Single-cell omic molecular profiling using capillary electrophoresis-mass spectrometry

Abstract

Tissues and other cell populations are highly heterogeneous at the cellular level, owing to differences in expression and modifications of proteins, polynucleotides, metabolites, and lipids. The ability to assess this heterogeneity is crucial in understanding numerous biological phenomena, including various pathologies. Traditional analyses apply bulk-cell sampling, which masks the potentially subtle differences between cells that can be important in understanding of biological processes. These limitations due to cell heterogeneity inspired significant efforts and interest toward the analysis of smaller sample sizes, down to the level of individual cells. Among the emerging techniques, the unique capabilities of capillary electrophoresis coupled with mass spectrometry (CE-MS) made it a prominent technique for proteomics and metabolomics analysis at the single-cell level. In this review, we focus on the application of CE-MS in the proteomic and metabolomic profiling of single cells and highlight the recent advances in sample preparation, separation, MS acquisition, and data analysis.

1. Introduction

There is a considerable amount of cellular heterogeneity in all tissue types, and this diversity can explain a wide breadth of biological phenomena. Variation arises even amongst cells of the same types, stemming from different stages in the natural cell cycle, diversity in extracellular environments, and cellular interactions [1]. Traditional bulk-cell analyses are efficient in highlighting differences between diseased and healthy tissues, however, more subtle differences in protein expression and potential low-abundance subpopulations are masked in the bulk analysis. Bulk-cell profiling approaches are limited to average measurements that are greatly affected by highly abundant cellular species. In fact, it has been reported that these average measurements may not be representative of any individual cell within the bulk sample [2]. In response to the limitations of traditional bulk-cell sampling approaches, great effort has been made over the past 10 years to push profiling toward the analysis of individual cells, driven by advances in sample preparation, separations, MS acquisition, and data analysis, which we will overview in this paper.

For years, genomics and transcriptomics (RNA-sequencing) methods have been able to profile and assess heterogeneity at the single-cell level [3], and since the first report published in 2009 [4] on since-cell transcriptomic profiling, they have become invaluable tools in understanding cellular heterogeneity [5]. The quick establishment of single-cell genomic and transcriptomic methodologies has been made possible by the availability of polynucleotide amplification, such as PCR and RT-PCR (RNA amplification), that can increase the amount of polynucleotide samples to easily detectable levels. Compared to nucleic acids, proteins are typically found at much higher abundances within a single cell in general. However, there are no comparable amplification methods to increase protein copy numbers across the proteome when performing analysis using extremely limited sample volumes.

To overcome the limitations due to the inability to amplify the copy numbers of low abundant proteins and proteoforms, extreme emphasis has been placed on minimizing sample losses and maximizing sensitivity [6-11]. The methodology behind sample preparation of trace samples and single cells is drastically different compared to techniques applied to bulk-cell samples, where protein amount is not limited. Sample preparation in the case of low nanogram or picogram level samples, as is often the case in single-cell analysis, must prioritize the minimization of sample loss and dilution. Efforts to downscale sample processing volumes and eliminate transfer steps for the cleanup of salts and detergents aim to reduce surface contact as much as possible, thereby diminishing overall sample loss. In recent years, a number of sample processing workflows have been developed for the analysis of single-cells and limited samples that aim to minimize sample losses, increase the signal-to-noise ratio, and boost proteome coverage [7, 8, 10]. With these workflows, the quantitation sensitivity has begun to reach a level where single-cell-omics analysis can begin to make an impact in driving biological discoveries and understanding human disease.

