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. Author manuscript; available in PMC: 2024 Feb 5.
Published in final edited form as: Anal Bioanal Chem. 2023 Aug 15;415(28):6961–6973. doi: 10.1007/s00216-023-04870-w

An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples

Jiaxue Li 1, Lushuang Huang 1, Yanting Guo 1, Kellye A Cupp-Sutton 1, Si Wu 1
PMCID: PMC10843549  NIHMSID: NIHMS1925941  PMID: 37581707

Abstract

Capillary electrophoresis mass spectrometry (CE-MS) is an emerging analytical tool for microscale biological sample analysis that offers high separation resolution, low detection limit, and low sample consumption. We recently developed a novel microsampling device, “spray-capillary,” for quantitative low-volume sample extraction (as low as 15 pL/s) and online CE-MS analysis. This platform can efficiently analyze picoliter samples (e.g., single cells) with minimal sample loss and no additional offline sample-handling steps. However, our original spray-capillary-based experiments required manual manipulation of the sample inlet for sample collection and separation, which is time consuming and requires proficiency in device handling. To optimize the performance of spray-capillary CE-MS analysis, we developed an automated platform for robust, high-throughput analysis of picoliter samples using a commercially available CE autosampler. Our results demonstrated high reproducibility among 50 continuous runs using the standard peptide angiotensin II (Ang II), with an RSD of 14.70% and 0.62% with respect to intensity and elution time, respectively. We also analyzed Ang II using varying injection times to evaluate the capability of the spray-capillary to perform quantitative sampling and found high linearity for peptide intensity with respect to injection time (R2 > 0.99). These results demonstrate the capability of the spray-capillary sampling platform for high-throughput quantitative analysis of low-volume, low-complexity samples using pressure elution (e.g., direct injection). To further evaluate and optimize the automated spray-capillary platform to analyze complex biological samples, we performed online CE-MS analysis on Escherichia coli lysate digest spiked with Ang II using varying injection times. We maintained high linearity of intensity with respect to injection time for Ang II and E. coli peptides (R2 > 0.97 in all cases). Furthermore, we observed good CE separation and high reproducibility between automated runs. Overall, we demonstrated that the automated spray-capillary CE-MS platform can efficiently and reproducibly sample picoliter and nanoliter biological samples for high-throughput proteomics analysis.

Keywords: CE-MS, Proteomics, Low-volume sampling, Mass-limited proteomics

Introduction

Proteomics allows the study of entire cellular proteomes at the macromolecular level to examine cellular processes under healthy and disease states. Typically, proteomics studies are performed using bulk cell analysis that combines proteins from thousands or millions of cells to obtain enough protein for analysis (μg-level for standard nano-LC–MS-based proteomics methods). More recently, however, analysis of limited-mass and single-cell proteomics methods that can analyze exceedingly small sample masses such as protein extracted from clinical specimens and individual cells have become increasingly relevant to bioanalytical researchers. The primary impediments to limited-mass and single-cell omics analysis include micro/nanoscale sample preparation, sensitivity of sample separation and analysis methods, low throughput, and limited reproducibility. Methods developed to address these issues and make limited-mass proteomics more accessible are briefly reviewed here.

High-volume, tube-based sample preparation methods result in high levels of sample loss, especially for limited-mass samples [1, 2]. To address issues with limited-mass sample preparation, a number of robust and high-throughput sample-handling approaches to quantitatively handle ultralow-volume samples (pL–nL) have been developed, and the current state of research in this area has been recently reviewed in the literature [1, 3-5]. Miniaturization of traditional tube-based methods has resulted in improved analysis of limited-cell and single-cell samples, and these methods are easily integrated into traditional bulk cell proteomics workflows [6-9]. However, there are also a number of methods that go beyond miniaturization to include small-volume liquid handling robots (stand alone or embedded) for automatic handling of nanoliter-level samples for single-cell omics. For example, chip-based devices designed for nanoscale sample-handling (~ 200 nL) nano-LC–MS analysis have been developed for analysis of limited-mass samples [10-17]. Other techniques such as microfluidics [18-20] and micro/nanoreactors [21-26] have also made considerable contributions toward high-throughput nano-LC–MS-based proteomics. While these methods focus on sample preparation at the nanoscale level, samples prepared with these methods must generally be diluted to microliter-level volumes due to limitations in sample loading mechanisms for LC–MS analysis. This dilution may result in additional sample loss and introduce possible sample-handling variation [1, 2].

As an alternative to LC–MS samples analysis to address issues of high-volume sample injection requirements, capillary electrophoresis (CE) has been implemented as a powerful separation platform that is particularly well-suited for nanoscale omics analysis (e.g., single-cell analysis) due to the high separation power and nanoliter-level sample requirement of CE separation [27-36]. Various ultralow-volume sample-handling approaches have been developed to facilitate the quantitative manipulation and injection of nanoscale samples for CE-MS omics analysis such as gravity flow injection [35, 37-41] and an inkjet droplet micro-chip system [42]. These methods, however, still may not be capable of analyzing extremely small volumes (e.g., nL–pL) of which CE separation is capable without additional handling steps or devices. In this vein, we recently developed a novel microsampling device, “spray-capillary,” for quantitative low-volume sample extraction (as low as ~ 15 pL/s) and online CE-MS analysis [43, 44]. The spray-capillary utilizes electrospray ionization (ESI) to provide a pressure difference between the sample end and the MS end of the capillary for sample loading onto the capillary. Without additional offline sample-handling steps, the spray-capillary CE-MS platform efficiently aspirates picoliter-volume samples (e.g., single cells) with minimal sample loss. The spray-capillary CE-MS platform addresses the issues of small-volume sample injection and low sensitivity previously associated with limited-mass analysis; however, as with many CE sampling methods, our original spray-capillary-based experiments required manual manipulation of the sample-inlet end between the column buffer vial and sample vials, which is time consuming and requires proficiency in device usage for reproducible results. Automation of online sampling, CE separation, and MS detection can greatly increase throughput and reproducibility.

