Over 10 000 Peptide Identifications from the HeLa Proteome by Using Single‐Shot Capillary Zone Electrophoresis Combined with Tandem Mass Spectrometry

Capillary zone electrophoresis (CZE)–tandem mass spectrometry (MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has so far fallen far below that of reversed‐phase liquid chromatography (RPLC)–MS/MS. The use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (ca. 300) is reported. This method was coupled to an Orbitrap Fusion mass spectrometer through an electrokinetically pumped sheath‐flow interface for the analysis of complex proteome digests. Single‐shot CZE–MS/MS lead to the identification of over 10 000 peptides and 2100 proteins from a HeLa cell proteome digest in approximately 100 min. This performance is nearly an order of magnitude better than earlier CZE studies and is within a factor of two to four of the state‐of‐the‐art nano ultrahigh‐pressure LC system.     

Heart‐cut nano‐LC–CZE–MS for the characterization of proteins on the intact level

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed‐phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well‐suited geometries/dimensions. Here, a heart‐cut nano‐LC–CZE–MS setup was developed utilizing for the first time a mechanical 4‐port valve as LC–CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart‐cut transfer of individual LC peaks and subsequent CZE–MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower μg/mL range were determined, which are considerably lower compared to traditional CZE–MS. In addition, this study represents the first application of an LC–CE–MS system for intact protein analysis. The nano‐LC–CZE–MS system is expected to be applicable to various other analytical challenges.     

Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE‐ESI‐MS/MS

Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post‐translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE‐ESI‐MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE‐ESI‐MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l ‐aspartic acid or d ‐aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI efficiency provided by CZE‐ESI‐MS/MS properties, and enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS‐based peptide analysis. Copyright © 2016 John Wiley & Sons, Ltd.     

Complementary middle‐down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis – mass spectrometry

High‐resolution capillary zone electrophoresis – mass spectrometry (CZE‐MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle‐down and intact CZE‐MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post‐translational modifications (PTMs) and glycosylation structures. Middle‐down and intact CZE separations were performed in an acidified methanol‐water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle‐down analysis of the IdeS‐digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X‐deamidated, 1X‐deamidated, and non‐deamidated forms at baseline resolution. In the course of the middle‐down CZE‐MS analysis, separation of glycoforms of the F_C/2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE‐MS². Incorporation of TCEP‐HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE‐MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X‐glycosylated, 1X‐glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE‐MS represents a complementary approach to the more conventional liquid‐chromatography – mass spectrometry‐based approaches.     

Therapeutic Fc‐fusion proteins: Current analytical strategies

Fc‐Fusion proteins represent a successful class of biopharmaceutical products, with already 13 drugs approved in the European Union and United States as well as three biosimilar versions of etanercept. Fc‐Fusion products combine tailored pharmacological properties of biological ligands, together with multiple functions of the fragment crystallizable domain of immunoglobulins. There is a great diversity in terms of possible biological ligands, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding constructs acting as cytokine traps. Due to their highly diverse structures, the analytical characterization of Fc‐Fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product‐specific methods over conventional generic/platform methods. This can be explained, for example, by the presence of numerous sialic acids, leading to high diversity in terms of isoelectric points and complex glycosylation profiles including multiple N‐ and O‐linked glycosylation sites. In this review, we highlight the wide range of analytical strategies used to fully characterize Fc‐fusion proteins. We also present case studies on the structural assessment of all commercially available Fc‐fusion proteins, based on the features and critical quality attributes of their ligand‐binding domains.     

