Electrospray ionization mass spectrometry from discrete nanoliter‐sized sample volumes

We describe a method for nanoelectrospray ionization mass spectrometry (nESI‐MS) of very small sample volumes. Nanoliter‐sized sample droplets were taken up by suction into a nanoelectrospray needle from a silicon microchip prior to ESI. To avoid a rapid evaporation of the small sample volumes, all manipulation steps were performed under a cover of fluorocarbon liquid. Sample volumes down to 1.5 nL were successfully analyzed, and an absolute limit of detection of 105 attomole of insulin (chain B, oxidized) was obtained. The open access to the sample droplets on the silicon chip provides the possibility to add reagents to the sample droplets and perform chemical reactions under an extended period of time. This was demonstrated in an example where we performed a tryptic digestion of cytochrome C in a nanoliter‐sized sample volume for 2.5 h, followed by monitoring the outcome of the reaction with nESI‐MS. The technology was also utilized for tandem mass spectrometry (MS/MS) sequencing analysis of a 2 nL solution of angiotensin I. Copyright © 2010 John Wiley & Sons, Ltd.     

Online nanoelectrospray/high‐field asymmetric waveform ion mobility spectrometry as a potential tool for discovery pharmaceutical bioanalysis

Nanoelectrospray ionization (nESI) coupled online with high‐field asymmetric waveform ion mobility spectrometry (FAIMS) for small molecule analysis in a discovery pharmaceutical setting was examined. A conventional capillary pump, autosampler and nESI source were used to introduce samples directly into the FAIMS device. The FAIMS device was used to separate gas‐phase ions on a timescale that was compatible with the mass spectrometer. The capability of the nESI‐FAIMS combination to efficiently remove metabolite interferences from the parent drug, and reduce ion suppression effects, was demonstrated. On average, 85% of the signal intensity obtained from a neat sample was preserved in the extracted plasma samples. Standard curves were prepared for several compounds. Linearity was obtained over approximately 3 to 4 orders of magnitude. Comparison of results from nESI‐FAIMS with those from conventional LC/MS for a mouse pharmacokinetic study yielded concentration values differing by no more than 30%. Copyright © 2009 John Wiley & Sons, Ltd.     

伴刀豆球蛋白亲和色谱柱的制备及其在糖蛋白核糖核酸酶B结构分析中的应用

通过对硅胶基质进行化学改性键合伴刀豆球蛋白(Con A),制备了对糖蛋白具有特异亲和作用的亲和色谱固定相;该固定相非特异性吸附弱,对于糖蛋白和糖肽的分离效果良好。对亲和色谱的分离条件进行了优化,以标准糖蛋白核糖核酸酶B(RNase B)为模型,对其进行了纯化;用糖苷酶切除糖链,并对切除糖链前后的RNase B用胰蛋白酶酶解;用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对亲和色谱分离得到的糖蛋白、糖链及糖肽进行了分析,确定了RNase B的一级结构、糖含量、糖基化位点及糖连接方式。该方法快速准确,适于糖蛋白和糖肽的分离表征。将其应用于血清中糖蛋白及酶解后血清中糖肽的分离富集,取得了很好的效果。     

Rapid analysis of atorvastatin calcium using capillary electrophoresis and microchip electrophoresis

In this work, a capillary electrophoretic method for the rapid quantitation of atorvastatin (AT) in a lipitor tablet was investigated and developed. Method development included studies of the effect of applied potential, buffer concentration, buffer pH, and hydrodynamic injection time on the electrophoretic separation. The method was validated with regard to linearity, precision, specificity, LOD, and LOQ. The optimum electrophoretic separation conditions were 25 mM sodium acetate buffer at pH 6, with a separation voltage of 25 kV using a 50 microm capillary of 33 cm total length. Sodium diclofenac was used as an internal standard. Analysis of AT in a commercial lipitor tablet by electrophoresis gave quite high efficiency, coupled with an analysis time of less than 1.2 min in comparison to LC. Once the separation was optimized on capillary, it was further miniaturized to a microchip platform, with linear imaging UV detection using microchip electrophoresis (MCE). Linear imaging UV detection allowed for real-time monitoring of the analyte movement on chip, so that the optimum separation time could be easily determined. This microchip electrophoretic method was compared to the CE method with regard to speed, efficiency, precision, and LOD. This work represents the most rapid and first reported analysis of AT using MCE.     