A series of local-built as well as commercial CE autosamplers have been developed to standardize CE sampling and improve reproducibility. For example, Peuchen et al. evaluated a PrinCE Next∣480 CE autosampler in conjunction with a commercial EMASS-II interface and found that RSDs were < 6% and 20% for migration time and intensity, respectively, with as little as 5 μL sample injection volume [45]. However, these autosamplers still use pressure-based or electrokinetic injection methods for sampling, which limits the sample volume to the nanoliter level. Here, we present an automated spray-capillary platform that couples the spray-capillary device with a commercially available CE autosampler for robust, high-throughput analysis of picoliter- and nanoliter-volume samples. This platform combines the reproducibility and throughput of the CE autosampler with the sub-nanoliter-level sampling offered by the spray capillary. We evaluated the precision of the device elution time and relative MS intensity and the durability of the device by performing and evaluating 50 automated, continuous runs. Additionally, we evaluated the quantitative accuracy of the sampling by varying the injection time (e.g., injection volume) and determining the quantitative mass of the injected sample. Our results demonstrated reproducible sampling and separation of a standard peptide (angiotensin II) over 50 continuous runs. The optimized system was further applied to the bottom-up proteomics analysis of ultralow-volume complex samples (Escherichia coli lysate digest). Overall, we demonstrated that the automated spray-capillary CE-MS platform can efficiently handle picoliter and nanoliter biological samples in high-throughput proteomics analysis.

Materials and methods

Chemicals and reagents

Angiotensin II (Ang II, A9525), LC–MS-grade HPLC water, acetonitrile (ACN), methanol (MeOH), formic acid (FA), ammonium bicarbonate (ABC) and hydrofluoric acid (HF, ≥ 48%, 30,107), hydrochloric acid (HCl), sodium hydroxide (NaOH), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless noted otherwise. Fused-silica capillaries were purchased from Polymicro Technologies (Phoenix, AZ). The standard peptide mixture used here was a solution of 10 μM Ang II (prepared in 0.1% FA in 45% ACN water solution). Trimethoxysilylpropyl-modified polyethyleneimine (50% PEI solution in MeOH, SSP-060, Gelest, Morrisville, PA) was used to coat the spray capillary. 0.1% formic acid was utilized as background electrolyte buffer (BGE) for all the experiments.

Escherichia coli cell lysate preparation

Escherichia coli (E. coli) cell lysate was prepared from Escherichia coli K-12 MG1655 cells (bacterial stock obtained from the lab of Dr. Tyrrell Conway) as previously mentioned [46]. Briefly, E. coli cells were cultured in LB solution at 37 °C for 12 h, collected, and pelleted through centrifugation at 10,000 × g, 4 °C for 20 min, followed by resuspension in 25 mM ammonium bicarbonate (ABC) buffer (5:1 ratio, w/w) and 0.1% (v/v) phenylmethylsul-phonyl fluoride (PMSF). A pressure-based approach was utilized for cell lysis and the soluble fraction was collected. To prepare E. coli digest, E. coli cell lysate was denatured in in an equal volume of 6 M urea followed by 200 mM DTT reduction at 37 °C for 1 h and 200 mM IAA alkylation at room temperature in the dark for 30 min. Excess IAA was neutralized by DTT after incubation. The sample was diluted to 1 mL followed by the addition of TPCK-Trypsin (Thermo Scientific, Rockford, IL) at a ratio of 1:50 (trypsin mass to protein mass), and incubation at 37 °C for 4 h. After 4 h, the same amount of trypsin was added for double digestion at 37 °C overnight. The digested E. coli was desalted using Strata™-C18-U columns (5 μm diameter, 300 Å pore size; Phenomenex, Torrance, CA), concentrated by SpeedVac, and resuspended into HPLC water. Protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). The E. coli lysate digest was aliquoted and stored at – 80 °C.

Fabrication of spray capillary

Commercially available fused-silica capillary (e.g., 360 μm O.D., 50 μm I.D., 100 cm in length) was purchased from Polymicro Technologies (Phoenix, AZ) and used to fabricate the spray-capillary device. A detailed fabrication process has been previously described [43]. Briefly, the outside polymer coating of the MS end (~ 3 cm) was removed by flame, and the exposed portion was etched using 48% HF to produce the porous segment ESI emitter of the sheathless interface. During the etching process, the capillary was continuously flushed with DI water at a flow rate of 0.2 μL/min to prevent etching of the inner wall. The tip shape and porosity of the MS end were evaluated using an inverted microscope. The sheathless CE-MS interface was assembled by inserting the etched MS end into a PEEK tee union through a short stainless steel tube (4 cm, 1/16″ O.D., 0.04″ I.D.) as discussed previously [43, 47]. The sample injection flow rate was monitored and recorded using a digital camera by measuring the movement rate of the boundary between the sample/column liquid interface (column liquid consisted of H2O and sample was 90% butanol) in the spray capillary during ESI injection [44].

PEI coating was conducted as previously reported [48]. Briefly, the bare spray capillary was pretreated with 0.1 M NaOH, 0.1 M HCl, and DDI water sequentially at a flow rate of 2 μL/min (50-min flush time for each solution). After flushing with DDI water, MeOH was flowed through the spray capillary at 2 μL/min. PEI stock solution, 300 μL, was dispensed into 10 mL MeOH to produce the coating solution. The spray-capillary was first flushed with the coating solution at a flow rate of 2 μL/min for 30 min. After flushing, the MS-inlet end of the spray-capillary was immersed into MeOH while the sample-inlet end was inserted into an Eppendorf tube containing PEI solution overnight. After incubation, MeOH was flowed through the coated spray-capillary at a flow rate of 2 μL/min for 15 min.

Automated spray-capillary platform setup and evaluation

A CE autosampler (ECE-001) from CMP Scientific Inc. (Brooklyn, NY) was adapted to automate the spray-capillary-based microsampling process (details in Fig. 1A). Two types of automated sample injection and MS detection schemes, (a) pressure elution and (b) CE-MS, were developed and optimized here.

Fig. 1.

