A simple sheathless CE-MS interface with a sub-micrometer electrical contact fracture for sensitive analysis of peptide and protein samples

Online coupling of capillary electrophoresis (CE) to electrospray ionization mass spectrometry (MS) has shown considerable potential, however, technical challenges have limited its use. In this study, we have developed a simple and sensitive sheathless CE-MS interface based on the novel concept of forming a sub-micrometer fracture directly in the capillary. The simple interface design allowed the generation of a stable ESI spray capable of ionization at low nanoliter flow-rates (45–90 nL/min) for high sensitivity MS analysis of challenging samples like those containing proteins and peptides. By analysis of a model peptide (leucine enkephalin), a limit of detection (LOD) of 0.045 pmol/μL (corresponding to 67 attomol in a sample volume of ∼15 nL) was obtained. The merit of the CE-MS approach was demonstrated by analysis of bovine serum albumin (BSA) tryptic peptides. A well-resolved separation profile was achieved and comparable sequence coverage was obtained by the CE-MS method (73%) compared to a representative UPLC-MS method (77%). The CE-MS interface was subsequently used to analyse a more complex sample of pharmaceutically relevant human proteins including insulin, tissue factor and α-synuclein. Efficient separation and protein ESI mass spectra of adequate quality could be achieved using only a small amount of sample (30 fmol). In addition, analysis of ubiquitin samples under both native and denatured conditions, indicate that the CE-MS setup can facilitate native MS applications to probe the conformational properties of proteins. Thus, the described CE-MS setup should be useful for a wide range of high-sensitivity applications in protein research.     

Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.     

Capillary electrophoresis coupled to mass spectrometer for automated and robust polypeptide determination in body fluids for clinical use

We describe the application of capillary electrophoresis (CE) coupled on-line to an electrospray ionization-time of flight-mass spectrometer (ESI-TOF-MS) to the analysis of human urine and serum for the identification of biomarkers for clinical diagnostics. CE-MS led to display > 1000 polypeptides present in complex biological samples within 45-60 min in a single analysis run. To extract the information of the CE-MS spectra in a timely fashion, a software was designed to automatically deconvolute and normalize the spectra. Both urine and serum contain several hundred polypeptides in samples from healthy individuals. Hence, it is possible to establish typical "normal urine" or "normal serum" polypeptide patterns. Samples from patients with different diseases display polypeptide patterns that differ significantly from those obtained from healthy individuals. Examining series of patients with the same disease allowed the establishment of polypeptide patterns typical for specific diseases. This permits the search for marker peptides specific for diseases. The data indicate that a single polypeptide present in all patients with the same disease, but absent in all healthy control individuals does not exist. The combination of several polypeptides found in either urine or serum or both are forming a specific pattern, which is indicative not only for the particular disease, but also for the stage of disease. CE-MS detects many polypeptides in single samples and the application of the software to the search of identical polypeptides excreted in urine allows the unbiased diagnosis based on a pattern and does not rely on single disease markers.     

Analyses of quaternary ammonium drugs in horse urine by capillary electrophoresis-mass spectrometry

A capillary electrophoresis-mass spectrometry (CE-MS) method for the analysis of quaternary ammonium drugs in equine urine was developed. Quaternary ammonium drugs were first extracted from equine urine by ion-pair extraction and then analysed by CE-MS in the positive electrospray ionization (ESI) mode. Within 12 min, eight quaternary ammonium drugs, each at 1 ng/mL in horse urine, could be detected. The confirmation of these drugs in urine samples was achieved by capillary electrophoresis tandem mass spectrometry (CE-MS/MS). A direct comparison of this method was made with existing liquid chromatography/mass spectrometry (LC-MS) methods in the detection and confirmation of glycopyrrolate and ipratropium bromide in horse urine. While the two drugs could be detected within the same CE-MS run at 1 ng/mL in urine, they could only be detected in separate LC-MS runs at 5 ng/mL in urine. In addition, CE-MS consumed a much smaller volume of extract; the analyte peak widths, in some cases, were much narrower; and as the quaternary ammonium ions were well separated electrophoretically from the mainly neutral urine matrix, a much cleaner background in the CE-MS total ion trace was observed.     

