Sheathless Capillary Electrophoresis Research Articles

Sheathless CESI-MS versus LC-MS: Results of qualitative and quantitative analyses of the primary and secondary metabolites of Pleioblastus amarus bamboo shoots.

The bamboo shoot of Pleioblastus amarus (Keng) Keng f. is a medicinal and edible resource in China. In this study, three separation techniques were applied to identify the primary and secondary metabolites component of P. amarus bamboo shoots, including sheathless capillary electrophoresis electrospray ionization-mass spectrometry (CESI-MS), reverse-phase liquid chromatography-MS (RPLC-MS), and hydrophilic interaction liquid chromatography-MS (HILIC-MS). A total of 201 metabolites were identified by the three methods. Among those metabolites, 146 were identified by RPLC-MS, 85 were identified by HILIC-MS, and 46 were identified by sheathless CESI-MS. These methods were complementary and had a linear coefficient. CESI-MS presented advantages in the identification of isomers, high sensitivity, very low sample usage, and good detection of polar and nonpolar metabolites, showing its unique applications in food analysis and prospects in metabolic research.

Protocol for Etching Bare-Fused Silica Capillaries for Sheathless Capillary Electrophoresis-Mass Spectrometry Coupling.

Homemade capillaries are a very common practice for the users of capillary electrophoresis (CE), notably in CE-UV. With the advent of the capillary electrophoresis-mass spectrometry coupling since the end of the 1980s, several interfaces have been developed. Among those interfaces, the porous tip sprayer allows great sensitivity at nano flow rates and has been used in numerous applications over the past few years. However, the homemade implementation of a suitable capillary for the porous tip sprayer is more challenging. The porous tip is created by etching the bare-fused silica capillary with hydrofluoric acid. Here we describe the complete process of etching bare-fused silica capillaries, from length cutting to quality control of the newly etched capillary.

Development of a novel sheathless CE-ESI-MS interface via a CO2 laser ablated opening

A new and easy to construct sheathless capillary electrophoresis electro spray ionization mass spectrometry (CE-ESI-MS) interface was developed that offers several advantages compared to traditional liquid junction interfaces. The fabrication of the device only requires a CO2 laser engraver that most groups working with microfluids have access to. It only takes a few seconds to create a CO2 laser ablated opening in the bare-fused silica capillaries and the opening can be placed as close as a few mm from the spray tip. The capillary is punctured through a silicone tube such that the opening is directly placed inside this tube, which also serves as a liquid reservoir for the make-up liquid. Electrical contact required for both CE separation and ESI is established via the liquid in this reservoir which is in contact with the electrode of an external high voltage power supply. The developed CE-ESI-MS interface is capable of analysing both small molecules and biomolecules such as peptides in physisorbed PEG polymer brush coated capillaries. Proof-of-principle of the interface was demonstrated by analysing a tryptic digest of BSA. Further, a range of drugs of abuse were also investigated. The examined small molecules (pethidine, nortriptyline, methadone, haloperidol and loperamide) have a quantification limit (LOQ) of 150 ng/mL and a detection limit (LOD) of 40 ng/mL (except for loperamide: LOD = 80 ng/mL). Finally, we used our novel CE-MS interface for the analysis of the Aβ40 peptide. This is a member of the beta-Amyloid peptide family, involved in the development of Alzheimer's disease. A LOQ of 9 μg/mL was obtained for Aβ40, corresponding to 23 fmoles in a sample volume of 11 nL.

EThcD and 213 nm UVPD for Top-Down Analysis of Bovine Seminal Plasma Proteoforms on Electrophoretic and Chromatographic Time Frames.

Seminal plasma is a critical and complex fluid that carries sperm to eggs to initiate the fertilization process. Here, we present a top-down mass spectrometry (TDMS) strategy for identifying proteins and posttranslational modifications (PTMs) in bovine seminal plasma. In this study, proteins were separated using sheathless capillary zone electrophoresis (CZE)-MS and reversed-phase liquid chromatography (LC)-MS, and then fragmented using electron-transfer/higher-energy collisional dissociation (EThcD) and 213 nm ultraviolet photodissociation (213 nm UVPD) to provide more comprehensive information about the proteomic landscape of this biological fluid. Four hundred and seventeen proteoforms were identified by sheathless CZE-MS, and one hundred and seventy-two species were unique to this method. LC-MS identified 3090 proteoforms, including 1707 unique species. All identifications were within ±10 ppm (mass error) and with a P-Score ≤1 × 10-04. Pooling results (triplicate measurements) from sheathless CZE-MS and LC-MS resulted in the identification of 1433 proteoforms (EThcD) and 2151 proteoforms (213 nm UVPD) with 612 species unique for EThcD and 1021 for 213 nm UVPD. The average sequence coverage was found to be higher for EThcD (28%) than for 213 nm UVPD (23%). The use of sheathless CZE-MS and LC-MS with EThcD and 213 nm UVPD provided complementary protein profiling and proteoform data that were more comprehensive than those of either method alone.

