Fabrication of internally tapered capillaries for capillary electrochromatography electrospray ionization mass spectrometry

In this study, we report a novel procedure for fabricating internally tapered capillary columns suitable for the coupling of capillary electrochromatography (CEC) to electrospray mass spectrometry (ESI-MS). The internal tapers were prepared by slowly heating the capillary end in a methane/O2 flame. Due to continuous self-shrinking of the inner channel of the capillary, the inside diameter of the opening was reduced to 7-10 microm. The procedure is easy to handle, with no requirement for expensive equipment as well as elimination of problematic grinding of the tip. Several advantages of these new internal tapers, as compared to using externally tapered columns, are described. First, the problems of poor durability and tip breakage associated with external tapering were successfully overcome with the internal taper. A comparison of the online CEC/ESI-MS between external versus internal tapers showed that the latter provides enhanced electrospray stability, resulting in significantly lower short-term noise and very short-term noise values. In turn, the more rugged design of internal tapers allows performing CEC/MS utilizing a harsh polar organic mobile phase, which was not previously successful using an external taper due to higher operating current and electrospray arcing. Next, data on the reproducibility of the internally tapered CEC/MS column using warfarin and beta-blockers as model analytes are presented. For example, when comparing the reproducibility for separation of warfarin under reversed-phase conditions, the internal taper demonstrated superior intraday % RSD (1.6-3.4) as compared to the external taper intraday % RSD (5-6). Last, the applicability of performing quantitative CEC/MS with internally tapered capillaries is demonstrated for simultaneous enantioseparation of beta-blockers. Impressive quantitative results include good linearity of calibration curves (e.g., R2 = 0.9940-0.9988) and limit of detection as low as 30 nM. The sensitive detection of a minor impurity of one enantiomer at the 0.1% level in a major chiral entity buttresses the suitability of compliance with FDA guidelines.     

Open-tubular capillary electrochromatography coupled with electrospray ionization mass spectrometry for peptide analysis

In this study, the open-tubular electrochromatographic (OT-CEC) migration behavior of various peptides has been characterized using etched and chemically (n-octadecyl- and cholesterol-) modified capillaries, interfaced to an electrospray ionization mass spectrometer through a sheath liquid configuration. The stationary phases were fabricated by etching the inner wall of the fused-silica capillary and then chemically modifying the new surface through a silanization/hydrosilation reaction. Unlike some other OT-CEC stationary-phase preparation methods, leaching of the immobilized stationary phase and subsequent contamination of the electrospray ion source was largely avoided with this novel surface modification technology. The influence of the immobilized organic phases and those of the buffer electrolytes (pH, the type and content of organic solvent) on the retention and separation of the selected peptides was investigated. Significant peptide retention was found even at very low pH with both types of stationary phases, under conditions whereby the electrophoretic migration dominated the separation process. Due to the effective coverage of the etched surface by a silanization/hydrosilation reaction, adverse adsorption of charged analytes onto the capillary wall was minimized. As a result, very efficient and highly reproducible peptide separations were achieved over a broad pH range. Moreover, peptide-specific multizoning effects were observed. The origin of this novel phenomenon was explored. Compared to capillary electrophoresis electrospray ionization mass spectrometry system, much higher detection sensitivity could be obtained, since a larger amount of sample could be injected and stacked at the head of the open-tubular capillary column without deteriorating the separation performance. On the basis of these observations, these procedures have been adapted to allow the analysis of tryptic peptides generated from proteins.     

Desorption electrospray ionization-mass spectrometry of proteins

Desorption electrospray ionization-mass spectrometry (DESI-MS) was evaluated for the detection of proteins ranging in molecular mass from 12 to 66 kDa. Proteins were uniformly deposited on a solid surface without pretreatment and analyzed with a DESI source coupled to a quadrupole ion trap mass spectrometer. DESI-MS parameters optimized for protein detection included solvent flow rate, temperature of heated capillary tube, incident and reflection angle, sheath gas pressure, and ESI voltage. Detection limits were obtained for all protein standards, and they were found to decrease with decreasing protein molecular mass: for cytochrome c (12.3 kDa) and lysozyme (14.3 kDa) a detection limit of 4 ng/mm2 was obtained; for apomyoglobin (16.9 kDa) 20 ng/mm2; for beta-lactoglobulin B (18.2 kDa) 50 ng/mm2; and for chymotrypsinogen A (25.6 kDa) 100 ng/mm2. The DESI-MS analysis of higher molecular mass proteins such as ovalbumin (44.4 kDa) and bovine serum albumin (66.4 kDa) yielded mass spectra of low signal-to-noise ratio, making their detection and molecular weight determination difficult. In this study, DESI-MS proved to be a rapid and robust method for accurate MW determination for proteins up to 17 kDa under ambient conditions. Finally, we demonstrated the DESI-MS detection of the bacteriophage MS2 capsid protein from crude samples with minimal sample preparation.     

