cell CE-ESI-MS was used to test the domain of metabolites from differential expression of the genome translates. 40 metabolites were identified from three different cell types with distinct tissue fates from 16-cell embryos of Xenopus laevis. The result demonstrated that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos [152]. Single cell analysis of metabolite difference is an important mean to understand cell heterogeneity. Sweedler et al. successfully employed whole-cell patch clamp to single cell analysis, combined with CE-MS analysis, they were able to detect 60 metabolites from 3 pL of cytoplasm. This method allowed the sample arisen from the interested structures of specified cell and it turned out to be effective to observe differences in the metabolome of heterogeneous cells in the brain [153]. Another achievement by the same research group was the combination of MALDI-MS and CE-ESI-MS via a custom liquid microjunction surface sampling probe. MALDI-MS is ideal for the detection of peptides and lipids because the interference from the MALDI matrix can be reduced due to their relatively large molecular masses. This technology can improve the throughput of profiling of single cells effectively. And it was used to rapidly analyze single rat pancreatic islet cells, classifying each cell into established cell types based on their peptide content [154]. Progress has also been made in direct metabolic analysis of embryo, which is challenging to assay due to its decreasing cell sizes and complex three-dimensional changes. Nemes et al. completed the direct metabolic analysis of live frog embryos by integrating capillary microsampling, microscale metabolite extraction, and CE-ESI-MS, resulting in the detection of 230 different molecular features [155]. This method raised new potentials for better understanding the development of embryos. As shown in Fig. 8a, the research group further improved this method by introducing a capillary microprobe, which ensured that a defined portion was extracted from a single identified cell in the frog embryo, leaving adjacent intact cells for subsequent analysis. By analyzing the cell content through a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer, metabolic cell heterogeneity can be better elucidated [156]. Another method (shown in Fig. 8b), integrating subcellular capillary microsampling, one-pot extraction/digestion of proteins, peptide separation by capillary electrophoresis, ionization by electrokinetically pumped nanoelectrospray, and detection by high-resolution Orbitrap MS, was able to directly perform proteomics detection in live single cells, namely vertebrate embryos. This was the first example of CE-MS

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Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Nos: 81872828 and 81573384). References

Fig. 8. Single cell analysis by CE-MS. (a) Metabolite detection by microprobe CE-ESIMS vs whole-cell dissection and electropherograms exemplifying metabolites identified in a V1R cell using whole-cell dissection and microsampling method [155]. Copyright© 2017 American Chemical Society. (b) Label-free single cell proteomics of an identified single cell in a live Xenopus laevis. Reprinted with permission from Ref. [157]. Copyright© 2019 American Chemical Society.

based label-free single-cell proteomics, which overcame the barrier that MS was incompatible with complicated tissues. About 750e800 protein groups were identified and quantified with high detection sensitivity and quantitative accuracy. This technology can be scalable to live zebrafish embryos, as demonstrated [157]. 4. Conclusion and outlook In this review, we summarize the recent advances in CE-MS for cell analysis, including different CE-MS interfaces, multiple CE modes, CE preconcentration methods and the applications of CEMS in cell analysis. In the early years, much effort has been paid on the development of robust, highly sensitive CE-MS interfaces. Nowadays, several commercially available interfaces with high stability and sensitivity have demonstrated their ability in routine analysis. Achieving high efficient separation by CE is important for analysis of complex biological samples. However, the MS incompatible electrolytes and additives limited the using of various CE modes. So the development of MS compatible CE methods like CEC, 2D CE or MEKC method with volatile surfactant to increase separation efficiency and selectivity is a direction in the future work. In cell analysis, the most difficult challenges are the low analyte concentration in the sample and small sample injection volumes of CZE, which hamper the sensitivity of CE-MS. Thus, it is vital to integrate proper sample preconcentration strategies such as SPE, dynamic pH junction and CITP into CE system to overcome this problem. In particular, it is interesting for investigation of highly selective SPE sorbents for enrichment of target analytes while cleaning the complex interferences in matrix. Single cell analysis is increased required for studying cellular heterogeneity in biological systems. The extremely small sample amounts in single cell analysis make CE be a perfect technique for application in this field, and MS can provide high sensitive detection with characterization property. However, research on single cell analysis is still at an early stage, sensitivity and sample complexity are the greatest challenges. Developing novel CE-MS method for more precise handling and sensitive analysis of single cell is highly expected.

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