Online preconcentration and two‐dimensional separation of cationic compounds via hyphenation of capillary zone electrophoresis with cyclodextrin‐modified micellar electrokinetic capillary chromatography

A novel preconcentration/separation approach, which online combined CZE with CD‐modified MEKC, was developed for simultaneous enhancing resolving power and detection sensitivity. CZE with cation‐selective exhaustive injection and transient ITP preconcentration was used as the first dimension, from which the effluent fractions were further analyzed by CD‐modified MEKC acting as the second dimension. As the key to successful integration of CZE with MEKC, a new interface was designed and electroaccumulation focusing strategy was employed to avoid analyte band diffusion at the interface. The comprehensive 2‐D system was successfully established with only one high voltage and four electrodes. The grouping of two orthogonal separation techniques, together with analytes preconcentration techniques, significantly enhanced resolution and sensitivity for 2‐D separation of cationic compounds. The resulting electrophoregram was quite different from that of either single CZE or MEKC. Up to 14 000‐ to 35 000‐fold improvement in sensitivity was obtained relative to conventional electrokinetic injection method. The limits of detection (S/N=3) were in the range of 0.03–0.1 μg/L. The number of theoretical plates was in the range of 103 000–184 000. This method was successfully applied to the analysis of trace cationic cardiovascular drugs in wastewater.     

Enantioselective column coupled electrophoresis employing large bore capillaries hyphenated with tandem mass spectrometry for ultra‐trace determination of chiral compounds in complex real samples

A new multidimensional analytical approach for the ultra‐trace determination of target chiral compounds in unpretreated complex real samples was developed in this work. The proposed analytical system provided high orthogonality due to on‐line combination of three different methods (separation mechanisms), i.e. (1) isotachophoresis (ITP), (2) chiral capillary zone electrophoresis (chiral CZE), and (3) triple quadrupole mass spectrometry (QqQ MS). The ITP step, performed in a large bore capillary (800 μm), was utilized for the effective sample pretreatment (preconcentration and matrix clean‐up) in a large injection volume (1–10 μL) enabling to obtain as low as ca. 80 pg/mL limits of detection for the target enantiomers in urine matrices. In the chiral CZE step, the different chiral selectors (neutral, ionizable, and permanently charged cyclodextrins) and buffer systems were tested in terms of enantioselectivity and influence on the MS detection response. The performance parameters of the optimized ITP – chiral CZE‐QqQ MS method were evaluated according to the FDA guidance for bioanalytical method validation. Successful validation and application (enantioselective monitoring of renally eliminated pheniramine and its metabolite in human urine) highlighted great potential of this chiral approach in advanced enantioselective biomedical applications.     

Field‐amplified sample injection combined with capillary electrophoresis for the simultaneous determination of five chlorophenols in water samples

A sensitive method of CZE‐ultraviolet (UV) detection based on the on‐line preconcentration strategy of field‐amplified sample injection (FASI) was developed for the simultaneous determination of five kinds of chlorophenols (CPs) namely 4‐chlorophenol (4‐CP), 2‐chlorophenol (2‐CP), 2,4‐dichlorophenol (2,4‐DCP), 2,4,6‐trichlorophenol (2,4,6‐TCP), and 2,6‐dichlorophenol (2,6‐DCP) in water samples. Several parameters affecting CZE and FASI conditions were systematically investigated. Under the optimal conditions, sensitivity enhancement factors for 4‐CP, 2‐CP, 2,4‐DCP, 2,4,6‐TCP, and 2,6‐DCP were 9, 27, 35, 43, and 43 folds, respectively, compared with the direct CZE, and the baseline separation was achieved within 5 min. Then, the developed FASI‐CZE‐UV method was applied to tap and lake water samples for the five CPs determination. The LODs (S/N = 3) were 0.0018–0.019 µg/mL and 0.0089–0.029 µg/mL in tap water and lake water, respectively. The values of LOQs in tap water (0.006–0.0074 µg/mL) were much lower than the maximum permissible concentrations of 2,4,6‐TCP, 2,4‐DCP, and 2‐CP in drinking water stipulated by World Health Organization (WHO) namely 0.3, 0.04, and 0.01 µg/mL, respectively, and thereby the method was suitable to detect the CPs according to WHO guidelines. Furthermore, the method attained high recoveries in the range of 83.0–119.0% at three spiking levels of five CPs in the two types of water samples, with relative standard deviations of 0.37–8.58%. The developed method was proved to be a simple, sensitive, highly automated, and efficient alternative to CPs determination in real water samples.     