LC-MS and CE-MS based -omics techniques have both been applied for the analysis of single-cell and mass-limited samples. However, the unique properties of capillary electrophoresis separations: the ability to separate molecules based on charge and hydrodynamic radius, open-tubular capillary geometry, and minimal sample consumption provide a number of benefits over traditional reverse-phase LC techniques for single-cell -omics analysis [12, 13]. These benefits have led to the implementation of CE-MS-based techniques for single-cell metabolomics analysis and single-cell proteomics analysis at both the peptide and intact protein levels [11, 14, 15]. In this review, we focus on the application of CE-MS for single-cell -omics analysis and the workflows that have enabled such analyses. As depicted in Fig. 1, recent advances in sample preparation methods, which can be used in downstream LC-MS or CE-MS analysis, will be covered, as sample processing is critical to maximizing the sensitivity and coverage at the single-cell level. The applications and working principles of CE-MS for single-cell proteomics at both the peptide and intact protein level, bottom-up and top-down approaches respectively, and single-cell metabolomics will be discussed. Additionally, challenges in single-cell CE-MS profiling and potential future directions will be briefly discussed.

Fig. 1. Recently developed workflows for limited sample and single-cell sample preparation for bottom-up proteomics analysis.

A) NanoPOTS workflow. Reprinted from Y. Zhu, P.D. Piehowski, R. Zhao, J. Chen, Y. Shen, R.J. Moore, A.K. Shukla, V.A. Petyuk, M. Campbell-Thompson, C.E. Mathews, R.D. Smith, W.J. Qian, R.T. Kelly, Nat [16]. B) OmSET workflow. Reprinted with permission from J.C. Kostas, M. Gregus, J. Schejbal, S. Ray, A.R. Ivanov, J Proteome Res 20 (2021) 1676. Copyright 2021 American Chemical Society [17]. C) SCoPE-MS workflow. Reprinted from B. Budnik, E. Levy, G. Harmange, N. Slavov, Genome Biol 19 (2018) 161 [10].

2. Capillary Electrophoresis

Capillary Electrophoresis (CE) is a nanoflow separation technique that is orthogonal to RP-LC,separating analytes on the basis of their charge and hydrodynamic volume. In capillary zone electrophoresis (CZE), analytes migrate through the capillary based on their electrophoretic mobilities under the applied electric field. [18]. Analytes with a smaller size-to-charge ratio generally migrate faster. Electroosmotic flow (EOF) is generated due to the movement of ions in the electrical double layer (formed at the solid-liquid interface of the the capillary wall and the ions present in the solution) induced by the applied potential and also impacts ion migration. EOF is also affected by the buffer pH and composition, as well as the presence or absence of any type of coating on the capillary inner wall [19-22], Hence, these factors play a crucial role in the separation efficiency of the analytes. CE shows high separation efficiency and great resolution, with much faster run times compared to conventional proteomics approaches. It also involves little to no sample preparation and minimal solvent consumption. Due to these reasons, CE has recently been utilized to analyze a variety of biological molecules [23-27]. CE can be coupled to several detection methods, including UV and fluorescence, but can also be interfaced with MS detection using electrospray ionization (ESI), the most frequently used ionization method, or matrix-assisted laser desorption ionization (MALDI) [28, 29].