Capillary liquid chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry

A miniaturized nebulizer chip for capillary liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (capillary LC-microchip APCI-MS) is presented. The APCI chip consists of two wafers, a silicon wafer and a Pyrex glass wafer. The silicon wafer has a DRIE etched through-wafer nebulizer gas inlet, an edge capillary insertion channel, a stopper, a vaporizer channel and a nozzle. The platinum heater electrode and pads for electrical connection were patterned on to the Pyrex glass wafer. The two wafers were joined by anodic bonding, creating a microchip version of an APCI-source. The sample inlet capillary from an LC column is directly connected to the vaporizer channel of the APCI chip. The etched nozzle in the microchip forms a narrow sample plume, which is ionized by an external corona needle, and the formed ions are analyzed by a mass spectrometer. The nebulizer chip enables for the first time the use of low flow rate separation techniques with APCI-MS. The performance of capillary LC-microchip APCI-MS was tested with selected neurosteroids. The capillary LC-microchip APCI-MS provides quantitative repeatability and good linearity. The limits of detection (LOD) with a signal-to-noise ratio (S/N) of 3 in MS/MS mode for the selected neurosteroids were 20-1000 fmol (10-500 nmol l(-1)). LODs (S/N = 3) with commercial macro APCI with the same compounds using the same MS were about 10 times higher. Fast heat transfer allows the use of the optimized temperature for each compound during an LC run. The microchip APCI-source provides a convenient and easy method to combine capillary LC to any API-MS equipped with an APCI source. The advantages and potentials of the microchip APCI also make it a very attractive interface in microfluidic APCI-MS.     

Some possibilities of an analysis of complex samples by a mass spectrometry with a sample pretreatment by an offline coupled preparative capillary isotachophoresis

This work deals with an analysis of biologically important compounds in complex matrices using preparative isotachophoresis (pITP) in column coupling configuration as a sample pretreatment technique followed by a direct infusion mass spectrometry with nano‐electrospray ionization (DI‐nESI‐MS). Busereline was chosen as a model analyte, and urine was chosen as an example of complex matrix. In pITP experiments, sodium cation (10 mmol/L concentration) was used as a leading ion and β‐alanine as terminating ion (20 mmol/L concentration). The fractions, obtained by pITP pre‐separation with the assistance of the mixture of discrete spacers, were finally analyzed by DI‐nESI‐MS. It was shown that pITP performed before DI‐nESI‐MS analysis can significantly simplify complex matrix, and, due to its concentration power, pITP can consequently decrease the concentration limit of detection. The concentration of buserelin in the urine samples analyzed by pITP‐DI‐nESI‐MS was 10 μg/L (reflecting at a 8.10^?9 mol/L concentration) in our work but from the ion intensities obtained in MS as well as MS/MS analyses, it is clear that this concentration level could be several orders of magnitude lower for reliable detection and identification of buserelin in urine analyzed using pITP with DI‐nESI‐MS detection.     

A New Approach to High Sensitivity Liquid Chromatography-Mass Spectrometry of Peptides using Nanoflow Solvent Assisted Inlet Ionization

Liquid chromatography (LC) solvent assisted inlet ionization (SAII) mass spectrometry (MS) was previously reported to give good chromatographic resolution and MS detection injecting 66 ng of a BSA tryptic digest. In analogy to nano-electrospray ionization (nESI), we extend SAII LC/MS to nano-SAII (nSAII) operating at nL min^–1 flow rates and demonstrate good quality ion chromatograms and mass spectra from injection of as little as 0.7 ng of BSA digest onto a capillary LC column. Data dependent fragmentation is demonstrated for injection of 7 ng of a BSA digest. This method has advantages over nESI in ease of use and low cost as it requires no voltage and is operational without the necessity of connectors or fragile nESI emitters, although similar constricted tips can be helpful in nSAII to stabilize the signal at low nanoliter flow. At a flow rate of 0.8 μL min^–1, the only requirement for nSAII is that the exit-end of the capillary LC column be adjusted near the aperture of the heated inlet of the mass spectrometer.     

基于Endo-M N175Q糖链转移反应与丙酮富集糖肽的LC-MS分析糖蛋白N-糖链

基于化学酶标记和丙酮富集糖肽方法,建立了一种可靠、有效、简单的糖蛋白N-糖链分析方法。以唾液酸糖肽(SGP)为模型糖肽,比较了样品中丙酮加入量对SGP的富集效果,最终选择加入样品体积5倍量的丙酮。用丙酮富集经胰蛋白处理的核糖核酸酶B(RNase B)酶解液中的糖肽,以富集分离得到的糖肽(糖基供体)和PDPZ-Boc-Asn-GlcNAc(糖基受体)作为酶反应底物,进行Endo-M N175Q的转糖基反应,得到PDPZBoc-Asn-GlcNAc-N-糖链标记物。采用YMC C18色谱柱为分析柱,10 mmol/L甲酸铵-乙腈为流动相梯度洗脱,经液相色谱-串联质谱(LC-MS)检测得到5种高甘露糖型糖链。结果表明,丙酮可有效地富集大量肽和少量糖肽混合溶液中的糖肽,Endo-M N175Q可将天然糖肽的糖链转移到PDPZ-Boc-Asn-GlcNAc受体上。将该方法应用于胎球蛋白N-糖链分析,检测到5种复杂型N-糖链。该研究为各种糖蛋白N-糖链检测提供了新的分析方法。     