Sensitive detection of benzophenone-type ultraviolet filters in plastic food packaging materials by sheathless capillary electrophoresis–electrospray ionization–tandem mass spectrometry

While they are commonly used as ultraviolet (UV) filters in plastic food packaging materials, benzophenones (BPs) are reported as environmental endocrine disruptors, and some of them possess significant estrogenic activity. Therefore sensitive determination of the content of those UV filters in plastic polymers is of vital importance in safety assessment of food packaging materials. Here, the sheathless capillary electrophoresis–electrospray ionization–tandem mass spectrometry (CE–ESI–MS/MS) method is applied for the first time to sensitively detect BP-type UV filters in plastic food packaging materials. We investigate and optimize a variety of factors that may affect ESI–MS efficiency and CE separation. Sensitive detection of six BP-type UV filters is achieved using sheathless CE–ESI–MS/MS in conjunction with accelerated solvent extraction and solid phase extraction, with the limit-of-detection of 7 pg/mL–2.4 ng/mL. The method exhibits excellent inter/intra-day repeatability along with the advantages of efficient separation, rapid analysis, low sample consumption and high sensitivity. Six BP-type UV filters in eight different brands of plastic films obtained from supermarkets are successfully analyzed using the method. Good recoveries of 81.3–104.1% at three levels of spiked concentrations are achieved with low RSDs (n = 5) of 2.5–8.7%. Our study shows that the sheathless CE–ESI–MS/MS is a robust and reliable method for sensitive and rapid analysis of UV filters, which would be of potential application in safety assessment of plastic food packaging materials.

Ultrasensitive and simultaneous determination of RNA modified nucleotides by sheathless interfaced capillary electrophoresis-tandem mass spectrometry.

A label-free ultrasensitive determination of eight RNA modified nucleotides simultaneously was first established based on a sheathless capillary electrophoresis-tandem mass spectrometry system. This system performed well using only 500 pg-5 ng practical RNA samples, and a downward trend of most target nucleotides in HCT 116 cells was observed with the increase of nickel concentration.

Dopant Enriched Nitrogen Gas Combined with Sheathless Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry for Improved Sensitivity and Repeatability in Glycopeptide Analysis.

Over the last years, numerous strategies have been proposed to enhance both ionization efficiency and spray stability in electrospray ionization (ESI), in particular for nanospray applications. In nano-liquid chromatography-mass spectrometry (nano-LC-ESI-MS), a better ESI performance has been observed when a coaxial gas flow is added around the ESI emitter. Moreover, enrichment of the gas with an organic dopant has led to an improved desolvation and ionization efficiency with an overall enhanced sensitivity. In this study, the use of a dopant enriched nitrogen (DEN)-gas combined with sheathless capillary electrophoresis (CE)-ESI-MS was evaluated for glycopeptide analysis. Using acetonitrile as a dopant, an increased sensitivity was observed compared to conventional sheathless CE-ESI-MS. Up to 25-fold higher sensitivities for model glycopeptides were obtained, allowing for limits of detection unachieved by state-of-the-art nano-LC-ESI-MS. The effect of DEN-gas on the repeatability and intermediate precision was also investigated. When compared to previously reported nano-LC-ESI-MS measurements, similar values were found for CE-ESI-MS with DEN-gas. The enhanced repeatability fosters the use of DEN-gas sheathless CE-ESI-MS in protein glycosylation analysis, where precision is essential. The use of DEN-gas opens new avenues for highly sensitive sheathless CE-ESI-MS approaches in glycoproteomics research, by significantly improving sensitivity and precision.