2.1. CE-MS interface

The successful coupling of CE with the MS often requires the integration of two closed electrical circuits (CE and ESI-MS) with a shared electrode. Two common categories of CE interfaces are sheath liquid and the sheathless interfaces [30] (Fig. 2). The former introduces a sheath liquid at the separation capillary outlet that forms a junction with the separation capillary [31]. The coaxial sheath liquid interface was developed in 1988 [32] and it utilizes sheath liquid flow around the capillary outlet encompassed by a metal tube on which voltage is applied to generate a closed circuit (Fig. 2A). Although these interfaces produce robust electrospray, the sheath liquid can cause sample dilution, especially at higher flow rates. First introduced by Lee et al., [33] liquid-junction interfaces use an ESI emitter separated from the CE capillary with a small gap (20-200 μm) filled in with a sheath liquid to close the circuit. Liquid-junction interfaces have significantly lower sheath liquid flow rates than those in coaxial interfaces and have been thus, used in microfluidic CE devices as well [34-36]. The electrokinetically pumped sheath liquid interface designed by Zhu, Sun, Dovichi et al. supports a robust nanospray with higher sensitivity [37-39]. The Nemes group has designed a CE-ESI sheath liquid interface optimized for nano sheath-flow (~100-300 nL/min) to further improve sensitivity [40]. Alternatively, sheathless CE-ESI-MS interfaces (Fig. 2B) achieve electrical contact without the need for sheath liquid, eliminating sample dilution problems. Moini et al. constructed a sheathless interface using a porous glass emitter chemically etched on the end of the separation capillary with hydrofluoric acid [41]. The electrical contact between the CE and the ESI-MS circuits is shared by the transportation of ions between the solution inside the capillary and the conductive liquid surrounding the emitter tip. However, the currently available sheathless interfaces do have their fair share of disadvantages. Spray instability can be observed depending on the quality and the technique of coating. The diameter of the capillary is also important as a very narrow tip opening can be prone to clogging, and a too big opening can negatively affect both the spray stability and EOF [42-44]. Thus, the flow rate, capillary and emitter diameter, capillary coating, and electric field strength need to be considered for stable spray and maximum ionization efficiency.

Fig. 2. Basic schematics of common CE-ESI interfaces.

A) Sheath liquid CE-ESI interfaces utilize contact between CE flow and a sheath liquid to produce electrospray. A coaxial sheath liquid interface design is represented here. B) Sheathless CE-ESI interfaces rely on a porous emitter tip to enable electrical contact between the conductive liquid and CE flow without mixing.

3. Single-Cell -Omics Sample Preparation Workflows

Herein we will discuss advances and approaches to proteomic and metabolomic analysis at the single-cell level that have enabled rapid progress in –omic capabilities, pushing the field towards the goal of translational applications. The vast majority of the progress in SCP workflows to date has centered on bottom-up proteomic approaches involving the digestion of proteins into peptides prior to analysis; however, over the past decade, there has been a push towards the analysis of intact proteins at all levels of proteomic analysis. In addition, the advances and approaches in metabolomic analysis at the single-cell level will also be discussed.

3.1. Bottom-up Proteomics Workflows

Bottom-up proteomics (BUP) analysis is a well-established tool for proteome-level characterization of complex biological samples. The term “bottom-up” in these analyses refers to the fact the analytes in these workflows are not the proteins themselves, but rather peptides produced by enzymatic digestion of the proteins, which can be used to infer the presence and quantity of their proteins of origin. In the first step of the BUP workflow, chaotropic agents and/or detergents are most often added to lyse cells and denature proteins, which are then typically reduced and alkylated to further disrupt tertiary structure and expose sites for proteolytic digestion. Traditionally, limitations in sensitivity made the bottom-up proteomic analysis of single cells largely inaccessible. However, new methodologies utilizing technological advances at all levels of the workflow have been developed that achieve the necessary sensitivity for in-depth single-cell analyses.

The nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) platform [9, 16] is one such method (Fig. 1A) that was developed, in which all processing steps could be performed in a single nanoliter-size droplet, reducing surface contact and transfer steps in order to minimize sample losses. On microsolid-phase extraction tip (OmSET) [17, 45] is another method (Fig. 1B) that similarly aims to minimize transfer steps and integrate all processing steps within a single device, incorporating the principles behind previously reported techniques such as filter-aided sample processing (FASP), [46] in-StageTip processing (iST) [47] and single-pot, solid-phase-enhanced sample preparation (SP3) [48] with adaptations for specific application to limited samples [49]. Another method, a single-cell workflow called SCoPE-MS (Single Cell ProtEomics by Mass Spectrometry), uses TMT labeling to multiplex single cells, with an additional channel containing a higher number of cells to boost the signal for peptide sequence identification (Fig. 1C) [6, 10, 50]. The concept of an additional carrier channel, combined with isobaric labeling, pioneered in the SCoPE-MS approach has been further refined to increase BUP identifications in single cells.

