新型纳升级毛细管刻蚀电喷雾质谱接口研究

对普通石英毛细管表面使用氢氟酸刻蚀技术进行刻蚀, 并与商品化鞘流液毛细管电泳-质谱接口(Sheathflow CE-MS interface)结合, 将其改装成一种新型的纳升级电喷雾质谱接口. 玻璃膜接口部分呈多孔结构, 壁厚约10 μm. 以细胞色素c对新型接口加以评价, 样品的流量最低可达到20 nL/min; 在50~500 nL/min流量范围内刻蚀接口具有较高的响应信号. 考察了接触电解质溶液对样品电离的影响; 比较微升级不锈钢接口和新型接口的蛋白质检测结果发现, 在流速为200 nL/min时, 检测灵敏度可以提高3.6倍.     

Porous polymer monolith assisted electrospray from a glass microdevice

The coupling of a lab-on-a-chip microfluidic device to a nanoelectrospray ionization mass spectrometer has the potential to automate many routine analytical procedures and produce a powerful analytical tool. However, past coupling strategies have relied on complex manufacturing steps including drilling and etching the device to attach a capillary or building a nanospray emitter directly into the device. This study shows that a nanospray emitter can be easily fabricated using a porous polymer monolith (PPM) at the end of a glass microdevice. These devices are able to obtain a stable electrospray at a variety of flow rates (50-500 nL/min) but optimal results are obtained at lower flow rates (50-100 nL/min) compatible with electroosmotic flow processes. The PPM is photo-patterned so that it can be placed in any position within the channel of the device with no dead volume. The porous character and the hydrophobic nature of the PPM both aid in development of a stable electrospray process. Total ion current traces for the constant infusion of leucine-enkephalin and PPG show relative standard errors as low as 4%, and produce mass spectra with good signal-to-noise (S/N 43) from only 2 fmol of material. In addition, multiple experiments in a given day show good repeatability with variability as low as 13%, and the multiple flow paths inherent in the PPM limit sprayer clogging.     

Surface roughening of a non-tapered open tubular emitter for improved electrospray ionization mass spectrometry performance at low flow rates

A non-tapered open tubular emitter with 75 microm internal diameter (i.d.) and 360 microm external diameter (o.d.) was developed by simply grinding the exit aperture of a fused-silica capillary. The roughened emitter, with a relatively large aperture, generates stable electrospray signals (generally <5% relative standard deviation (RSD) for most conditions studied) at less than 500 nL/min flow rates, and was characterized with atomic force microscopy. The surface treatment greatly extends the operational range of an open tubular emitter to lower flow rates, compared to that of a cleaved capillary with similar dimensions. The stabilized nanoelectrospray is attributed to the increased surface roughness and modified wetting characteristics of the emitter exit resulting from grinding. Electrospray performance was evaluated, and as a result of the enhanced sensitivity from a roughened emitter, five femtomoles of leucine enkephalin were detected at a 50 nL/min flow rate with a signal to noise (S/N) ratio of 48. Furthermore, trypsin-digested bovine serum albumin (BSA) was used to demonstrate the application of the emitter in protein identification, giving a sequence coverage of 60%. These emitters are robust, and may become a facile alternative to tapered emitters at moderate nano flow rates (e.g. 50 to 500 nL/min).     

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.     

Efficient electrospray ionization from polymer microchannels using integrated hydrophobic membranes

A simple process for realizing stable and reliable electrospray ionization (ESI) tips in polymer microfluidic systems is described. The process is based on the addition of a thin hydrophobic membrane at the microchannel exit to constrain lateral dispersion of the Taylor cone formed during ESI. Using this approach, ESI chips are shown to exhibit well-defined Taylor cones at flow rates as low as 80 nL min(-1) through optical imaging. Furthermore, stable electrospray current has been measured for flow rates as low as 10 nL min(-1) over several hours of continuous operation. Characterization of the electrospray process by optical and electrical monitoring of fabricated ESI chips is reported, together with mass spectrometry validation using myoglobin as a model protein. The novel process offers the potential for low-cost, direct interfacing of disposable polymer microfluidic separation platforms to mass spectrometry.     