Fig. 1

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Integration of the automated CE system with the spray-capillary platform. A Schematic of the automated spray-capillary platform; the dotted line indicates that the capillary can be automatically moved between the sample vial and the BGE vial. B Sequence designed for automated spray-capillary microsampling with pressure elution. C Sequence designed for automated spray-capillary microsampling with capillary electrophoresis separation

  1. Development of automated spray-capillary microsampling with pressure-based sample elution (Fig. 1B). In this sequence, the following steps were performed. (1) Preconditioning (~0.5 min): With the sampling end of the spray capillary placed in the BGE vial, pressure (e.g., 690 mbar) was applied to the BGE vial to fill the column with BGE buffer until a droplet formed at the MS end of the spray capillary. (2) Arm transfer (~ 0.1 min): The sampling end of the spray capillary was automatically moved from the BGE vial to the sample vial. (3) Spray-capillary injection (Tinj): ESI voltage was applied to the MS end of the spray capillary for sample injection for the required injection time (Tinj). (4) Arm transfer (~ 0.1 min): The sampling end of the spray capillary was moved from the sample vial to the BGE vial. (5) Pressure elution (~ 6 min): Pressure (e.g., 690 mbar) was applied in the BGE vial to elute the sample for online MS detection.

    Standard peptide Ang II (10 μM in 0.1 FA and 45% ACN) was used to evaluate the robustness, quantification, and reproducibility of the automatic spray-capillary microsampling with pressure elution. A bare spray-capillary (360 μm O.D., 50 μm I.D., 100 cm in length) was used for the following experiments. Fifty continuous analyses of Ang II were performed using fully automated spray-capillary pressure elution and a 6 s spray-capillary injection time (Tinj = 6 s, sample injection ESI voltage = 3.0 kV). The spray-capillary was pressure flushed (e.g., 690 mbar) with BGE between runs to ensure no carry-over from the previous sample injection. In addition, we evaluated the spray-capillary quantitative microsampling using the automated system with various sample injection times (Tinj = 6 to 90 s, sample injection ESI voltage = 3.0 kV).

  2. Automatic spray-capillary microsampling with online CE separation (Fig. 1C). In this sequence, we included the following steps. (1) Preconditioning (~ 0.5 min): With the sampling end of the spray-capillary placed in the BGE vial, pressure (e.g., 690 mbar) was applied to the BGE vial to fill the column with BGE buffer until a droplet formed at the MS end of the spray capillary. (2) Arm transfer (~ 0.1 min): The sampling end of the spray capillary was moved from the BGE vial to the sample vial. (3) Spray-capillary injection (Tinj): ESI voltage was applied to the MS end of the spray-capillary for sample injection for the required injection time (Tinj). (4) Arm transfer (~ 0.1 min): The sampling end of the spray-capillary was moved from the sample vial to the BGE vial. (5) CE separation (Tseparation): High separation voltage (e.g., – 30 kV for PEI-coated spray-capillaries) was applied on the sampling end of the spray-capillary for online CE separation and MS detection.

    E. coli cell digest (1 μg/μL in 0.1% FA and 45% ACN) spiked with 10 μM Ang II was used to evaluate the automatic spray-capillary microsampling performance with online CE separation. A PEI-coated spray-capillary (360 μm O.D., 50 μm I.D., 100 cm in length) was prepared for all CE-MS/MS experiments. We used the automated system to quantitatively inject E. coli cell lysate digest onto the spray capillary with various sample injection times (Tinj = 6 to 90 s, sample injection ESI voltage is 3 kV). After injection, a – 30 kV separation voltage was applied to the sampling end of the spray-capillary for online CE separation and MS detection.

Mass spectrometry analysis and data analysis

An LTQ Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with LTQ Velos as the detector was utilized for spray-capillary microsampling with pressure-based elution for standard Ang II. The MS1 m/z range was 100–2000. The MS-inlet capillary temperature was set to 275 °C. 3E4 and 50 ms was utilized as AGC target and maximum injection time, respectively.

For spray-capillary microsampling with online CE analysis, an Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used with the following parameters: the MS-inlet capillary temperature was 275 °C, 3.0 kV of ESI voltage was used for sample injection, and 2.4 kV was used for CE separation. MS1 scans were acquired at 120,000 resolution with 2 microscans from 350–1350 m/z range. Auto maximum injection time mode was selected for MS1. The top 10 precursor ions with charge 2–6 were selected for fragmentation with 30% as the normalized HCD collision energy. MS2 scans were collected at 30,000 resolution with 2 microscans and 200 ms maximum injection time from 350 to 1350 m/z. The isolation window was 2 m/z and the RF lens was 70%. AGC was 1E6 for MS1 and 1E5 for MS2.

Peak extraction was done manually in Xcalibur. Max-Quant (version 2.1.3.0) was utilized for peptide identification against the annotated E. coli protein database (UniProt 2019–10-13, 4519 species). A decoy database was used with a false discovery rate (FDR) of 1%. Trypsin was used for digestion. Carbamidomethyl on cysteine (C) was set as a fixed modification; oxidation on methionine (M) and acetylation on the N-terminal were set as variable modifications. The other parameters were set as default. Results were processed and plotted using Excel or GraphPad Prism.

Results

Automated spray-capillary sample injection platform setup and performance evaluation

Our previous work described the spray-capillary sample injection platform for ultralow-volume sampling and online CE-MS analysis [44]. This platform demonstrated quantitative and reproducible ultralow-volume sample handling, high separation efficiency, and ultrasensitive detection. However, this platform required manual operation for most steps including moving the sampling end of the spray-capillary between the sample and the BGE vials, operating and timing the ESI voltage for sample injection, switching the CE HV on/off, and tuning N2 pressure for pressure elution. Although the microsampling process was robust, as previously described [49], the manual manipulation required by this platform iteration limited the precision and accuracy (e.g., absolute elution time for direct injection, migration time for online CE separation, and peak intensity) as well as sample throughput. Furthermore, the platform (e.g., air/nitrogen flow generator) was uniquely constructed in the lab and attempts to reproduce the manual platform could suffer from interlaboratory variation. In this work, we present an improved, automated spray-capillary platform to perform high-throughput microsampling and online CE-MS/MS by incorporating the spray-capillary device into a commercially available CE autosampler through a collaboration with CMP Scientific Inc. as shown in Fig. 1.