Sensitive CE–MS analysis of potentially genotoxic alkylation compounds using derivatization and electrokinetic injection

A CE–MS method has been developed to detect trace levels of potentially genotoxic alkyl halides. After derivatization of the target components with 4-dimethylaminopyridine (DMAP) or butyl 1-(pyridinyl-4yl) piperidine 4-carboxylate (BPPC), the natively positively charged derivatives are pre-concentrated by applying electrokinetic injection and separated by a highly efficient CZE method using a background electrolyte (BGE) consisting of 100 mM of TRIS adjusted to pH 2.5 with phosphoric acid. Using a sheath liquid interface, subsequent MS detection allows highly specific and sensitive analysis of alkyl halides.     

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.     

Design of Sensitive Biocompatible Quantum‐Dots Embedded in Mesoporous Silica Microspheres for the Quantitative Immunoassay of Human Immunodeficiency Virus‐1 Antibodies

A novel sandwich‐type electrochemiluminescence (ECL) immunosensor was developed to enable the sensitive detection of HIV‐1 antibodies. This system incorporated mesoporous silica (mSiO₂) complexed with quantum dots (QDs) and nano‐gold particles, which were assembled to enhance signal detection. Magnetic beads were used by immobilizing the secondary anti‐IgG antibody. This was first employed to capture HIV‐1 antibody (Ab) to form a Fe₃O₄/anti‐IgG/Ab complex. A high loading and signal‐enhanced nanocomposite (hereafter referred to as Au‐mSiO₂‐CdTe) was used as a HIV‐1 antigen label. The Au‐mSiO₂‐CdTe nanocomposite was conjugated with the Fe₃O₄/anti‐IgG/Ab complex to form an immunocomplex (hereafter referred to as Fe₃O₄/anti‐IgG/Ab/HIV‐1/CdTe‐mSiO₂‐Au). This complex could be further separated by an external magnetic field to produce ECL signals. Due to the large specific surface area and pore volume of mSiO₂, the loading of the CdTe QDs was markedly increased. Thus, the loaded QDs released a powerful chemiluminescent signal with a concordantly increased sensitivity of the immunosensor. The immunosensor was highly sensitive, and displayed a linear range of responses for HIV‐1 antibody across a dilution range of 1 : 1500 through 1 : 50 with the detection limit of 1 : 4500. The immunoassay can be a promising candidate in early diagnosis of HIV infection.     

Separation of Enantiomers by On‐Line Capillary Isotachophoresis‐Capillary Zone Electrophoresis

The ability of capillary zone electrophoresis (CZE) coupled on‐line with capillary isotachophoresis (ITP) sample pretreatment in the column‐coupling capillary electrophoresis equipment to separate trace enantiomers present in samples of complex ionic matrices and enantiomers present in their mixtures at significantly differing concentrations has been studied. Enantiomers of 2,4‐dinitrophenyl labeled norleucine (DNP‐Nleu) and tryptophan enantiomers were employed as model analytes in this work while urine and mixtures of tryptophan enantiomers of differing concentrations served as model samples. Experiments performed with urine samples spiked with the DNP‐Nleu racemate at sub‐μmol/L concentrations demonstrated excellent sample pretreatment capabilities of ITP (concentration of the analytes, in‐column and post‐column sample clean up) when coupled on‐line with chiral CZE separations. In the CZE separations of enantiomers present in the samples at trace concentrations the sample pretreatment could be performed in both achiral and chiral ITP electrolyte systems. The use of a chiral electrolyte system was found to be essential in the ITP pretreatment of the samples containing the enantiomers at very differing concentrations. For example, a 2×10^–7 mol/L concentration of L‐tryptophan could be detected in the CZE separation stage of the ITP‐CZE combination in samples containing about a 10⁴ excess of D‐tryptophan only when the ITP pretreatment was carried out in the electrolyte system providing the resolution of enantiomers (α‐cyclodextrin served for this purpose in the present work). A post‐column ITP sample clean up was found effective in enhancing the destacking rate of the trace enantiomer in the CZE stage when the migration configuration of the enantiomers was less favorable (the trace constituent migrating behind the major enantiomer).     