A sheath-flow nanospray interface for capillary electrophoresis/mass spectrometry

We have developed a novel sheath-flow interface for low-flow electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The interface is composed of two capillaries. One is a tapered fused-silica ESI emitter suitable for microliter and nanoliter flow rate electrospray and the other is a tail-end gold-coated CE separation column that is inserted into the emitter. A sheath liquid is supplied between the column and the emitter capillaries. The gold coating and the sheath liquid are used as the conducting media for ESI and the CE circuit. This novel design was initially evaluated by an infusion ESI-MS analysis of the most common antiretroviral dideoxynucleosides, followed by CE/MS coupling analysis of several antidepressant drugs. With infusion studies, the effects of the sheath liquid and the sample flow rates on detection sensitivity and signal stability were investigated. For an emitter with an internal diameter of 30 microm, the optimum flow rates for the sheath and the sample were 200 and 300 nL/min, respectively. The main improvement of this approach in comparison with conventional sheath liquid approaches using an ionspray interface is the gain in sensitivity. Sensitivities were three times better for dideoxynucleosides analyzed by infusion and 12 times higher for antidepressant drugs analyzed by CE/MS with this interface compared with ionspray. The emitter is durable, disposable, and simple to fabricate.     

Capillary high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry using a novel nanoflow gradient generator

A type of high-performance liquid chromatography (HPLC) based on a novel nanoflow gradient generator (Asymptotic-Trace-10-Port-Valve (AT10PV) nanoGR generator) was developed and coupled with an electrospray ion trap time-of-flight mass spectrometer (ESI-IT-TOF MS). Stability of the nanoflow GR HPLC system was tested at flow rates of 20 and 50 nL/min by using a nanoflow meter. Average flow rates in a 2-h run were 51.2 nL/min with RSD 0.7% and 21.0 nL/min with RSD 1.8%. Repeatability of analysis of the nanoHPLC/ESI-IT-TOF MS system was also tested by injecting 1.0 microL of trypsin digested bovine serum albumin (BSA) (100 fmol) into a monolithic silica-ODS column (30 microm i.d., 150 mm in length) through a packed silica-ODS trapping column (particle size 5 microm, 150 microm i.d., 10 mm in length). At a flow rate of 50 nL/min, the result demonstrated a reasonably good repeatability of peak retention times (RSD: 0.32-1.1%) and base-ion peak areas (RSD: 4.4-6.6%).     

A heated electrospray source for mass spectrometry of analytes from aqueous solutions

An electrospray interface is described that provides high sensitivity and signal stability for mass spectrometric detection of analytes in solvents with high water content including 100% water. The electrospray capillary tip section is heated close to the boiling point of the solvent. An approximately 20°C hotter airstream, with flow coaxial to the electrospray tip and codirectional to the electrospray.. is also used. With this arrangement, the analyte signal sensitivity and stability obtained with neat water is equal to that obtained with neat methanol. The heated electrospray also affords the use of a wide range of flow rates: 1–100 μL/min.     

High sensitivity analysis of phenylthiohydantoin amino acid derivatives by electrospray mass spectrometry

A new methodology has been developed for high sensitivity electrospray ionization mass spectrometric analyses of phenylthiohydantoin (PTH) amino acid derivatives. Key components of the methodology are the use of a solvent system consisting of methanol/dichloromethane (1:1 v/v) containing 5-mM lithium triflate, a stainless steel electrode having a relatively large surface area, and a microscale electrospray nozzle that provides for stable electrospray at flow rates in the range of 100–500 nL/min. A linear response for the absolute signal intensity of the protonated molecule was observed for a number of derivatives over the concentration range of 50–1000 fmol/µL. For all except the arginine derivative, there was a decrease in the signal intensity with increasing flow rate with 100–300 nL/min being optimum. Collision induced dissociation (CID) product ion spectra were obtained for 21 derivatives including carboxymethyl cysteine and dehydrothreonine. Leucine and isoleucine can be distinquished on the basis of their CID product ion spectra. A subfemtomole detection limit was demonstrated for the phenylalanine PTH derivative in a selected reaction monitoring (SRM) experiment. Samples from an automated Edman microsequencer run have been analyzed using the new technique and compared to results obtained by conventional high-performance liquid chromatography analysis with UV detection. This work demonstrates the feasibility of using mass spectrometry to identify and quantitate the products generated by automated protein microsequencing using standard Edman degradation chemistry.     

On-line CE-MS using pressurized liquid junction nanoflow electrospray interface and surface-coated capillaries

A simple and cost-effective laboratory-made liquid junction interface was used for coupling of CE with MS. In this device the capillary column and the spray tip were positioned in the electrode vessel containing appropriate spray liquid. The electrospray potential was applied on the electrode inside the liquid junction. A stable electrospray was produced at nanoliter per minute flow rates generated in the emitter tip without using an external pump. This arrangement provided high durability of the spray tip and independent optimization of the CE separation (use of coated capillaries) and ESI conditions. CE-MS analysis of mixtures of drugs, peptides, tryptic digests of proteins and biological fluids was optimized with respect to the effects of the distance between the separation capillary and electrospray tip and pressure applied on the liquid junction. The sensitivity of the system, in terms of the LOD (base peak monitoring) was below 10 ng/mL for the beta-blocker drugs and below 200 ng/mL for peptide analysis.     