To evaluate the reproducibility of the sampling using the automated spray-capillary platform, 50 continuous analyses of Ang II were performed using fully automated spray-capillary microsampling and pressure elution (Tinj = 6 s, sample injection ESI voltage is 3.0 kV) according to the scheme given in Fig. 1B. A bare spray capillary (360 μm O.D., 50 μm I.D., 100 cm in length) was used to perform these experiments. Reproducibility and precision were evaluated via the peak intensity and elution time. The estimated flow rate was ~ 300 pL/s using the video-monitoring method developed previously [49]. For each run, the running time was 7.2 min, the sample injection volume was 1800 pL, and the estimated injection amount of Ang II was ~ 1.8E – 15 mol (~ 0.19 fg). Figure 2A displays base peak chromatograms (BPCs) for these 50 continuous injections, demonstrating highly similar peak intensity, elution time, and peak shape for all 50 runs. Additionally, 10 randomly selected runs from the 50 continuous runs were overlayed in Fig. 2B to demonstrate the reproducibility of the spray-capillary sampling and pressure elution process. The average elution time and peak intensity for Ang II in these 50 continuous injections were 4.03 min (RSD: 0.62%) and 4.73E+08 (RSD: 14.70%), respectively (Fig. 2C, D). Our results demonstrated that the spray-capillary injection is highly reproducible for handling picoliter-to nanoliter-level samples. The integrated automated system significantly reduced variation in Tinj and pressure elution, making it more robust for large-scale sample analysis.

Fig. 2.

Fig. 2

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Demonstration of reproducibility of 50 automated analyses. A Base peak chromatograms (BPCs) from 50 continuous analyses of Ang II using fully automated spray-capillary pressure elution and 6 s spray-capillary sample injection time. B 10 overlapped MS chromatograms from fully automated spray-capillary platform with 6 s spray-capillary sample injection. C Elution time of 50 continuous runs. D Peak intensity of 50 continuous runs

Quantitative spray-capillary microsampling and pressure elution MS analysis

We have previously reported that the spray-capillary device can quantitatively aspirate ultralow-volume samples by adjusting the spray-capillary injection time. A bare spray capillary (360 μm O.D., 50 μm I.D., 100 cm in length) was used to evaluate the quantitative sampling of the automated spray-capillary platform; we varied the Tinj from 6 to 90 s (N = 3, Fig. 3A). The total injection volume ranged from 1800 pL to 27 nL based on our observed capillary flow rates (~ 300 pL/s) using camera monitoring. The estimated sample injection amount was 0.19 to 2.7 fg. It should be noted that while total injection volume/mass was very low (e.g., nL or pL level), the total sample volume was limited to the microliter-level or higher in this study by the selection of sample vials/well plates compatible with the CMP CE autosampler. In this case, we used low-volume vials that hold up to 200 μL of sample. The datasets for this calibration curve were collected automatically without user oversight. The sample injection time was plotted against the area under the curve and was fit with an R2 of 0.9964 as shown in Fig. 3B. The linearity of the calibration curve indicates that the automated spray-capillary platform is capable of performing precise, automated, and quantitative microsampling. The sample injection time is included in the elution time, so the elution time increased incrementally as the injection time increased. For example, the elution time is 5.46 min for Tinj = 90 s, and the elution time is 4.46 min for Tinj = 30 s. The correlation between injection time and Ang II elution time is plotted in Fig. 3C, and this correlation can be used to normalize elution time for run-to-run comparison. These results demonstrate that the fully automated spray-capillary microsampling platform is capable of performing reproducible, high-throughput microsampling analysis with a wide range of injection volumes. Figure 3D displays total ion chromatograms (TICs) of triplicate runs of Ang II with Tinj = 6 s; an RSD for elution time of 0.3% was observed between these runs. Low RSD values of < 0.5% were observed for all sample injection time points, demonstrating high run-to-run reproducibility with the automatic spray-capillary microsampling and pressure elution. These results demonstrated the feasibility of coupling spray-capillary microsampling and pressure elution for direct infusion measurement of low-volume and low-complexity samples in a rapid and automated fashion, which can be easily adapted to large-scale sample analysis. Additionally, as discussed below, the spray-capillary device can be directly used for CE separation of more complex protein mixtures.

Fig. 3.

Fig. 3

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Evaluation of automated spray-capillary quantitative microsampling. A Base peak chromatograms (BPCs) of automated pressure elution analyses of Ang II at various spray-capillary elution times (6–90 s). B Calibration curve displaying peak intensity as a function of injection time (N = 3). Error bars indicate the standard deviation. C Correlation between Ang II migration time and spray-capillary injection time (N = 3). Error bars indicate the standard deviation. D Triplicate BPCs of Ang II automated runs with 6-s injection time

Performance evaluation of automated spray-capillary CE-MS platform

A primary advantage of the spray-capillary device is that it can directly serve as the CE separation column for online CE separation and MS analysis with no additional sampling devices [44, 49]. This makes the spray-capillary a very attractive device that can be adapted to any commercially available CE autosampler for ultralow-volume sampling and online CE-MS analysis. Here, we developed and optimized an automated spray-capillary sample injection and online CE-MS platform using the CMP autosampler (example sequence in Fig. 1C). A PEI-coated spray-capillary (360 μm O.D., 50 μm I.D., 100 cm in length) was implemented to evaluate system performance. The coating process was performed as discussed in the experimental section [48]. It has been reported that PEI coating can promote highly efficient CE separation and provide a more stable electroosmotic flow (EOF) for high-throughput CE separation, which is beneficial for reducing run-to-run variation in large-scale, automatic sample analysis [48].

Here, we evaluated the automated, high-throughput spray-capillary-based CE-MS analysis of E. coli lysate digest (1 μg/mL) spiked with 10 μM Ang II. Figure 4A shows the base peak chromatogram (BPC) of a replicate using Tinj = 15 s with 3 arbitrarily chosen examples of identified E. coli peptides. The peaks’ full width at half maximum (FWHM) for all three peptides were less than 5 s, resulting in the average plate number among the three peptides of ~ 30,000, which suggested good column separation efficiency for a fast separation (Table 1). The reproducibility of the automated spray-capillary CE-MS platform for analysis of E. coli cell lysate was demonstrated using four replicate injections with 15-s injection times (Fig. 4B).