3.2. Top-down Proteomics Workflows

Top-down proteomics (TDP) is the analysis of intact proteins, rather than protein digests, and can more accurately represent protein identifications by distinguishing specific proteoforms that are present in the cell. The term proteoform describes protein variants resulting from post-translational modifications (PTMs), alternative splicing, and genetic mutation that all originate from the same gene [51]. It is difficult to characterize distinct proteoforms using a BUP approach, because the digestion step(s) breaks the protein into peptides prior to analysis. While several modifications may be detected at the peptide level, it is impossible to determine the exact molecular composition of the full-length proteoform [52]. Understanding these proteoform shas been demonstrated to reveal links to certain diseases and their underlying mechanisms [53, 54].

However, MS analysis of intact proteins is complicated by the diversity of ionization products for a single proteoform, resulting in multiple charge states and adducts that cause signal dilution and contribute to spectral overlap. Additionally, the broad dynamic range of protein abundances and molecular masses makes it difficult to detect low-abundance species and makes it impossible to cover all proteins using a single analytical platform. Data processing presents an additional challenge, as many top-down analysis software packages were not designed to handle complex ‘omics’ data, and there is currently no universal data evaluation platform. There is potential for the same sample preparation technology employed in BUP to be translated to TDP analysis at the single-cell level, greatly sensitivity and the depth of proteome coverage [55]. However, historically the number of proteins capable to be analyzed by TDP at the single-cell level is limited to only a few highly abundant proteins [56-59].

3.3. Metabolomics Workflows

Metabolomics is the study of the metabolites present in cells and tissues, which are the final downstream product of the transcriptome, and therefore provide the most accurate and direct representation of the condition and activity of the cell [60, 61]. The study of the cellular metabolome presents a unique opportunity to track phenotypic and genotypic changes and differences among cells, with the added potential of being able to perform these analyses in real-time and without destroying the cell (live-cell mass spectrometry) [25, 62-64]. However, the study of metabolites presents a number of analytical challenges due to a broad range of metabolite physicochemical properties, low concentration of metabolites within the cell, difficulty in extracting small sample volumes from the cell for analysis, the wide dynamic range of metabolites, and the quick turnover rate of metabolites within the cell [60, 61, 65]. Additionally, a foundational problem of metabolomics analysis is the difficulty in the identification of metabolites due to a large number of potential isoforms and isobaric compounds [60]. Mass spectrometry has subsequently emerged as the most prominent tool for the analysis of intra- and extracellular metabolites from single cells due to the high sensitivity, specificity, label-free detection, and multi-level fragmentation that can increase compound identification offered by modern mass spectrometers.

One of the main challenges in single-cell metabolomics (SCM) workflows is the extraction of down to picolitre scale volumes from cells for the analysis of intracellular metabolites. Several common techniques for the direct sampling of cellular contents for metabolomics use a micropipette, capillary, or nano-spray tip to aspirate small volumes directly from the cell using negative pressure [64, 66, 67]. This method largely minimizes disturbances to the cell, a benefit as disturbances to cellular conditions can alter the cellular metabolome on the timescale of seconds [60, 61], and is suitable for cells of various sizes, as long as they can be isolated and targeted under a microscope. The micropipette/capillary used for extraction can then be directly sprayed into the mass spectrometer, or the aspirated sample can be processed (e.g., by employing extraction of polar or non-polar analytes via liquid-liquid extraction) to be analyzed via capillary electrophoresis coupled to high-resolution mass spectrometry. The use of capillary electrophoresis as an online upfront separation technique for SCM greatly increases the sensitivity, and the depth of metabolome coverage compared to direct sample introduction approaches. However, a drawback to upfront capillary electrophoresis approaches is the current need for sample preparation steps, and a longer analysis time associated with CE-MS analysis compared to direct infusion-based MS approaches, leading to an overall lower throughput. The throughput of capillary electrophoresis-based approaches can be increased by future advances in the automation of sample preparation, handling, and introduction, in addition to the development of sampling techniques that utilize the separation capillary for cellular sampling [11, 58, 68, 69].