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.     

A high-pressure interconnect for chemical microsystem applications

A PEEK interface for use in microfluidic applications is designed, fabricated and tested. The interface allows for the facile, non-permanent coupling of standard capillary tubing to silicon/glass micromixer chips. Importantly, the interface provides for a secure connection between capillary lines and chip reservoirs without the need for any adhesive materials. Furthermore, when used in conjunction with silicon/glass micromixer chips fluidic transport is stable over a wide range of volumetric flow rates (1-1500 microL min(-1)), and the entire construct can be rapidly assembled and disassembled at any time during the course of experimentation.     

Flow-rate characterization of microfabricated polymer microspray emitters

Microfabricated polymer microspray emitters are characterized in terms of applicable flow rates, temporal and spectral signal-to-noise ratios (SNRs), and solution composition. First, microspray emitters can be operated with 50% methanol/49% water/1% acetic acid from 250 nL/min up to 7 microL/min, with better SNRs above 1 microL/min. Interestingly, even at the lowest flow rates tested, they compare well with nanospray capillaries in terms of mass spectral performances. Secondly, they can be operated with acetonitrile from 10 up to 99% (v/v), with flow rates from 250 nL/min up to 4 microL/min. Even if the mass spectral performances (especially the spectral SNRs) vary with the acetonitrile content, this study validates such microfabricated microspray emitters as interfaces between liquid-phase separations and electrospray mass spectrometers.     

Development of an ultra-low volume flow cell for surface plasmon resonance detection in a miniaturized capillary electrophoresis system

A miniaturized capillary electrophoresis system coupled to a surface plasmon resonance (SPR) sensor on a microfluidic platform fabricated from PDMS is detailed. A previously described split-flow injection technique is first utilized to manipulate sample into the microfluidic chip, followed by separation within the fused-silica capillary and final off-capillary detection of analytes via SPR. Instead of using commercial SPR flow cells requiring relatively large detection volumes, samples of less than 1 nL volume are utilized. The interface between the CE system and SPR sensor made it possible to detect minute volumes of sample with minimal dispersion. The flow cell has the potential to be applicable to miniaturized flow-injection (FI) systems where submicroliter volumes of sample are frequently only available for analysis. The components present in solution, but not bound to the sensor surface, were also investigated. The sensitivity of the CE-SPR system was similar to that found in UV-spectrometric instruments and nonchromophoric components could also be measured.     

Negative mode sheathless capillary electrophoresis electrospray ionization-mass spectrometry for metabolite analysis of prokaryotes

Capillary electrophoresis (CE) was coupled to negative mode electrospray ionisation-mass spectrometry (MS) for separation and detection of phosphorylated and acidic metabolites in extracts of prokaryotes. Unlike previous CE-MS systems for metabolite analysis, a sheathless interface was used to improve sensitivity. To accomplish this, the separation capillary was modified by creating a porous junction near the outlet where the electrospray voltage and cathodic voltage for CE were applied. The outlet of the capillary was pulled to a 5 microm inner diameter to form an electrospray emitter and had a frit fabricated near the exit to prevent clogging. During analysis pressure was applied at the inlet of the separation column to create sufficient flow towards the detector. Limits of detection for 19 metabolites in full scan mode ranged from 20 nM for ADP ribose to 2.5 microM for alpha-ketoglutarate for 40 nL injections. Extracts of Escherichia coli, strain DH5-alpha, were analyzed using this system. In full scan mode, 118 different metabolites were detected. Tandem mass spectrometry was also employed to attempt identification. Reproducible fragmentation of 19 parent peaks was found and 10 of these produced spectra that were consistent with identification obtained from matching to compounds in the MetaCyc database. These results demonstrate the utility of a sensitive CE-MS system for large scale metabolite detection in biological samples.     

Picoelectrospray Ionization Mass Spectrometry Using Narrow-Bore Chemically Etched Emitters

Electrospray ionization mass spectrometry (ESI-MS) at flow rates below ~10 nL/min has been only sporadically explored because of difficulty in reproducibly fabricating emitters that can operate at lower flow rates. Here we demonstrate narrow orifice chemically etched emitters for stable electrospray at flow rates as low as 400 pL/min. Depending on the analyte concentration, we observe two types of MS signal response as a function of flow rate. At low concentrations, an optimum flow rate is observed slightly above 1 nL/min, whereas the signal decreases monotonically with decreasing flow rates at higher concentrations. For example, consumption of 500 zmol of sample yielded signal-to-noise ratios ~10 for some peptides. In spite of lower MS signal, the ion utilization efficiency increases exponentially with decreasing flow rate in all cases. Significant variations in ionization efficiency were observed within this flow rate range for an equimolar mixture of peptide, indicating that ionization efficiency is an analyte-dependent characteristic for the present experimental conditions. Mass-limited samples benefit strongly from the use of low flow rates and avoiding unnecessary sample dilution. These findings have important implications for the analysis of trace biological samples.