Fig. 4.

Fig. 4

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Examples of identified E. coli peptides with A BPC, EIEs, and isotope distribution. B Replicate runs (N = 4) with 15 s injection time, demonstrating the reproducibility of automated spray-capillary CE-MS separation. C Reproducibility evaluation among replicates with various injection times (6–90 s)

Table 1.

Summary of FWHM and plate number for three peptide examples

Peak P0CE48 P0AB71 P0A7U3
FWHM (min) 0.08 0.06 0.08
Avg. FWHM (min) 0.073 ± 0.012
Migration time (min) 4.75 5.29 5.96
Plate number 19,531 43,064 30,748
Avg. plate number 31,114 ± 9611
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Quantitative microsampling and CE separation efficiency were also investigated by varying the sample injection time from 6 to 90 s (N = 4). The total injection volume ranged from 1800 pL to 27 nL, estimated based on our calculated capillary flow rates (~ 300 pL/s) observed from camera monitoring. The estimated sample injection amount ranged from 1800 pg to 27 ng. All analyses were performed continuously without interruption. Most peptides eluted between 5 and 9 min after sample injection. The reproducibility among replicated runs was demonstrated using correlation plots for all injection times (6–90 s) (Fig. 4C). In total, 749 unique peptides from 170 protein groups among all 20 runs (4 replicates for each injection time point, 5 injection time points in total) were identified. The number of identified peptides observed here is lower than generally observed using bottom-up LC–MS/MS for mass-limited samples due to shorter separation gradients and sharper peaks associated with CE. The number of identified peptides can potentially be improved with the implementation of data independent acquisition (DIA)-MS/MS [50] and online ion mobility mass spectrometry methods such as FAIMS [51].

Peak shape was consistent between different sampling times, and peak intensity increased proportionally with sampling time (Fig. 5A). Extracted ion electropherograms (EIEs) of Ang II (523.75–524.81 m/z) across different injection times are also displayed in Fig. 5B. The relative abundance of Ang II with respect to Tinj fits to a linear curve as shown in Fig. 5C with R2 = 0.9705. The linearity of this fit demonstrates scalable quantitative analysis using the automated spray-capillary-based CE-MS platform. The CE-MS migration times were highly reproducible with low standard deviation and varied from (RSD = 0.38%) to (RSD = 1.69%) for Tinj 6–90 s for Ang II. Furthermore, the extracted ion intensities for Ang II in replicate runs were also reproducible with RSDs ranging from 3 to 32% for Tinj 6–90 s. The relative abundance of three arbitrarily chosen peptides with respect to Tinj also demonstrated high linearity (R2 > 0.97) (Fig. 5C). This further indicates the capability of quantitative sample analysis in complex biological samples using the automated spray-capillary-based CE-MS platform. The EIE migration time was also plotted against the injection time for the three peptides as well as Ang II in Fig. 5D and again shifted proportionally with increased Tinj. The standard deviation varied from 0.33 to 1.37%, demonstrating high reproducibility.

Fig. 5.

Fig. 5

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A Base peak chromatograms (BPCs) of E. coli cell lysate with Ang II (DRVYIHPF) spiked in using various fully automated spray-capillary injection times. B Extracted ion electropherograms (EIEs) of Ang II at various spray-capillary injection times. C Correlation between abundance and spray-capillary-based injection times (6–90 s). D Correlation between migration time and spray-capillary-based injection times (6–90 s). Error bars represent standard deviation

In summary, the automated spray-capillary CE-MS platform demonstrated quantitative proteomic analysis in complex biological samples with high reproducibility.

Discussion

The previously reported spray-capillary CE-MS platform was simple and robust for the analysis of simple peptide mixtures and complex metabolomics [44, 49]. However, manual manipulation of the spray capillary could be challenging, and improper operation could lead to experimental discrepancies. Here, we have improved the spray-capillary platform using a commercially available CE autosampler and a PEI-coated capillary for automated spray-capillary-based microsampling, highly efficient CE separation, and direct MS detection. We demonstrated that this platform could perform 50 continuous pressure elution analyses overnight without interruption or supervision. Both elution time and peak intensity were consistent among each of the 50 runs with low RSD which demonstrates highly consistent sampling. Additionally, scalable, quantitative microsampling was performed and evaluated by varying the sample injection time. The relative peak intensity demonstrated a linear correlation with the injection time, indicating that the automated spray-capillary device is capable of highly reproducible and quantitative automated sampling. We also demonstrated that automated quantitative CE-MS on low-volume samples can be reproducibly performed using this automated spray-capillary platform by varying the sample injection time and observing the area under the curve for each peptide in a complex sample such as E. coli digest. Though current CE autosamplers cannot facilitate such a low volume of sample for injection, future development of autosampler technology to adapt ultralow-volume sample containers such as Cellenion’s cellenCHIP (e.g., 50–500 nL working volumes) could allow for low nanoliter-volume sample analysis using the spray capillary.

Automation and capillary coating improved the performance of the spray-capillary platform; however, there is still room for further improvement. The fabrication of the ESI tip is a labor-intensive process done by hand. This process may limit the use and dissemination of this platform. Furthermore, the automated spray-capillary CE-MS platform parameters, such as (1) capillary coating and (2) capillary inner diameter, may be optimized for various experimental purposes including bottom-up/top-down proteomics, metabolomics, lipidomics, etc. Here, we utilized a PEI coating as it has been shown to improve EOF, reduce adsorption to the capillary wall, and shorten migration times [48]. While the decrease in migration time may negatively affect separation efficiency for PEI compared with a bare capillary [52]. PEI coating results in robust and reproducible separation, improved sensitivity, and narrower signal windows [53]. Other coatings including neutral capillary coatings such as linear polyacrylamide (LPA) have also been applied to CE-MS-based proteomics [54, 55] and even for top-down proteomics [56] to eliminate EOF and extend separation windows for improved separation. These neutral capillary coatings may also be evaluated to improve separation efficiency for the spray-capillary platform. Decreasing capillary inner diameter has also been shown to improve CE separation and sensitivity [57] and decrease sample loading volume [58]. For example, the Dovichi group used an uncoated, 10-μm-I.D. capillary to identify approximately 250 peptides from 16 pg of E. coli lysate [59]. As such, integrating a smaller-inner-diameter capillary to the automated spray-capillary platform may improve peptide identification and sensitivity. The spray-capillary platform may be optimized for the separation of anionic molecules/metabolites [60, 61] such as carboxylic acids [62], N-glycans [63], carbohydrates [64], etc. Overall, we believe that this automated spray-capillary platform has the potential to benefit the high-throughput omics analysis of picoliter-to-nanoliter complex biological samples that are currently challenging the field.