Structural characterization of antibody drug conjugate by a combination of intact, middle-up and bottom-up techniques using sheathless capillary electrophoresis – Tandem mass spectrometry as nanoESI infusion platform and separation method

Antibody-drug conjugates (ADCs) represent a fast growing class of biotherapeutic products. Their production leads to a distribution of species exhibiting different number of conjugated drugs overlaying the inherent complexity resulting from the monoclonal antibody format, such as glycoforms. ADCs require an additional level of characterization compared to first generation of biotherapeutics obtained through multiple analytical techniques for complete structure assessment. We report the development of complementary approaches implementing sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) to characterize the different aspects defining the structure of brentuximab vedotin. Native MS using sheathless CE-MS instrument as a nanoESI infusion platform enabled accurate mass measurements and estimation of the average drug to antibody ratio alongside to drug load distribution. Middle-up analysis performed after limited IdeS proteolysis allowed to study independently the light chain, Fab and F(ab')2 subunits incorporating 1, 0 to 4 and 0 to 8 payloads respectively. Finally, a CZE-ESI-MS/MS methodology was developed in order to be compatible with hydrophobic drug composing ADCs. From a single injection, complete sequence coverage could be achieved. Using the same dataset, glycosylation and drug-loaded peptides could be simultaneously identified revealing robust information regarding their respective localization and abundance. Drug-loaded peptide fragmentation mass spectra study demonstrated drug specific fragments reinforcing identification confidence, undescribed so far. Results reveal the method ability to characterize ADCs primary structure in a comprehensive manner while reducing tremendously the number of experiments required. Data generated showed that sheathless CZE-ESI-MS/MS characteristics position the methodology developed as a relevant alternative for comprehensive multilevel characterization of these complex biomolecules.

Improvement of Mitochondria Extract from Saccharomyces cerevisiae Characterization in Shotgun Proteomics Using Sheathless Capillary Electrophoresis Coupled to Tandem Mass Spectrometry.

In this work, we describe the characterization of a quantity-limited sample (100 ng) of yeast mitochondria by shotgun bottom-up proteomics. Sample characterization was carried out by sheathless capillary electrophoresis, equipped with a high sensitivity porous tip and coupled to tandem mass spectrometry (CESI-MS-MS) and concomitantly with a state-of-art nano flow liquid chromatography coupled to a similar mass spectrometry (MS) system (nanoLC-MS-MS). With single injections, both nanoLC-MS-MS and CESI-MS-MS 60 min-long separation experiments allowed us to identify 271 proteins (976 unique peptides) and 300 proteins (1,765 unique peptides) respectively, demonstrating a significant specificity and complementarity in identification depending on the physicochemical separation employed. Such complementary, maximizing the number of analytes detected, presents a powerful tool to deepen a biological sample's proteomic characterization. A comprehensive study of the specificity provided by each separating technique was also performed using the different properties of the identified peptides: molecular weight, mass-to-charge ratio (m/z), isoelectric point (pI), sequence coverage or MS-MS spectral quality enabled to determine the contribution of each separation. For example, CESI-MS-MS enables to identify larger peptides and eases the detection of those having extreme pI without impairing spectral quality. The addition of peptides, and therefore proteins identified by both techniques allowed us to increase significantly the sequence coverages and then the confidence of characterization. In this study, we also demonstrated that the two yeast enolase isoenzymes were both characterized in the CESI-MS-MS data set. The observation of discriminant proteotypic peptides is facilitated when a high number of precursors with high-quality MS-MS spectra are generated.

Ultrasensitive determination of 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA by sheathless interfaced capillary electrophoresis-mass spectrometry.

A newly developed sheathless interface for capillary electrophoresis-mass spectrometry, using a porous tip sprayer, was first applied for highly sensitive determination of cytosine modifications. The system performed well in identification and quantification of both 5-methylcytosine and 5-hydroxymethylcytosine using only 125 pg (∼20 cells) genomic DNA samples.

Sheathless capillary electrophoresis electrospray ionization‐mass spectrometry interface based on poly(dimethylsiloxane) membrane emitter and thin conducting liquid film

This study develops a sheathless CE-MS interface using a robust PDMS membrane emitter and liquid-film electric conduction. A 3D mold was constructed for casting the device by using a one-step casting procedure. The interface consisted of a 125 μm-thick triangular emitter with a 50 μm-diameter microchannel, a conducting reservoir, and a 375 μm-diameter channel for assembling the separation capillary. The separation capillary was inserted into the 375 μm channel and connected to the emitter through the conducting reservoir. The electric contact for the CE outlet was established through a conductive liquid film in the space between the capillary terminus and the 375 μm channel. The one-step casting procedure and using a membrane emitter instead of a tapered emitter produced an easily fabricated and robust interface. A stable electrospray was obtained from 30 to 350 nL/min. Analyzing a five-peptide mixture in low-EOF (60 nL/min) and high-EOF (210 nL/min) conditions demonstrated the utility of the interface.

Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes

Development of nano-electrospray (nanoESI) sources allowed to increase significantly the sensitivity which is often lacking when studying biological noncovalent assemblies. However, the flow rate used to infuse the sample into the mass spectrometer cannot be precisely controlled with nanoESI and the robustness of the system could represent an issue. In this study, we have used a sheathless capillary electrophoresis-mass spectrometry (CESI) prototype as a nanoESI infusion device. The hydrodynamic mobilization of the capillary content was characterized and the ability of the system to generate a stable electrospray under controlled flow rate conditions ranging from 4 up to 900 nL/min was demonstrated. The effect of the infusing flow rate on the detection of an intact model protein analyzed under native conditions was investigated. Results demonstrated a significant increase in sensitivity of 46-fold and a signal-to-noise ratio improvement of nearly 5-fold when using an infusing flow rate from 456.9 down to 13.7 nL/min. The CESI prototype was further used to detect successfully the β ring homodimer in its native conformation. Obtained results were compared with those achieved with conventional ESI. Intensity signals were increased by a factor of 5, while sample consumption decreased 80 times. β ring complexed with the P14 peptide was also studied. Finally, the CESI interface was used to observe the quaternary structure of native hemocyanins from Carcinus maenas crabs; this high molecular complex coexisting under various degrees of complexation and resulting in masses ranging from 445 kDa to 1.34 MDa.

Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry

Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) – tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.

Low-Flow Sheathless Capillary Electrophoresis–Mass Spectrometry for Sensitive Glycoform Profiling of Intact Pharmaceutical Proteins

Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (∼5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-β allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars.

Improving the Comprehensiveness and Sensitivity of Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry for Proteomic Analysis

We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) procedure which employs a high sensitivity porous electrospray ionization (ESI) sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency, we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity.

The development of a sheathless capillary electrophoresis electrospray ionization–mass spectrometry interface based on thin conducting liquid film

A simple two column sheathless CE/MS interface was constructed using polydimethylsiloxane to fabricate a microdevice allowing facile column alignment and electrical connection. One conducting reservoir, two holes and one 1mm length microchannel between the holes were fabricated on the microdevice. The two holes were used for connecting separation capillary and ESI sprayer. The hole for ESI sprayer was fabricated at the edge of the conducting reservoir. The ESI sprayer was inserted through the reservoir to the hole so allowing it to be aligned with the separation column. Because the size of the hole was fabricated slightly larger than the outer diameter of the ESI sprayer, the electrical conduction was established through the thin conductive liquid film formed in the space between the ESI sprayer and the hole. The interface design presented was both easy to fabricate and operate and demonstrated good performance. The dead volume did not significantly affect operation as indicated by a demonstrated preservation of separation resolution. The intra-day precision and inter-day precision for peak areas and migration times observed using this interface were found to be less than 12% and 5%, respectively.

Characterization of drug-lysozyme conjugates by sheathless capillary electrophoresis–time-of-flight mass spectrometry

Drug-protein conjugates have been widely used for the cell-specific targeting of drugs to cells that can bind and internalize the proteinaceous carrier. For renal drug targeting, lysozyme (LZM) can be used as an effective carrier that accumulates in proximal tubular cells. We used capillary electrophoresis–time-of-flight mass spectrometry (CE–TOF-MS) for the characterization of different drug-LZM conjugates. A recently developed prototype porous tip sprayer was employed for sheathless electrospray ionization (ESI) CE–MS interfacing. In order to prevent adsorption of LZM conjugates to the capillary wall, a positively charged polyethylenimine capillary coating was used in combination with a low-pH background electrolyte. Drug-LZM products had been prepared by first coupling BOC- l-methionine hydroxysuccinimide ester (BOCmet) to lysine residues of LZM followed by conjugation with the kinase inhibitors LY364947, erlotinib, or Y27632 via a platinum(II)-based linker. CE–TOF-MS of each preparation showed narrow symmetrical peaks for the various reaction products demonstrating that drug-LZM conjugates remained stable during the CE analysis and subsequent ESI. Components observed in the drug-LZM products were assigned based on their relative migration times and on molecular mass as obtained by TOF-MS. The TOF-MS data obtained for the individual components revealed that the preparations contained LZM carrying one or two drug molecules, next to unmodified and BOCmet-modified LZM. Based on relative peak areas (assuming an equimolar response for each component) a quantitative conjugate profile could be derived for every preparation leading to drug loading values of 0.4–0.6 mol drug per mole protein.