Comparison of sheathless and sheath-flow electrospray interfaces for the capillary electrophoresis-electrospray ionization-mass spectrometry analysis of peptides

Capillary electrophoresis coupled to mass spectrometry via an electrospray interface provides a powerful system for separation and characterization of a high number of biomolecules. The present paper describes a home-made sheathless interface and compares it with a commercial sheath-flow interface, using a separation method based on a peptide hormone mixture of therapeutic interest. In a previous work, we optimized the parameters involved in a sheath-flow interface and obtained good results in sensitivity and reproducibility. The sheathless interface is performed with a graphite-coated electrospray ionisation (ESI) tip attached to the separation capillary. We demonstrate that electrolyte composition is the main parameter affecting signal sensitivity and separation resolution. The effect of the nature and concentration of the organic solvent added to the separation electrolyte is carefully studied. Furthermore, a general comparison of both interfaces is made in terms of separation, reproducibility, and sensitivity obtained under the optimized conditions described. Advantages and disadvantages of both coupling setups have been evaluated.     

Preparation of Durable Emitter of Electrospray Mass Spectrometry by Covalently Coating the Fused‐Silica Capillary Tip with Carbon‐Nanotube Sol‐Gel Composite Material

A simple approach for the preparation of emitter of electrospray ionization mass spectrometry by covalently coating the fused‐silica capillary tip with the conductive carbon‐nanotube sol‐gel composite material (CNTSCM) is described. The CNTSCM was prepared by dispersing single‐walled carbon nanotubes in the sol composed of a mixture of 3‐glycidoxypropyltrimethoxysilane and 3‐aminopropyltrimethoxysilane, and ethanol. The long‐term stability of the prepared ESI emitters was demonstrated by at least 180 h of continuous use. Signal intensity obtained by the prepared emitter was mass‐flux sensitive when the flow rate was lower than 500 nL/min, while the signal intensity performed a concentration dependence when the flow rate was in the range of 500–800 nL/min. The usefulness of such a prepared emitter was demonstrated by the analysis of various types of samples such as organic small molecular drugs, oligosaccharide, peptide, and protein.     

Microchip capillary electrophoresis–electrospray ionization–mass spectrometry of intact proteins using uncoated Ormocomp microchips

We present rapid (<5 min) and efficient intact protein analysis by mass spectrometry (MS) using fully microfabricated and monolithically integrated capillary electrophoresis–electrospray ionization (CE–ESI) microchips. The microchips are fabricated fully of commercial inorganic–organic hybrid material, Ormocomp, by UV-embossing and adhesive Ormocomp–Ormocomp bonding (CE microchannels). A sheath-flow ESI interface is monolithically integrated with the UV-embossed separation channels by cutting a rectangular emitter tip in the end with a dicing saw. As a result, electrospray was produced from the corner of chip with good reproducibility between parallel tips (stability within 3.8–9.2% RSD). Thanks to its inherent biocompatibility and stable (negative) surface charge, Ormocomp microchips enable efficient intact protein analysis with up to ∼10⁴ theoretical separation plates per meter without any chemical or physical surface modification before analysis. The same microchip setup is also feasible for rapid peptide sequencing and mass fingerprinting and shows excellent migration time repeatability from run to run for both peptides (5.6–5.9% RSD, n = 4) and intact proteins (1.3–7.5% RSD, n = 3). Thus, the Ormocomp microchips provide a versatile new tool for MS-based proteomics. Particularly, the feasibility of the Ormocomp chips for rapid analysis of intact proteins with such a simple setup is a valuable increment to the current technology.     

脉冲电喷雾现象的初步研究

生物质谱方法的发展得益于生物质谱技术的发展,众所周知,电喷雾与基体辅助激光解吸技术的发展,极大地改善了生物质谱的分析性能,对于复杂生命体系的研究,现有的生物质谱面临着新的挑战,复杂体系要求以很少量的样品（nL-μL级）得到功能蛋白质组的许多信息,如各蛋白质的分子量、肽谱、氨基酸序列以及各类转译后修饰结构等,而现有的生物质谱大多要求足够大的样品量（1－10μL）,因此对低流量样品的高通量信息需求是生物质谱分析领域中的难题。