4. Novel applications

4.1. Proteomics

The microanalytical CE platforms and CE-ESI interfaces designed in recent years [13, 14, 26, 70] have been instrumental in the transcriptomic, proteomic, and metabolomic profiling of single cells ex vivo [63, 71, 72], in situ [73], and in vivo [25, 62]. They have enabled significant progress in developmental neurobiology using the embryos of Xenopus laevis, the South African frog as model systems. The Nemes group has devised a protocol for direct proteomic profiling of single cells embedded in tissues without dissociation [74]. This method was used to profile blastomeres in 4-, 8-, and 16-cell embryos from Xenopus laevis. The cells are first identified using morphological cues and then probed using microfabricated capillaries, followed by bottom-up CE-MS. They have recently integrated patch-clamp electrophysiology with proteomics at the subcellular level using the above-mentioned CE-HRMS protocol [26]. This aided the functional characterization of neurons based on their neuronal activity. About 1 picogram of protein digest, or approximately 0.25% of the total proteome of the cell was characterized, yielding 157 different proteins from the somas of three different neurons. Hence, proteomic profiling was achieved at a subcellular level. The previously mentioned electrokinetically pumped sheath liquid interface by the Dovichi group has enabled them to analyze proteins using single-shot CZE-MS/MS from Escherichia coli digests up to 1 ng, which approaches single-cell levels. They were able to identify 142 protein groups and 627 peptides, which were higher than those obtained using UPLC- MS/MS [72].

Nearly a decade ago, the Ramsey group showed the potential of their microfluidic chip-based electrophoresis (still in its infancy) to analyze the hemoglobin content of single red blood cells (RBCs), achieving on-chip cell lysis using rapid buffer exchange and high electric fields [58]. However, TDP of limited samples and single cells remains a challenge. Since CE is an open-tubular separation, it is possible to inject intact cells and lyse them directly in the separation column, thereby eliminating any sample losses that occur during sample processing and transfer steps. Our group has recently developed a method for the direct processing of intact cells on the separation capillary coupled to online CE-MS/MS analysis for proteome-level TDP analysis of <10 mammalian cancer cells, down to the single-cell level. Applying our novel method for single-cell proteomics (SC-TDP), we were able to identify 40 proteins and 50 proteoforms from a single HeLa cell, which is a significant improvement in proteome coverage compared to previously published single-cell intact protein analyses, which were only able to characterize 1-2 proteins from a single cell (Fig. 2)[57].

4.2. Metabolomics

The analysis of the metabolome of a single cell requires proper sampling and efficient metabolite extraction. The addition of a separation method such as CE at the front end of the workflow further enables researchers to simplify the metabolome, reduce interferences, and help in the identification of complex metabolites. The Nemes group has used their microprobe capillary method to analyze the metabolome directly from a live X. laevis embryo cell [25]. The microprobe capillary could aspirate about ~10 nL portion from the cell under the guidance of an imaging system. The collected sample is then analyzed by CE-ESI coupled to a high-resolution MS. This method can be used to identify the metabolic differences in the cells between the different developmental stages of the embryo. This sampling method of aspiration is fast (a few seconds), scalable for smaller cells, and leaves the adjacent cells intact for further analysis. The Si Wu group recently developed an electrospray-assisted device called the spray-capillary for low-volume (pL-nL) sample collection from single cells that can be directly used for CE separation and online MS detection, with microsampling and CE-MS analysis was successfully demonstrated using a single onion cell [75] .