Performance of a sheathless porous tip sprayer for capillary electrophoresis–electrospray ionization-mass spectrometry of intact proteins

The performance of a prototype porous tip sprayer for sheathless capillary electrophoresis–mass spectrometry (CE–MS) of intact proteins was studied. Capillaries with a porous tip were inserted in a stainless steel needle filled with static conductive liquid and installed in a conventional electrospray ionization (ESI) source. Using a BGE of 100 mM acetic acid (pH 3.1) and a positively charged capillary coating, a highly reproducible and efficient separation of four model proteins (insulin, carbonic anhydrase II, ribonuclease A and lysozyme) was obtained. The protein mass spectra were of good quality allowing reliable mass determination of the proteins and some of their impurities. Sheath-liquid CE–MS using the same porous tip capillary and an isopropanol–water–acetic acid sheath liquid showed slightly lower to similar analyte responses. However, as noise levels increased with sheath-liquid CE–MS, detection limits were improved by a factor 6.5–20 with sheathless CE–MS. The analyte response in sheathless CE–MS could be enhanced using a nanoESI source and adding 5% isopropanol to the BGE, leading to improved detection limits by 50-fold to 140-fold as compared to sheath liquid interfacing using the same capillary – equivalent to sub-nM detection limits for three out of four proteins. Clearly, the sheathless porous tip sprayer provides high sensitivity CE–MS of intact proteins.

Simplifying CE−MS Operation. 2. Interfacing Low-Flow Separation Techniques to Mass Spectrometry Using a Porous Tip

A robust, reproducible, and single-step interface design between low flow rate separation techniques, such as sheathless capillary electrophoresis (CE) and nanoliquid chromatography (nLC), and mass spectrometry (MS) using electrospray ionization (ESI), is introduced. In this design, the electrical connection to the capillary outlet was achieved through a porous tip at the capillary outlet. The porous section was created by removing 1-1.5 in. of the polyimide coating of the capillary and etching this section by 49% solution of HF until it is porous. The electrical connection to the capillary outlet is achieved simply by inserting the capillary outlet containing the porous tip into the existing ESI needle (metal sheath) and filling the needle with the background electrolyte. Redox reactions of water at the ESI needle and transport of these small ions through the porous tip into the capillary provides the electrical connection for the ESI and for the CE outlet electrode. The etching process reduces the wall thickness of the etched section, including the tip of the capillary, to 5-10 microm, which for a 20-30 microm i.d. capillary results in stable electrospray at approximately 1.5 kV. The design is suitable for interfacing a wide range of capillary sizes with a wide range of flow rates to MS via ESI, but it is especially useful for interfacing narrow (<30 microm i.d.) capillaries and low flow rates (<100 nL/min). The advantages of the porous tip design include the following: (1) its fabrication is reproducible, can be automated, and does not require any mechanical tools. (2) The etching process reduces the tip outer diameter and makes the capillary porous in one step. (3) The interface can be used for both nLC-MS and CE-MS. (4) If blocked or damaged, a small section of the tip can be etched off without any loss of performance. (5) The interface design leaves the capillary inner wall intact and, therefore, does not add any dead volume to the CE-MS or nLC-MS interface. (6) Bubble formation due to redox reactions of water at the high-voltage electrode is outside of the separation capillary and does not affect separation or MS performances. The performance of this interface is demonstrated by the analyses of amino acids, peptide, and protein mixtures.

Design and performance of a sheathless capillary electrophoresis/mass spectrometry interface by combining fused‐silica capillaries with gold‐coated nanoelectrospray tips

A simple sheathless capillary electrophoresis (CE)/mass spectrometry (MS) interface was constructed by combining widely used nanospray needles with fused-silica capillaries and it was successfully applied for the separation of peptides. The end of the CE capillary was pulled to a taper, etched and then fitted into the metal-coated nanospray borosilicate capillary. The nanospray needle can be used for several CE runs, but it can be easily and rapidly changed in the case of accidental breakage or evaporation of the coating. A fast capillary electrochromatographic method was also developed for MS analysis of peptides containing numerous basic amino acids.