Analysis of carbonic anhydrase in human red Blood cells using capillary electrophoresis/ electrospray ionization-mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of human red blood cells (RBCs) using the split-flow technique for interfacing CE to MS. By using a long (approximately125-cm) and narrow (approximately 15-microm-i.d.) capillary, the four major proteins of the RBC, which are hemoglobin (Hb, alpha- and beta-chains, 900 amol/chain), carbonic anhydrase I (CAI, approximately 7 amol/cell), and carbonic anhydrase II (CAII, approximately 0.8 amol/cell), were separated from each other and detected at low-attomole levels in one run and minimal sample preparation. Under these conditions, the detection limits for CAI and CAII in lysed RBCs were approximately 20 and approximately 44 amol, respectively. The approximately 20-amol detection limit of CAI was confirmed by the CE/ESI-MS analysis of three intact RBCs that had been drawn into the capillary under a microscope. A shorter capillary (approximately 55 cm long) provided faster analysis time but did not separate CAII from the beta-chain of hemoglobin, causing the CAII signal to be masked by the background chemical noise generated by the approximately 1,000 x molar excess of the beta-chain. Under this condition, the CAII detection limit increased to approximately 500 amol. From three methods of sample introduction (injection of lysed blood, injection of intact cells under microscope, and injection of intact cells suspended in saline solution), injection of lysed blood provided the optimum sensitivity. It was found that a background electrolyte (BGE) containing 0.1% acetic acid in water worked best for the analysis of intact cells, while a BGE containing 0.1% acetic acid in water + acetonitrile (50/50 by volume) worked best for the analysis of lysed blood.     

Coupling capillary electrochromatography with electrospray Fourier transform mass spectrometry for characterizing complex oligosaccharide pools

To deal with the complexity of the glycan mixtures released from glycoproteins, an efficient form of micro-column separations, capillary electrochromatography, has been combined with high-performance mass spectrometry (Fourier transform ion cyclotron resonance). Contour plots have been generated from the mixtures of O-linked oligosaccharides originated from bovine mucin and bile salt-stimulated lipase, a large glycoprotein enzyme.     

Analysis of bile acids and their conjugates by capillary electrochromatography/electrospray ion trap mass spectrometry

Different macroporous, monolithic capillary columns were prepared to separate various bile acid mixtures through capillary electrochromatography (CEC) at high efficiency. These columns are shown to be ideally suitable for coupling to an electrospray ionization/ion trap mass spectrometer. Detection and structural identification of different bile acid derivatives in either the positive- or negative-ion mode necessitated column technologies with different polarities and the capabilities of a reversed electroosmotic flow. High column efficiencies (610,000 theoretical plates/meter for glycocholic acid in normal-phase separation) were preserved in the coupling to mass spectrometry (MS), with the detection limits of approximately 40 femtomole (for cholic acid) and identification through CEC/MS/MS.     

Ultra-low flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics

The potential benefits of ultra-low flow electrospray ionization (ESI) for the analysis of phosphopeptides in proteomics was investigated. First, the relative flow dependent ionization efficiency of nonphosphorylated vs multiplyphosphorylated peptides was characterized by infusion of a five synthetic peptide mix with zero to four phophorylation sites at flow rates ranging from 4.5 to 500 nL/min. Most importantly, similar to what was found earlier by Schmidt et al., it has been verified that at flow rates below 20 nL/min the relative peak intensities for the various peptides show a trend toward an equimolar response, which would be highly beneficial in phosphoproteomic analysis. As the technology to achieve liquid chromatography separation at flow rates below 20 nL/min is not readily available, a sheathless capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) strategy based on the use of a neutrally coated separation capillary was used to develop an analytical strategy at flow rates as low as 6.6 nL/min. An in-line preconcentration technique, namely, transient isotachophoresis (t-ITP), to achieve efficient separation while using larger volume injections (37% of capillary thus 250 nL) was incorporated to achieve even greater sample concentration sensitivities. The developed t-ITP-ESI-MS strategy was then used in a direct comparison with nano-LC-MS for the detection of phosphopeptides. The comparison showed significantly improved phosphopeptide sensitivity in equal sample load and equal sample concentration conditions for CE-MS while providing complementary data to LC-MS, demonstrating the potential of ultra-low flow ESI for the analysis of phosphopeptides in liquid based separation techniques.     