集成化液膜萃取-反萃取-毛细管电泳联用芯片

我们设计并制作了集成有支持液膜萃取-反萃取试样预处理的毛细管电泳(SLMEBE-CCE)微流控芯片. 分别以荧光素钠和丁基罗丹明B作为模型待测物和共存物, 在该芯片上进行了在线试样预处理与毛细管电泳联用的初步实验.     

On-line selective enrichment and ion-pair reaction for structural determination of sulfated glycopeptides by capillary electrophoresis-mass spectrometry

We describe a new capillary electrophoresis-mass spectrometry (CE-MS)-based technique for analyzing sulfated glycopeptides. The proposed method performs selective enrichment of sulfated glycopeptides from a complex mixture of peptides based on field-enhanced sample injection and ion-pair reaction with a basic ion-pair reagent (Lys-Lys-Lys; KKK) at the exit end of a capillary in a single analysis, which permits successful fragmentation of sulfated glycopeptides in positive-ion mode at the MS/MS stage for comprehensive structural analysis. In this study, the method was verified using a model sulfated monosaccharide, N-acetyl-d-galactosamine 4-sulfate (GalNAc 4S). As an example of an application of this method, sulfated glycopeptides were selectively enriched from the enzymatic digest of thyroid stimulating hormone, affording approximately 500-fold sensitivity enhancement, and structural information was successfully obtained via on-line ion-pair complexation reaction.     

Detection of Copper Ion with Laser-Induced Fluorescence in a Capillary Electrophoresis Microchip

A capillary electrophoresis microchip coupled with a confocal laser-induced fluorescence (LIF) detector was successfully constructed for the analysis of trace amounts of heavy metals in environmental sources. A new fluorescence dye, RBPhOH, synthesized from rhodamine B, was utilized in a glass microchip to selectively determine copper with high sensitivity. A series of factors including running buffer concentration, detection voltage, and sample loading time were optimized for maximum LIF detector response and, hence, method sensitivity.     

Capillary zone electrophoresis of glycopeptides under controlled electroosmotic flow conditions coupled to electrospray and matrix-assisted laser desorption/ionization mass spectrometry

Defined conditions of EOF along with different pH values of the BGE were compared for the purpose of analyzing glycopetides by CZE coupled to MS (CZE-MS). Hyphenation to MS involved ESI as well as MALDI, and single-stage and multistage MS were applied. Variation of the EOF was accomplished by selecting appropriate coatings for the capillary, namely hexadimethrine bromide (HDMB) and HDMB/dextran sulfate. A high and reproducible anodic and cathodic EOF, respectively, was obtained in both approaches, allowing the detection of analytes with net positive as well as negative charge in one single run. Thus, a fast and sensitive determination of the glycopeptides in a tryptic digest of antithrombin, chosen as a test sample, was achieved. Ionization suppression effects, a phenomenon typically observed with glycopeptides in MS analysis, were minimized thanks to separation from other peptides present. The high stability of the coatings permitted the generation of mass spectra without interfering peaks originating from the coating polymers. The high EOF generated by the coatings facilitated the maintenance of a stable spray when coupling to ESI-MS, and a stable CZE current when working with a sheath flow-assisted analyte deposition onto MALDI targets, respectively. In conclusion, CZE-MS could be demonstrated as a robust complementary method to capillary RP-HPLC-MS in combination with both soft-ionization techniques, ESI and MALDI, generally, and particularly in the context of glycopeptide analysis.     

Evaluation of analytical performance and reliability of direct nanoLC‐nanoESI‐high resolution mass spectrometry for profiling the (xeno)metabolome

Mass spectrometry (MS) profiling techniques are used for analysing metabolites and xenobiotics in biofluids; however, detection of low abundance compounds using conventional MS techniques is poor. To counter this, nanoflow ultra‐high‐pressure liquid chromatography‐nanoelectrospray ionization‐time‐of‐flight MS (nUHPLC‐nESI‐TOFMS), which has been used primarily for proteomics, offers an innovative prospect for profiling small molecules. Compared to conventional UHPLC‐ESI‐TOFMS, nUHPLC‐nESI‐TOFMS enhanced detection limits of a variety of (xeno)metabolites by between 2 and 2000‐fold. In addition, this study demonstrates for the first time excellent repeatability and reproducibility for analysis of urine and plasma samples using nUHPLC‐nESI‐TOFMS, supporting implementation of this platform as a novel approach for high‐throughput (xeno)metabolomics. Copyright © 2014 John Wiley & Sons, Ltd.     