Classification of gasoline data obtained by gas chromatography using a piecewise alignment algorithm combined with feature selection and principal component analysis

A fast and objective chemometric classification method is developed and applied to the analysis of gas chromatography (GC) data from five commercial gasoline samples. The gasoline samples serve as model mixtures, whereas the focus is on the development and demonstration of the classification method. The method is based on objective retention time alignment (referred to as piecewise alignment) coupled with analysis of variance (ANOVA) feature selection prior to classification by principal component analysis (PCA) using optimal parameters. The degree-of-class-separation is used as a metric to objectively optimize the alignment and feature selection parameters using a suitable training set thereby reducing user subjectivity, as well as to indicate the success of the PCA clustering and classification. The degree-of-class-separation is calculated using Euclidean distances between the PCA scores of a subset of the replicate runs from two of the five fuel types, i.e., the training set. The unaligned training set that was directly submitted to PCA had a low degree-of-class-separation (0.4), and the PCA scores plot for the raw training set combined with the raw test set failed to correctly cluster the five sample types. After submitting the training set to piecewise alignment, the degree-of-class-separation increased (1.2), but when the same alignment parameters were applied to the training set combined with the test set, the scores plot clustering still did not yield five distinct groups. Applying feature selection to the unaligned training set increased the degree-of-class-separation (4.8), but chemical variations were still obscured by retention time variation and when the same feature selection conditions were used for the training set combined with the test set, only one of the five fuels was clustered correctly. However, piecewise alignment coupled with feature selection yielded a reasonably optimal degree-of-class-separation for the training set (9.2), and when the same alignment and ANOVA parameters were applied to the training set combined with the test set, the PCA scores plot correctly classified the gasoline fingerprints into five distinct clusters.     

Simulation and optimization of peptide separation by capillary electrophoresis-mass spectrometry

The potential of capillary electrophoresis (CE) for the separation of peptides has been extensively demonstrated in the last decade. Their correct characterization and sequenciation is a difficult task that can be accomplished using CE-mass spectrometry (CE-MS). An important limitation of CE-MS is the buffer choice since it should provide an adequate CE separation without ruining the MS signal. In this work, a new strategy is used to help to solve this limitation based on the combination of two different methodologies. Namely, an ab initio semiempirical model that relates electrophoretic behavior of peptides to their sequence is first used to obtain in a fast and easy way adequate CE buffers compatible with MS analysis. Next, CE-MS is used to separate and characterize peptides via the determination of their relative molecular masses. The usefulness of this procedure is demonstrated analyzing in a single CE-MS run a group of 10 standard peptides of very different nature (i.e., relative molecular masses ranging from 132 to 1037 and isoelectric points ranging from 5.69 to 10.62). It is concluded that the use of this strategy can help to overcome the buffer limitation in CE-MS.     

Bottom-up proteomics of envelope proteins extracted from spinach chloroplast via high organic content CE-MS

A high organic content CE-MS/MS (HOCE-MS/MS) method was developed for the proteomic analysis of envelope proteins extracted from spinach leaves. Separation was performed in a 1-m long hydroxypropyl cellulose coated capillary, using 8% (v/v) formic acid in 70% (v/v) methanol and 22% water as the BGE. A flow-through microvial interface was used to couple the CE system with an Orbitrap Fusion Lumos mass spectrometer, and field-amplified sample stacking was used to improve the concentration sensitivity. Using this optimized method, 3579 peptides and 1141 proteins were identified using the Proteome Discoverer software with a 1% false discovery rate at the protein level. Relative to conventional aqueous CE, HOCE-MS did a better job of discovering hydrophobic peptides and provided more peptide and protein identifications. Relative to nano-LC-MS, it achieved comparable peptide and protein identification performance and detected peptides not identified by LC-MS: of the full set of peptides identified using the two techniques, 19% were identified only using HOCE-MS. It also outperformed nano-LC-MS with respect to the detection of low molecular weight peptides.     