Rapid analysis of constituents of Radix Cyathulae using hydrophilic interaction‐reverse phase LC‐MS

A hydrophilic interaction chromatography (HILIC) and reverse‐phase liquid chromatography (RPLC) coupled with electrospray TOF MS method was developed for the analysis and characterization of constituents in the radix of Cyathula officinalis Kuan. Separation parameters of HILIC such as buffer pH, mobile phase strength, and organic modifier were evaluated. Fructose, glucose, and sucrose were identified by HILIC‐ESI/TOF MS. Reverse‐phase liquid chromatography‐ESI/TOF MS were applied for quick and sensitive identification of major saponins in Cyathula officinalis. In‐source collision‐induced dissociation has been performed to elucidate the fragmentation pathways of oleanane‐, hederagenin‐, and gypsogmin‐type saponins. Twelve saponins were characterized in this plant for the first time, and four of them were presumed to be new compounds. In addition, one phytoecdysteroid (cyasterone) and one coumarin (6,7‐dimethoxycoumarin) were detected at the same time. The present method was capable of rapid characterizing and providing structure information of constituents from herbal drugs.     

Identification of CpG methylation of the SNRPN gene by methylation‐specific multiplex PCR

In this article, we show that methylation‐specific multiplex PCR (MS‐multiplex PCR) is a sensitive and specific single assay for detecting CpG methylation status as well as copy number aberrations. We used MS‐multiplex PCR to simultaneously amplify three sequences: the 3′ ends of the SNRPN gene (for unmethylated sequences), the KRITI gene (as internal control), and the promoter of the SNRPN gene containing CpG islands (for methylated sequences) after digestion with a methylation‐sensitive restriction enzyme (HhaI). We established this duplex assay for the analysis of 38 individuals with Prader–Willi syndrome, 2 individuals with Angelman syndrome, and 28 unaffected individuals. By comparing the copy number of the three regions, the methylation status and the copy number changes can be easily distinguished by MS‐multiplex PCR without the need of bisulfite treatment of the DNA. The data showed that MS‐multiplex PCR allows for the estimation of the methylation level by comparing the copy number aberrations of unknown samples to the standards with a known methylated status. The in‐house‐designed MS‐multiplex PCR protocol is a relatively simple, cost‐effective, and highly reproducible approach as a significant strategy in clinical applications for epigenetics in a routine laboratory.     

CE‐MS with electrokinetic supercharging and application to determination of neurotransmitters

Electrokinetic supercharging (EKS) is known as one of the most effective online electrophoretic preconcentration techniques, though pairing with it with mass spectrometry has presented challenges. Here, EKS is successfully paired with ESI‐MS/MS to provide a sensitive and robust method for analysis of biogenic amines in biological samples. Injection parameters including electric field strength and the buffer compositions used for the separation and focusing were investigated to achieve suitable resolution, high sensitivity, and compatibility with ESI‐MS. Using EKS, the sensitivity of the method was improved 5000‐fold compared to a conventional hydrodynamic injection with CZE. The separation allowed for baseline resolution of several neurotransmitters within 16 min with LODs down to 10 pM. This method was applied to targeted analysis of seven biogenic amines from rat brain stem and whole Drosophila tissue. This is the first method to use EKS with CE‐ESI‐MS/MS to analyze biological samples.     

Isotachophoretic focusing and mass spectrometry detection as tools for improving the determination of aromatic sulfonates in capillary electrophoresis

We explored isotachophoresis-capillary zone electrophoresis (ITP-CZE) with diode array detection on a single capillary to find out how to increase the injection volume and decrease the detection limits of aromatic sulfonates in CZE. The ITP was performed by applying a negative voltage in conjunction with hydrodynamic backpressure programming, and the terminating buffer was removed before the CZE separation, which resulted in highly sensitive determinations. The ITP increased the signal response of conventional hydrodynamic injection by a factor of 100, whereas the separation efficiency was unaffected. The limits of detection of the method were between 3 and 5 nugL(-1). The method was successfully used to determine these compounds in water samples. Experimental conditions for capillary electrophoresis-mass spectrometry were optimized and applied to determine aromatic sulfonates in water samples. These techniques enables the 2-naphthalenesulfonate to be determined in water samples.     