Tube radial distribution phenomenon observed in an aqueous micellar solution of non-ionic surfactant fed into a microspace and an attempt of capillary chromatographic application

A new type of tube radial distribution phenomenon was observed in an aqueous micellar solution of non-ionic surfactant that was fed into a microspace. A homogeneous aqueous solution containing 2 wt % Triton X-100 and 2.0 M sodium chloride was fed into a microchannel (40 μm in depth and 200 μm in width) in a microchip at a flow rate of 4.0 μL/min, where the microchip was maintained at a temperature of 34°C. The homogeneous aqueous solution changed to a heterogeneous solution with two phases in the microchannel; the surfactant-rich phase was generated around the middle of the channel, while the aqueous phase containing little surfactant was formed near the wall. The radial distribution of the surfactant was observed through Rhodamine B dissolved in the aqueous micellar solution with a bright-field microscope — CCD camera system. An open-tubular capillary chromatographic system was also tried to develop using the fusedsilica capillary tube (75 μm inner diameter and 120 cm length) as a separation column and the aqueous micellar solution as a carrier.     

Coupling a microchip with electrospray ionization quadrupole time-of-flight mass spectrometer for peptide separation and identification

The present study reports a simple method of coupling a glass microchip to an electrospray ionization (ESI) quadrupole time-of-flight mass spectrometer (QTOF-MS) for separation and identification of peptides. A sheath-flow electrospray interface was constructed based on attaching a short fused-silica capillary to the microchip. The dead volume at the interface was effectively reduced by wet etching an approximate flat-bottom capillary insertion channel coaxial to the end of separation microchannel and using a wire-controlled epoxy-blocking attachment method. The makeup liquid and neb gas were coaxially pumped through two stainless-steel tees to maintain a stable and efficient electrospray. The coupled microchip/ESI-QTOF-MS system was successfully used to carry out electrophoresis separation of peptides and ESI-QTOF-MS identification.     

Analysis of phospholipase A2 glycosylation patterns from venom of individual bees by capillary electrophoresis/electrospray ionization mass spectrometry using an ion trap mass spectrometer

A method based on tryptic digestion, ultrafiltration and capillary electrophoresis/mass spectrometry (CE/MS) has been developed for the analysis of the glycosylation pattern in the phospholipase A2 (PLA) of individual honeybees. Without reducing the disulfide bonds, PLA was digested with trypsin and filtered with a 3 kDa molecular weight (MW) cut-off membrane. With this procedure, the glycopeptides could be isolated from the nonglycosylated peptides. After tryptic digestion and ultrafiltration, the disulfide bonds were reduced before analysis by CE. To reduce the adsorption, CE separation was performed on successive multiple ionic-polymer (SMIL) polybrene (PB) coated capillary columns. The SMIL-PB columns allowed partial separation of the glycopeptides and eight glycopeptides were identified by on-line coupling of CE with electrospray ionization (ESI) mass spectrometry. The analysis of phospholipase A2 from the venom of individual bees indicated that the variation and relative abundances of different glycopeptides were similar between the younger and the older bees.     

Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.     

Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS

Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-specific characterisation of densely glycosylated mucin-type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region containing a range of O-linked, tumour-associated carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing number of tandem repeats were studied. In addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously determined, while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterisation of mucin-type glycopeptides containing high-density O-linked glycan clusters, using accessible and relative low-resolution/low-mass accuracy IT MS instrumentation.     

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.     

蛋白质/多肽的微流控二维芯片电泳*

在微流控芯片上构建多维分离系统,为蛋白质组学研究提供了一个有发展前景的高效分离分析技术平台。本文介绍了二维芯片电泳系统耦联模式选取及正交性评价的方法;综述了针对蛋白质/多肽分离分析的各种耦联模式微流控二维芯片电泳分析系统,如胶束电动力学色谱（MEKC）与毛细管区带电泳（CZE）,开管电色谱（OECE）与CZE,等电聚焦（IEF）与CZE, IEF与SDS毛细管凝胶电泳（CGE）, SDS-CGE与MEKC等。特别对二维电泳芯片切换接口的类型进行了分类,探讨了用于微流控二维芯片电泳系统的检测技术,并展望了微流控二维电泳芯片在蛋白质组学研究中的应用前景和发展方向。