Conduct-as-cast polymer monoliths as separation media for capillary electrochromatography

We have developed porous polymer monoliths (PPMs) that are versatile and robust reversed-phase chromatography media. The PPMs are cast-to-shape, UV-cured polymers that form uniform packings within pretreated glass capillaries and fused-silica chips. No applied pressure is ever needed to flush the PPMs since they support electroosmotic flow as cast. Such characteristics make the PPMs useful for chip-based devices. Our results show efficiencies greater than or equal to 150,000 plates/m for both capillary and chip-based separations of polycyclic aromatic hydrocarbons. By changing the monomers, the hydrophobicity of the polymers, and the direction of the electroosmotic flow can be altered without degrading chromatographic performance. We describe here the development of these acrylate-based materials along with both physical and chromatographic characterization.     

Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated fused-silica capillary

A simple procedure was developed for preparing a carbon-coated fused-silica capillary for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The tapered capillary tip was smeared with a marker pen before coating with carbon using a soft pencil. The layer from the ink of the marker pen was critical to the preparation of the carbon-coated capillary. The fabrication of a carbon-coated fused-silica capillary tip requires less than 1 min. The stability of this carbon-coated fused-silica capillary is examined, and its utility in on-line sheathless CE/ESI-MS is demonstrated with the separation of berberine, coptisine, and palmatine chlorides. Although the carbon-coated fused-silica capillary tip is not as rugged as a gold-coated capillary, it is durable enough for sheathless CE/ESI-MS applications. Moreover, it is easy to refurbish the column once the performance of the tip is degraded.     

Simultaneous separation of acidic, basic, and neutral organic compounds, including strong and moderate acids and bases, by capillary electrochromatography

The separation of strongly basic, moderately basic, weakly basic, strongly acidic, moderately acidic, weakly acidic, and neutral compounds in a single run using capillary electrochromatography (CEC) is presented. This is accomplished using a 3-μm CEC Hypersil C8 capillary with high organic content acetonitrile/phosphate (pH 2.5) mobile phases containing hexylamine. Fifteen basic, acidic, and neutral drugs of forensic interest are resolved using a step gradient. Strong and moderately basic drugs separate before t(o), apparently by a combination of free zone electrophoresis (CZE) and chromatographic phenomena. Weak bases separate after t(o), also by a combination of CZE and chromatographic processes. Due to large selectivity differences between CEC and CZE for bases, there is evidence that the stationary phase is playing a significant role in the separation of these solutes. The CEC approach presented offers unique selectivity, expanded peak capacity, and the ability to solubilize both hydrophilic and hydrophobic solutes in an injection solvent that is compatible with the chromatographic system.     

Sheathless capillary electrophoresis/electrospray ionization mass spectrometry using a pulled bare fused-silica capillary as the electrospray emitter

It has always been assumed that electrical contact at the capillary outlet is a necessary requirement when coupling capillary electrophoresis (CE) with electrospray ionization mass spectrometry (ESI-MS). In this study, we used a pulled bare-capillary tip as the ESI emitter, but neither was it coated with any electrically conductive materials nor was a high external voltage applied on its outlet. In this paper, we demonstrate that this straightforward approach may be used to generate multiply charged ions of proteins and peptides through electrospray ionization. Our results indicate that peptides and proteins, including bradykinin, cytochrome c, myoglobin, and tryptic digest products that elute from a pulled bare-capillary tip can be detected directly by ESI-MS using the tapered bare-capillary interface. Thus, we have demonstrated that CE and ESI-MS may be combined successfully without the need to modify the outlet of the capillary tip with an electrically contacting material.     