The work of Xiangtang Li et al. applied microchip-based electrophoresis for the real-time analysis of chiral metabolites, D- and L-serine, in single PC-12 neuron cells [68, 69]. This homebuilt microchip approach allowed for fast (1-sec timescale) lysis of the cells on the microchip, decreasing sample losses, and the inclusion of a microchamber with the chip allowed for examining changes in metabolic expression after hypoxic conditions or chemical stimulation. The same group later expanded upon this work to use the same system for the analysis of dopamine and glutamic acid concentration in the intracellular environment of single PC-12 neuronal cells, with similar experiments performed utilizing variable chemical conditions in the microchip microchamber. The Sweedler group also developed a novel MALDI MS-guided liquid microjunction extraction technique for CE-MS analysis that allowed for the identification of pancreatic islet alpha and beta cells using unique peptide signatures [14]. Kawai et al. utilized a thin-walled tapered emitter to aspirate cellular contents from single HeLa cells that were then dried and reconstituted in 1 M hydrochloric acid for subsequent CE-MS analyses on a custom interface, where they analyzed the concentration and detection limits for 20 natural amino acids [66].

5. Conclusions and Future Perspectives

Over the past decade, there have been considerable advances in all aspects of single-cell -omics analysis, driven by increased efforts to understand the inherent heterogeneity of biological systems and how this heterogeneity can be leveraged to better understand human health and disease. In this review, we have summarized recent advances in sample preparation workflows and technologies that have greatly enhanced single-cell -omics capabilities, with a focus on proteomics and metabolomics analysis. General workflows for bottom-up, top-down, and metabolomics analysis at the single-cell level were also discussed. Lastly, novel applications of CE-MS techniques for single-cell -omics analysis were highlighted, emphasizing the advancements that have been made over the past decades and the increasing potential for single-cell analysis to increase the fundamental understanding of biological systems, human health, and disease.

Despite the advances in CE-MS techniques for analysis at the single-cell level, there are still a number of challenges that must be overcome to push the field forward. There is a clear need for the development and improvement of technologies to increase sample throughput, enable simplified sample handling and preparation, increase analyte detection and sensitivity, and analyze the complex data sets obtained by these analyses. Herein we will briefly discuss and highlight potential advancements that could further improve single-cell -omics capabilities.

The automation of sample handling, preparation, and introduction to the capillary electrophoresis system is highly desirable in order to increase sample throughput and make single-cell analysis more feasible in a clinical or diagnostic setting where those analyses can be of the most benefit. There have been great strides made in recent years in the automation of the sample handling and preparation for single-cell -omics analysis; however, there are still improvements to be made in the full automation of these platforms and the robustness of their connections to separation instruments such as capillary electrophoresis. Improvements in microchip-based electrophoretic separations also have the potential to drastically increase the throughput of SCP and SCM analysis owing to the greatly reduced run times compared to traditional capillary electrophoresis analysis and the potential for these microchip platforms to be used on a variety of mass spectrometers. However, the technology for microchip-based capillary electrophoresis instrumentation is still relatively new compared to the established silica capillaries and there still need to be improvements in their robustness, sensitivity, and sample consumption to fully realize their potential benefits in single-cell analysis. In recent years, substantial efforts have been made to increase the detection sensitivity of mass spectrometers by developing various ion traps and by employing a combination of mass analyzers [77]. However, greater advancements could be made for maximizing sensitivity at the single-cell level by further optimizing the ionization efficiency. The development of data analysis software suited to single-cell analysis will be an important step in furthering the applications of SCP and SCM analysis in settings beyond academia. In particular, the development of a robust data processing software and pipeline for SCM analysis could greatly improve the detection and identification of metabolites, which is currently a major difficulty in metabolomic analysis. The integration of genomic, transcriptomic, proteomic, and metabolomic data at the single-cell level presents the potential for a comprehensive and holistic understanding of cellular physiology, heterogeneity, development, and dynamics in an effort to increase the understanding of human health and further improve the treatment and diagnosis of relevant diseases.

In summary, SCP and SCM analysis by capillary electrophoresis coupled to mass spectrometry has experienced exponential growth in the past decade, with advancements at all levels of the experimental workflow. Recent advances and applications have shown the ability of these analyses to provide valuable information on cellular pathways, physiology, health, and development. Given the increasing interest in the field of single-cell analysis and the continued push towards personalized treatments and healthcare, we expect an increasing number of improvements and breakthroughs in the CE-MS-based technologies, methods, and applications for single-cell -omics analysis.