Electrolyte system strategies for anionic isotachophoresis with electrospray‐ionization mass‐spectrometric detection. 1. Regular isotachophoresis and free‐acid isotachophoresis

The subject of this work is the definition of a simple model based on general ITP theory that allows describing and predicting the behavior of ITP systems compatible with ESI‐MS detection. The model is exemplified by anionic ITP of weak acids that represent an interesting potential application field of ITP‐ESI‐MS. Suitable ESI‐compatible electrolyte systems of very simple composition are proposed including a special free‐acid ITP arrangement. The properties of these systems are discussed using illustrative diagrams of their stacking windows. The use of anionic ITP‐ESI‐MS in negative‐ion ESI mode is reported for the first time and its suitability for sensitive trace analysis is demonstrated. The presented ITP‐ESI‐MS application example comprises a free‐acid ITP system formed of formic and propionic acids and direct injection analysis of ibuprofen and diclofenac in waters with quantitation limits of the order 10^?10 M.     

Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical technique

Fragmentation in protein‐based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size‐exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE‐SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein‐based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab‐related low molecular weight fragments, a non‐reducible thio‐ether linked 75 kDa HL fragment was also identified.     

Window optimization in isotachophoresis superimposed on capillary zone electrophoresis

A sample stacking procedure to which a specific combination of electrolyte solutions is applied is isotachophoresis (ITP) superimposed on capillary zone electrophoresis (CZE), a so-called ITP/CZE system. In ITP/CZE some components migrate in an ITP fashion on top of a background electrolyte, and the other analytes migrate in a zone electrophoretic manner. For such a system, the leading electrolyte consists of a mixture of an ionic species, L1, of high mobility (the leading ion of the ITP system), an ionic species, L2, of low mobility (the coions of the CZE system), and a buffering counter-ionic species, whereas the terminating solution only contains the ionic species L2 and the buffering counterions. The zones of the components migrating in the ITP/CZE mode are sharp owing to the self-correcting properties of the zones and the concentrations of the L1 ions of the system. Mobility windows can be calculated, indicating which ions can migrate in the ITP/CZE mode. In this article mobility windows are calculated by applying both strong and weak acids as L1 and L2 ions and it appears that mobility windows can be optimized by chosing different ratios of L1 and L2 as well as different pH values. It is possible to construct very narrow mobility windows, and thereby choose which component of a sample solution can be concentrated, and to what concentration, in a very selective way. The big advantage of ITP/CZE compared with applications such as transient ITP and transient stacking is that the stacked sample ionic species migrate in the ITP mode during the whole experiment; furthermore, they do not destack. Experimentally obtained electropherograms validate the calculated mobility windows for the ITP/CZE mode.     

Capillary zone electrophoresis tandem mass spectrometry detects low concentration host cell impurities in monoclonal antibodies

We have evaluated CZE‐ESI‐MS/MS for detection of trace amounts of host cell protein impurities in recombinant therapeutics. Compared to previously published procedures, we have optimized the buffer pH used in the formation of a pH junction to increase injection volume. We also prepared a 5‐point calibration curve by spiking 12 standard proteins into a solution of a human mAb. A custom CZE‐MS/MS system was used to analyze the tryptic digest of this mixture without depletion of the antibody. CZE generated a ～70‐min separation window (～90‐min total analysis duration) and ～300‐peak capacity. We also analyzed the sample using ultra‐performance LC‐MS/MS. CZE‐MS/MS generated approximately five times higher base peak intensity and more peptide identifications for low‐level spiked proteins. Both methods detected all proteins spiked at ～100 ppm level with respect to the antibody.