Acknowledgements

This work was partly supported by grants from OCAST HR23-169, NIH NIAID R01AI141625, and NIH NIH/NIAID2U19AI062629.

We also thank the OU Protein Production and Characterization Core (PPC) facility and Dr. Philip Bourne for supporting E. coli protein extraction. The PPC is supported by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (Grants P20GM103640 and P30GM145423), the OU Vice President for Research and Partnerships, and the OU College of Arts and Sciences.

Biographies

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Jiaxue Li received a master’s in chemistry from the University of Oklahoma, Norman, with Dr. Si Wu in 2022. He is now a Scientist I at Genentech working in mass spectrometry, liquid chromatography, capillary electrophoresis, and antibody engineering.

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Lushuang Huang received his Ph.D. in chemistry from the University of Oklahoma, Norman, with Dr. Si Wu in 2021. He is now Senior Scientist at Regeneron Pharmaceuticals, Inc. working on using liquid chromatography-mass spectrometry as a tool to support drug development at different stages and utilizing novel technologies for the development of MS-based analytical methods for antibody characterization.

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Yanting Guo received her Ph.D. degree from the Department of Chemistry and Biochemistry at the University of Oklahoma, Norman, with Dr. Si Wu in 2023. During her Ph.D. program, she majorly focused on optimizing novel analytical methods for high-throughput top-down proteomics to quantify the intact proteome in complex biological samples.

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Kellye A. Cupp-Sutton received her Ph.D. in chemistry from the University of Oklahoma, Norman, with Dr. Michael T. Ashby in 2018. She subsequently began her postdoctoral research in the lab of Dr. Si Wu at the University of Oklahoma, and her postdoctoral research focuses on the development and application of high-throughput quantitative, top-down mass spectrometry-based proteomics approaches to study functional proteomics. Currently, she serves on the ACS Analytical Chemistry Education committee and the FEMs communication committee.

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Si Wu received her Ph.D. in 2006 from Washington State University under the direction of Prof. James Bruce working on developing novel MS tools for studying proteins in complex samples. She then worked as a postdoctoral fellow at the Pacific North-west National Laboratory with Drs. Ljiljiana Pasa-Tolic and Richard D. Smith on top-down proteomics and became a research scientist at PNNL in 2008. In 2015, she moved to the University of Oklahoma where she is currently Associate Professor in the Department of Chemistry. She was named a “2017 ASMS Emerging Investigator” by JASMS and received the USHUPO “Robert J. Cotter New Investigator Award” in 2020. Her current research is focused on developing and applying high-throughput quantitative top-down and functional proteomics techniques to address important clinical and biological questions.

Footnotes

Competing interests The authors declare that they have no known competing interests to declare that are relevant to the content of this article.