Evaluation of opiate separation by high-resolution electrospray ionization-ion mobility spectrometry/mass spectrometry

The separation of opiates and the primary metabolites was evaluated with ESI-IMS/MS. Seven opiate molecules were analyzed, and spectra were shown for each compound. The IMS separation of two isomers (morphine and norcodeine) was shown with baseline separation. Differences in the mobilities were found for the nonacetylated, monoacetylated, and biacetylated compounds. In this study, two primary findings are reported. First, IMS can easily separate metabolic isomers, and second, the two-dimensional separation capability of IMS/MS can be employed to confidently identify and separate both the opiates and metabolites. Although previous IMS studies have shown the separation of isomers, this is the first example to show the capability of IMS to separate metabolic isomers (within 70 s), a significant advantage in high-throughput screening for pharmacokinetic studies. Second, the monoacetylated and biacetylated compounds were found to form more compact ions for the sodium adducts in comparison to the protonated molecular ions. On the basis of the mobilities, information on structures and conformation can be deduced when sodium and protonated ions are compared.     

Combined partial acid hydrolysis and electrospray ionization-mass spectrometry for the structural determination of oligosaccharides

A general oligosaccharide acid hydrolysis method, amenable to electrospray ionization mass spectrometry (ESI-MS), is described that allows for hydrolysis of glycosidic bonds for both hexose- and N-acetylhexosamine-containing oligosaccharides. The partial acid hydrolysis of oligosaccharides is obtained by using an acid-exchange resin as the acid catalyst. A ladder sequence of the glycan is produced in solution that is directly analyzed by ESI tandem mass spectrometry, employing both ion trap and Fourier transform ion cyclotron resonance mass spectrometers, to provide sequence and linkage information. Unlike traditional acid hydrolysis procedures, there is minimal degradation of monosaccharide residues or deacetylation of N-acetylhexosamines by employing this technique. It is further demonstrated that the stereochemistry of the released monosaccharides and the anomeric configuration within disaccharides is determined by direct derivatization of the hydrolysate with Zn(dien)-Cl2 followed by ESI-MS/MS.     

Negative-ion electrospray mass spectrometry of neutral underivatized oligosaccharides

Negative-ion electrospray mass spectrometry (ES-MS) with collision-induced dissociation (CID) and MS/MS scanning on a quadrupole-orthoganal time-of-flight instrument provide a sensitive means for structural analysis of neutral underivatized oligosaccharides. Molecular mass information can be readily obtained from the dominant [M - H]- ions in the ES mass spectrum formed with subnanomole amounts of oligosaccharides, and similar sensitivity is available from CID-MS/MS to give structural details. The CID spectra of 14 oligosaccharides demonstrated that sequence and partial linkage information can be derived and isomeric structures can be differentiated. Series of C-type fragment ions give sequence information while the double glycosidic D-type cleavage of a 3-linked GlcNAc or Glc and the saccharide ring fragmentation of the 0,2A-type from 4-linked GlcNAc or Glc can provide partial linkage information. The distinctive D- and A-cleavages are important for differentiation of oligosaccharide type 1 and type 2 chains and to define the blood group H, Le(a), Le(x), Le(b), and Le(y) determinants carried by their fucosylated analogues.     

Monolithic silica-based capillary reversed-phase liquid chromatography/electrospray mass spectrometry for plant metabolomics

Application of C18 monolithic silica capillary columns in HPLC coupled to ion trap mass spectrometry detection was studied for probing the metabolome of the model plant Arabidopsis thaliana. It could be shown that the use of a long capillary column is an easy and effective approach to reduce ionization suppression by enhanced chromatographic resolution. Several hundred peaks could be detected using a 90-cm capillary column for LC separation and a noise reduction and automatic peak alignment software, which outperformed manual inspection or commercially available mass spectral deconvolution software.     

Metabolic differentiation of neuronal phenotypes by single-cell capillary electrophoresis-electrospray ionization-mass spectrometry

Single-cell mass spectrometry (MS) is a rapidly emerging field in metabolic investigations. The inherent chemical complexity of most biological samples poses analytical challenges when using MS platforms to measure sample content without prior chemical separation. Here, a single-cell capillary electrophoresis (CE) system was coupled with electrospray ionization (ESI) MS to enable the simultaneous measurement of a vast array of endogenous compounds in over 50 identified and isolated large neurons from the Aplysia californica central nervous system. More than 300 distinct ion signals (m/z values) were detected from a single neuron in the positive ion mode, 140 of which were selected for chemometric data analysis. Metabolic features were evaluated among six different neuron types (B1, B2, left pleural 1 (LPl1), metacerebral cell (MCC), R2, and R15) chosen for their various physiological functions. The results indicated chemical similarities among some neuron types (B1 to B2 and LPl1 to R2) and distinctive features for others (MCC and R15 cells). The quantitative nature of the MS platform allowed the comparison of metabolite levels for specific neurons. The CE-ESI-MS approach for examination of individual nanoliter-volume cells as described herein is readily adaptable to other volume-limited samples.     