多模型共识偏最小二乘法用于近红外光谱定量分析

将多模型共识偏最小二乘法用于近红外光谱定量分析。利用随机抽取的训练子集建立一系列偏最小二乘模型,选取其中性能较好的部分模型作为成员模型,用这些成员模型来预测未知样品。将该方法用于一组生物样本的近红外光谱与样品中人血清白蛋白、γ-球蛋白以及葡萄糖含量之间的建模研究,并与单模型偏最小二乘法了进行比较。结果 PLS对独立测试集中三种组分进行50次重复预测的平均RMSEP分别为0.1066,0.0853和0.1338,RMSEP的标准偏差分别为0.0174,0.0144和0.0416;而本方法重复预测的平均RMSEP分别为0.0715,0.0750和0.0781,RMSEP的标准偏差分别为0.0033,0.2729×10-4和0.0025。     

Screening and analytical confirmation of sulfonamide residues in milk by capillary electrophoresis-mass spectrometry

A new methodology is proposed to automate the monitoring of sulfonamide residues in milk samples. It combines a screening unit for the total amount of sulfonamide with capillary electrophoresis-mass spectrometry (CE-MS) equipment for processing the samples containing a detectable level of sulfonamide. The screening unit consists of continuous-flow system (CFS) to precipitate the proteins connected on-line to the CE-MS equipment, in which a common characteristic ion of all sulfonamides was monitored with the MS detector by flushing the sample through the capillary. The confirmatory method is based on the purification and preconcentration of sulfonamides in a CFS unit and posterior analysis by CE-MS. The sample treatment unit was also on-line connected to the CE-MS equipment. In order to increase sensitivity, the flow rate of the sheath liquid was diminished from 0.5 to 0.2 microL.min(-1) by increasing the content in water from 0 to 50% and the formic acid from 0.5 to 1.5% in this liquid and by applying an overimposed pressure of 5 mbar during the electrophoretic separation. The method allowed the analysis of 30 samples per hour.     

Topological shape and size of peptides: identification of potential allele specific helper T cell antigenic sites

A database of primary sequences of 28 immunogenic peptides, known to elicit T cell response, derived from five different haplotypes was compiled to identify allele specific helper T cell antigenic sites using a rule based graph-theoretical method. The prediction was based on the identification of allele specific patterns in the form of "topological shape and size" present in the peptides. Indices computed from weighted connected graph models of amino acid side chains and peptides were used in this purpose. The system was trained by 10 Ad and 10 non-Ad restricted peptide sequences, assigned actives and inactives, respectively, chosen randomly from the database, and four Ad and four non-Ad restricted sequences were kept as test peptides. This allowed the system to learn about "topological shape and size" specific for Ad restricted peptides from the differences, if any, they had with the inactive peptides in that respect. The system made 100% correct prediction for the training set peptides and misclassified only one inactive peptide of the test set. The system also identified crucial residues for lambda repressor 12-24 and insulin A-chains. This identification also shows that activity related/crucial residues could be located at varying distances from the peptide terminals. To our knowledge, the method is unique of its kind in the literature and may find application in the rational design of synthetic vaccines and other peptides of immunological importance.     