References

  • 1.Sun B, Kumar S. Protein adsorption loss—the bottleneck of single-cell proteomics. J Proteome Res. 2022;21(8):1808–15. [DOI] [PubMed] [Google Scholar]
  • 2.Wu R, Xing S, Badv M, Didar TF, Lu Y. Step-wise assessment and optimization of sample handling recovery yield for nanoproteomic analysis of 1000 mammalian cells. Anal Chem. 2019;91(16):10395–400. [DOI] [PubMed] [Google Scholar]
  • 3.Kelly RT. Single-cell proteomics: progress and prospects. Mol Cell Proteomics. 2020;19(11):1739–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Slavov N. Single-cell protein analysis by mass spectrometry. Curr Opin Chem Biol. 2021;60:1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Cupp-Sutton KA, Fang M, Wu S. Separation methods in single-cell proteomics: RPLC or CE? Int J Mass Spectrom. 2022;481:116920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Zhang P, Gaffrey MJ, Zhu Y, Chrisler WB, Fillmore TL, Yi L, et al. Carrier-assisted single-tube processing approach for targeted proteomics analysis of low numbers of mammalian cells. Anal Chem. 2019;91(2):1441–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Whittal RM, Keller BO, Li L. Nanoliter chemistry combined with mass spectrometry for peptide mapping of proteins from single mammalian cell lysates. Anal Chem. 1998;70(24):5344–7. [DOI] [PubMed] [Google Scholar]
  • 8.Sun L, Zhu G, Dovichi NJ. Integrated capillary zone electrophoresis–electrospray ionization tandem mass spectrometry system with an immobilized trypsin microreactor for online digestion and analysis of picogram amounts of RAW 264.7 cell lysate. Anal Chem. 2013;85(8):4187–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Sun L, Zhu G, Li Y, Yang P, Dovichi NJ. Coupling methanol denaturation, immobilized trypsin digestion, and accurate mass and time tagging for liquid-chromatography-based shotgun proteomics of low nanogram amounts of RAW 264.7 cell lysate. Anal Chem. 2012;84(20):8715–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Li Z-Y, Huang M, Wang X-K, Zhu Y, Li J-S, Wong CCL, et al. Nanoliter-scale oil-air-droplet chip-based single cell proteomic analysis. Anal Chem (Washington, DC, U S). 2018;90(8):5430–8. [DOI] [PubMed] [Google Scholar]
  • 11.Zhu Y, Piehowski PD, Zhao R, Chen J, Shen Y, Moore RJ, et al. Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells. Nat Commun. 2018;9(1):882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Liang Y, Acor H, McCown MA, Nwosu AJ, Boekweg H, Axtell NB, et al. Fully automated sample processing and analysis workflow for low-input proteome profiling. Anal Chem. 2021;93(3):1658–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Woo J, Williams SM, Markillie LM, Feng S, Tsai C-F, Aguilera-Vazquez V, et al. High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip. Nat Commun. 2021;12(1):6246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Williams SM, Liyu AV, Tsai C-F, Moore RJ, Orton DJ, Chrisler WB, et al. Automated coupling of nanodroplet sample preparation with liquid chromatography–mass spectrometry for high-throughput single-cell proteomics. Anal Chem. 2020;92(15):10588–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Specht H, Harmange G, Perlman DH, Emmott E, Niziolek Z, Budnik B, et al. Automated sample preparation for high-throughput single-cell proteomics. bioRxiv Syst Biol. 2018;399774. [Google Scholar]
  • 16.Budnik B, Levy E, Harmange G, Slavov N. SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation. Genome Biol. 2018;19(1):161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Petelski AA, Emmott E, Leduc A, Huffman RG, Specht H, Perlman DH, et al. Multiplexed single-cell proteomics using SCoPE2. Nat Protoc. 2021;16(12):5398–425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Hughes AJ, Lin RK, Peehl DM, Herr AE. Microfluidic integration for automated targeted proteomic assays. Proc Natl Acad Sci. 2012;109(16):5972–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Martin JG, Rejtar T, Martin SA. Integrated microscale analysis system for targeted liquid chromatography mass spectrometry proteomics on limited amounts of enriched cell populations. Anal Chem. 2013;85(22):10680–5. [DOI] [PubMed] [Google Scholar]
  • 20.Xue M, Wei W, Su Y, Kim J, Shin YS, Mai WX, et al. Chemical methods for the simultaneous quantitation of metabolites and proteins from single cells. J Am Chem Soc. 2015;137(12):4066–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Huang EL, Piehowski PD, Orton DJ, Moore RJ, Qian W-J, Casey CP, et al. SNaPP: simplified nanoproteomics platform for reproducible global proteomic analysis of nanogram protein quantities. Endocrinology. 2016;157(3):1307–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Yan L, Qiao L, Ji J, Li Y, Yin X, Lin L, et al. In-tip nanoreactors for cancer cells proteome profiling. Anal Chim Acta. 2017;949:43–52. [DOI] [PubMed] [Google Scholar]
  • 23.Hughes CS, Foehr S, Garfield DA, Furlong EE, Steinmetz LM, Krijgsveld J. Ultrasensitive proteome analysis using paramagnetic bead technology. Mol Syst Biol. 2014;10(10):757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Yang Z, Zhang Z, Chen D, Xu T, Wang Y, Sun L. Nanoparticle-aided nanoreactor for nanoproteomics. Anal Chem. 2021;93(30):10568–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Zhang Z, Dubiak KM, Huber PW, Dovichi NJ. Miniaturized filter-aided sample preparation (MICRO-FASP) method for high throughput, ultrasensitive proteomics sample preparation reveals proteome asymmetry in Xenopus laevis embryos. Anal Chem. 2020;92(7):5554–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kostas JC, Greguš M, Schejbal J, Ray S, Ivanov AR. Simple and efficient microsolid-phase extraction tip-based sample preparation workflow to enable sensitive proteomic profiling of limited samples (200 to 10,000 cells). J Proteome Res. 2021;20(3):1676–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.DeLaney K, Sauer CS, Vu NQ, Li L. Recent Advances and New Perspectives in Capillary Electrophoresis-Mass Spectrometry for Single Cell “Omics.” Molecules. 2019;24(1):42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Onjiko RM, Portero EP, Moody SA, Nemes P. In situ microprobe single-cell capillary electrophoresis mass spectrometry: metabolic reorganization in single differentiating cells in the live vertebrate (Xenopus laevis) embryo. Anal Chem. 2017;89(13):7069–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lombard-Banek C, Moody SA, Manzini MC, Nemes P. Microsampling capillary electrophoresis mass spectrometry enables single-cell proteomics in complex tissues: developing cell clones in live Xenopus laevis and zebrafish embryos. Anal Chem. 2019;91(7):4797–805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kawai T, Ota N, Okada K, Imasato A, Owa Y, Morita M, et al. Ultrasensitive single cell metabolomics by capillary electrophoresis-mass spectrometry with a thin-walled tapered emitter and large-volume dual sample preconcentration. Anal Chem. 2019;91(16):10564–72. [DOI] [PubMed] [Google Scholar]