Pressurized electrochromatography coupled with electrospray ionization mass spectrometry for analysis of peptides and proteins

Pressurized capillary electrochromatography (pCEC) was coupled with electrospray ionization mass spectrometry (ESI-MS) using a coaxial sheath liquid interface. It was used for separation and analysis of peptides and proteins. The effects of organic modifier and applied voltage on separation were investigated, and the effects of pH value of the mobile phase and the concentration of the electrolyte on ESI-MS signal were investigated. The resolution and detection sensitivity with different separation methods (pCEC, capillary high-performance liquid chromatography) coupled on-line with mass spectrometry were compared for the separation of a peptide mixture. To evaluate the feasibility and reliability of the experimental setup of the system, tryptic digests of cytochrome c and modified protein as real samples were analyzed by using pCEC-ESI-MS.     

Direct ionization of large proteins and protein complexes by desorption electrospray ionization-mass spectrometry

Desorption electrospray ionization-mass spectrometry (DESI-MS) has advantages for rapid sample analysis with little or no sample pretreatment, but performance for large biomolecules has not been demonstrated. In this study, liquid sample DESI, an extended version of DESI used for analysis of liquid samples, was shown to have capabilities for direct ionization of large noncovalent protein complexes (>45 kDa) and proteins (up to 150 kDa). Protein complex ions (e.g., superoxide dismutase, enolase, and hemoglobin) desorbed from solution by liquid sample DESI were measured intact, indicating the capability of DESI for preserving weak noncovalent interactions. Doping the DESI spray solvent with supercharging reagents resulted in protein complex ions having increased multiple charging without complex dissociation. Ion mobility measurements of model protein cytochrome c showed that the supercharging reagent favored the more compact conformation for the lower charged protein ions. Liquid sample DESI of hydrophobic peptide gramicidin D suggests that the ionization mechanism involves a droplet pick-up mixing process. Measurement of liquid samples significantly extends the mass range of DESI-MS, allowing the analysis of high-mass proteins such as 150 kDa immunoglobulin G (IgG) and thus represents the largest protein successfully ionized by DESI to date.     

Surface scanning analysis of planar arrays of analytes with desorption electrospray ionization-mass spectrometry

The analysis of analytes deposited on, separated on, or otherwise distributed about a planar surface using desorption electrospray ionization mass spectrometry in a surface scanning sampling mode was investigated. The physical regions of the surface-impacting solvent/gas jet desorption/ionization plume were described. Under the conditions typical for desorption electrospray ionization used here, the impact plume formed an elliptical desorption/ionization region on the surface. Most effective desorption/ionization was obtained from a smaller elliptical area within the larger impact region that was centered on a point on-axis from the sprayer tip to the surface. Maximum signal from a given amount of material on a surface was observed with proper plume and sample alignment when the diameter of the sample spot was less than the diameter of the central high-efficiency desorption/ionization region of the impact plume. Solvent and gas flow out of this high-efficiency desorption/ionization region was found to limit analyte accessibility to this region via a "washing effect" when analytes were on smooth surfaces or on surfaces for which the analyte had little affinity. As such, the direction of surface scanning and scan speed during an analysis was found to be important for maximizing signal levels and signal reproducibility on particular surface types. Overall, the results presented illustrate means to improve analysis of sample spots on various types of surfaces using desorption electrospray ionization mass spectrometry in a surface scanning mode.     

Separation of neutral saccharide mixtures with capillary electrochromatography using hydrophilic monolithic columns

While developing a combination of capillary electrochromatography (CEC) with tandem mass spectrometry (MS) for the benefit of characterizing complex oligosaccharide mixtures, we needed highly efficient CEC columns operating in an "MS-friendly" mode. We demonstrate here novel types of polar, monolithic CEC columns that separate effectively complex mixtures of saccharides with the use of mobile phases containing acetonitrile/dilute ammonium formate buffers. Using the positive-ion mode of detection for neutral saccharides, the detection conditions were optimized down to the low-femtomole sensitivities with the use of an ion trap mass spectrometer. This column technology provides a nearly universal system that can separate a wide range of carbohydrates: mono- and oligosaccharides with the intact reducing end, as well as saccharide alditols. Even the anomers formed due to mutarotation could be resolved with a high content of organic phase.