Fig. 3. Overview of intact cell injection workflows and cell suspension droplets.

(A) Schematic of spray voltage injection workflow. The separation capillary is prepared for cell lysis and CE-MS/MS analysis by filling in first with BGE and then with a plug of lysis buffer. The capillary inlet is positioned in cell suspension (500 nL) pipetted on a glass slide (the zoomed capillary inlet in the droplet is shown in the red-frame insert), and electrospray voltage is applied at the MS inlet, drawing cells into the capillary. (B) Schematic of manual injection of single cells with hydrodynamic loading using height difference between the capillary inlet and outlet to generate flow and draw in the cell. In the injection flow panel, a 75% FA plug, (I) a single cell, (II) and another 75% FA plug (III) were loaded in this order for single-cell injection and lysis. (C) Representative image cell suspension droplet used for single-cell loading. Reprinted with permission from K.R. Johnson, Y. Gao, M. Greguš, A.R. Ivanov, Analytical Chemistry 94 (2022) 14358. Copyright 2022 American Chemical Society [11].

Fig 4. Interdisciplinary strategy enabling in vivo subcellular proteo-metabolomic systems biology with demonstrated compatibility for cell, neurodevelopmental, and behavioral biology using Xenopus laevis as the biological model.

As an example, the left dorsal-animal (L-D1) and left ventral-animal (L-V1) cells were identified and the content of each cell rapidly microsampled twice using clean capillaries each time. The collected protein and metabolite samples were analyzed using custom-built ultrasensitive capillary electrophoresis (CE) nano/microflow electrospray ionization (ESI) high-resolution orbitrap and time-of-flight (TOF) mass spectrometry (HRMS). The tadpoles developing from the embryos were characterized for survival, anatomy, and behavior. Scale bars=250 mm (black), 1 mm (white). Reprinted with permission from C. Lombard-Banek, J. Li, E.P. Portero, R.M. Onjiko, C.D. Singer, D.O. Plotnick, R.Q. Al Shabeeb, P. Nemes, Angewandte Chemie International Edition 60 (2021) 12852. [76]. Copyright 2021 Wiley-VCH GmbH

Highlights –

• Significance of single-cell analysis for elucidating cellular heterogeneity.

• Capillary electrophoresis coupled to electrospray ionization (ESI) mass spectrometry (CE-MS) is an attractive orthogonal technique to current methods for single-cell analysis such as reversed-phase liquid chromatography (RPLC) alone and coupled to MS, direct infusion MS, and matrix-assisted laser desorption ionization (MALDI)-MS

• Novel applications in single-cell proteomics and metabolomics using CE-MS.

Abbreviations –

BGE

background electrolyte

BUP

bottom-up proteomics

CE-MS

capillary electrophoresis coupled to mass spectrometry

CZE

capillary zone electrophoresis

EOF

electroosmotic flow

ESI

electrospray ionization

FASP

filter aided sample processing

HRMS

high resolution mass spectrometry

iST

in-stage tip processing

LC-MS

liquid chromatography coupled to mass spectrometry

MALDI

matrix-assisted laser desorption ionization

MS

mass spectrometry

nanoPOTS

nanodroplet processing in one pot for trace samples

OmSET

on microsolid-phase extraction tip

PCR

polymerase chain reaction

PTM

post-translational modification

RPLC

reversed-phase liquid chromatography

RT-PCR

reverse transcription polymerase chain reaction

SCM

single-cell metabolomics

SCoPE-MS

Single-Cell ProtEomics by Mass Spectrometry

SCP

single-cell proteomics

SP3

single-pot, solid-phase-enhanced sample preparation

TDP

top-down proteomics

TMT

tandem mass tags

TOF

time-of-flight

UPLC

ultra-high performance liquid chromatography