  • 31.Liu L, Chen D, Wang J, Chen J. Advances of Single-Cell Protein Analysis Cells. 2020;9(5):1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Johnson KR, Gao Y, Greguš M, Ivanov AR. On-capillary cell lysis enables top-down proteomic analysis of single mammalian cells by CE-MS/MS. Anal Chem. 2022;94(41):14358–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Onjiko RM, Portero EP, Moody SA, Nemes P. In situ microprobe single-cell capillary electrophoresis mass spectrometry: metabolic reorganization in single differentiating cells in the live vertebrate (Xenopus laevis) embryo. Anal Chem. 2017;89(13):7069–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lombard-Banek C, Choi SB, Nemes P. Single-cell proteomics in complex tissues using microprobe capillary electrophoresis mass spectrometry. Methods Enzymol. 2019;628:263–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lombard-Banek C, Moody SA, Manzini MC, Nemes P. Microsampling capillary electrophoresis mass spectrometry enables single-cell proteomics in complex tissues: developing cell clones in live Xenopus laevis and zebrafish embryos. Anal Chem (Washington, DC, U S). 2019;91(7):4797–805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Marie A-L, Ray S, Ivanov AR. Highly-sensitive label-free deep profiling of N-glycans released from biomedically-relevant samples. Nat Commun. 2023;14(1):1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Stuart JN, Sweedler JV. Single-cell analysis by capillary electrophoresis. Anal Bioanal Chem. 2003;375(1):28–9. [DOI] [PubMed] [Google Scholar]
  • 38.Lombard-Banek C, Moody SA, Nemes P. Single-cell mass spectrometry for discovery proteomics: quantifying translational cell heterogeneity in the 16-cell frog (Xenopus) embryo. Angew Chem Int Ed Engl. 2016;55(7):2454–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Onjiko RM, Moody SA, Nemes P. Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo. Proc Natl Acad Sci. 2015;112(21):6545–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Lombard-Banek C, Moody SA, Nemes P. Single-cell mass spectrometry for discovery proteomics: quantifying translational cell heterogeneity in the 16-cell frog (Xenopus) embryo. Angew Chem, Int Ed. 2016;55(7):2454–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Nemes P, Baxi AB, Pade LR. Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo. JoVE. 2022;182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Zeng H, Weng Y, Ikeda S, Nakagawa Y, Nakajima H, Uchiyama K. Accurate and highly reproducible picoliter injection system for capillary electrophoresis. Anal Chem. 2012;84(24):10537–42. [DOI] [PubMed] [Google Scholar]
  • 43.Huang L, Wang Z, Cupp-Sutton KA, Smith K, Wu S. Spray-capillary: an electrospray-assisted device for quantitative ultralow-volume sample handling. Anal Chem. 2020;92(1):640–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Huang L, Fang M, Cupp-Sutton KA, Wang Z, Smith K, Wu S. Spray-capillary-based capillary electrophoresis mass spectrometry for metabolite analysis in single cells. Anal Chem. 2021;93(10):4479–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Peuchen EH, Zhu G, Sun L, Dovichi NJ. Evaluation of a commercial electro-kinetically pumped sheath-flow nanospray interface coupled to an automated capillary zone electrophoresis system. Anal Bioanal Chem. 2017;409(7):1789–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Guo Y, Yu D, Cupp-Sutton KA, Liu X, Wu S. Optimization of protein-level tandem mass tag (TMT) labeling conditions in complex samples with top-down proteomics. Anal Chim Acta. 2022;1221:340037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Cao P, Moini M. A novel sheathless interface for capillary electrophoresis/electrospray ionization mass spectrometry using an incapillary electrode. J Am Soc Mass Spectrom. 1997;8(5):561–4. [DOI] [PubMed] [Google Scholar]
  • 48.Santos M, Ratnayake C, Fonslow B, Guttman A. A covalent, cationic polymer coating method for the CESI-MS analysis of intact proteins and polypeptides. SCIEX Separations application note. 2015. [Google Scholar]
  • 49.Huang L, Wang Z, Cupp-Sutton KA, Smith K, Wu S. Spray-capillary: an electrospray-assisted device for quantitative ultralow-volume sample handling. Anal Chem. 2020;92(1):640–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Ludwig C, Gillet L, Rosenberger G, Amon S, Collins BC, Aebersold R. Data-independent acquisition-based SWATH-MS for quantitative proteomics: a tutorial. Mol Syst Biol. 2018;14(8):e8126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Johnson KR, Greguš M, Ivanov AR. Coupling high-field asymmetric ion mobility spectrometry with capillary electrophoresis-electrospray ionization-tandem mass spectrometry improves protein identifications in bottom-up proteomic analysis of low nanogram samples. J Proteome Res. 2022;21(10):2453–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Faserl K, Sarg B, Gruber P, Lindner HH. Investigating capillary electrophoresis-mass spectrometry for the analysis of common post-translational modifications. Electrophoresis. 2018;39(9–10):1208–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Cheng J, Morin GB, Chen DDY. Bottom-up proteomics of envelope proteins extracted from spinach chloroplast via high organic content CE-MS. Electrophoresis. 2020;41(5–6):370–8. [DOI] [PubMed] [Google Scholar]
  • 54.Gomes FP, Yates JR III. Recent trends of capillary electrophoresis-mass spectrometry in proteomics research. Mass Spectrom Rev. 2019;38(6):445–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Zhu G, Sun L, Dovichi NJ. Thermally-initiated free radical polymerization for reproducible production of stable linear polyacrylamide coated capillaries, and their application to proteomic analysis using capillary zone electrophoresis–mass spectrometry. Talanta. 2016;146:839–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Lubeckyj RA, McCool EN, Shen X, Kou Q, Liu X, Sun L. Single-shot top-down proteomics with capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for identification of nearly 600 Escherichia coli proteoforms. Anal Chem. 2017;89(22):12059–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Wahl JH, Goodlett DR, Udseth HR, Smith RD. Use of small-diameter capillaries for increasing peptide and protein detection sensitivity in capillary electrophoresis-mass spectrometry. Electrophoresis. 1993;14(1):448–57. [DOI] [PubMed] [Google Scholar]
  • 58.Jankowski JA, Tracht S, Sweedler JV. Assaying single cells with capillary electrophoresis. TrAC, Trends Anal Chem. 1995;14(4):170–6. [Google Scholar]
  • 59.Sun L, Zhu G, Zhao Y, Yan X, Mou S, Dovichi NJ. Ultrasensitive and fast bottom-up analysis of femtogram amounts of complex proteome digests. Angew Chem Int Ed. 2013;52(51):13661–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Kok MGM, Somsen GW, de Jong GJ. Comparison of capillary electrophoresis-mass spectrometry and hydrophilic interaction chromatography-mass spectrometry for anionic metabolic profiling of urine. Talanta. 2015;132:1–7. [DOI] [PubMed] [Google Scholar]
  • 61.Ramautar R, Somsen GW, de Jong GJ. CE–MS for metabolomics: developments and applications in the period 2014–2016. Electrophoresis. 2017;38(1):190–202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Soga T, Heiger DN. Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry. Anal Chem. 2000;72(6):1236–41. [DOI] [PubMed] [Google Scholar]
  • 63.Gennaro LA, Salas-Solano O. On-line CE–LIF–MS technology for the direct characterization of N-linked glycans from therapeutic antibodies. Anal Chem. 2008;80(10):3838–45. [DOI] [PubMed] [Google Scholar]
  • 64.Maxwell EJ, Ratnayake C, Jayo R, Zhong X, Chen DDY. A promising capillary electrophoresis–electrospray ionization–mass spectrometry method for carbohydrate analysis. Electrophoresis. 2011;32(16):2161–6. [DOI] [PubMed] [Google Scholar]

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