毛细管电泳-质谱联用技术及其在蛋白质组学中的应用

蛋白质组学研究在生物学、精准医学等方面发挥着重要的作用。然而研究面临的巨大挑战来自生物样品的复杂性,因此在质谱（MS）鉴定技术不断革新的同时,发展分离技术以降低样品复杂度尤为重要。毛细管电泳（CE）技术具有上样体积小、分离效率高、分离速度快等优势,其与质谱的联用在蛋白质组学研究中越来越受到关注。低流速鞘流液和无鞘流液接口的发展及商品化推动了CE-MS技术的发展。目前毛细管区带电泳（CZE）、毛细管等电聚焦（CIEF）、毛细管电色谱（CEC）等分离模式已与质谱联用,其中CZE-MS应用最广泛。目前被广泛采用的蛋白质组学研究策略主要是基于酶解肽段分离鉴定的"自下而上（bottom-up）"策略。首先,CE-MS技术对酶解肽段的检测灵敏度高达1 zmol,已成功应用于单细胞蛋白质组学;其次,毛细管电泳技术与反相液相色谱互补,为疏水性质相近的肽段（尤其是翻译后修饰肽段）的分离鉴定提供了新的途径。基于整体蛋白质分离鉴定的自上而下"top-down"策略可以直接获得更精准、更完整的蛋白质信息。CE技术在蛋白质大分子的分离方面具有分离效率高、回收率高的优势,其与质谱的联用提高了整体蛋白质的鉴定灵敏度和覆盖度。非变性质谱（native MS）是一种在近生理条件下从完整蛋白质复合物水平上进行分析的质谱技术。CE与非变性质谱联用已被尝试用于蛋白质复合体的分离鉴定。该文引用了与CE-MS和蛋白质组学应用相关的93篇文献,综述了以上介绍的CE-MS的研究进展以及在蛋白质组学分析中的应用优势,并总结和展望了其应用前景。     

Capillary electrophoresis-mass spectrometry: recent advances to the analysis of small achiral and chiral solutes

We describe here the state-of-the-art development of on-line capillary electrophoresis-mass spectrometry (CE-MS) over the last two years. Technological developments included are novel designs of new interfaces and ionization sources, new capillary coatings, buffers, and micelles as well as application of various modes of CE-MS published in the recent literature. The areas of CE-MS application in analysis of small achiral and chiral solutes are covered in sections that highlight the recent advances and possibilities of each mode of CE-MS. Application areas reviewed in this paper include achiral and chiral pharmaceuticals, agrochemicals, carbohydrates, and small peptides. The separation of enantiomers using micellar electrokinetic chromatography (MEKC)-MS with molecular micelles and capillary electrochromatography (CEC)-MS using pack tapered columns appears to provide good tolerance to electrospray stability for routine on-line CE-MS. These two modes seem to be very suitable for sensitive detection of chiral pharmaceuticals in biological samples, but their use will probably increase in the near future. Overall, it seems that one mode of CE-MS, in particular capillary zone electrophoresis (CZE)-MS, is now recognized as established technique for analysis of small charged solutes, but other modes, such as MEKC-MS and CEC-MS, are still within a period of development in terms of both MS-compatible pseudostationary phases and columns as well as applications.     

Determination of peptides and proteins in human urine with capillary electrophoresis-mass spectrometry, a suitable tool for the establishment of new diagnostic markers

The on-line coupling of capillary electrophoresis (CE) with electrospray-time-of-flight mass spectrometry (MS) has been used to obtain patterns of peptides and proteins present in the urine of healthy human individuals. This led to the establishment of a "normal urine polypeptide pattern", consisting of 247 polypeptides, each of which was found in more than 50% of healthy individuals. Applying CE-MS to the analysis of urine of patients with kidney disease revealed differences in polypeptide pattern. Twenty-seven polypeptides were exclusively found in samples of patients. Another 13, present in controls, were missing. These data indicate that CE-MS can be applied as powerful tool in clinical diagnostics.     

Simplifying capillary electrophoresis-mass spectrometry operation: eliminating capillary derivatization by using self-coating background electrolytes

To simplify capillary electrophoresis-mass spectrometry (CE-MS) operation, a background electrolyte (BGE) containing a polymer additive is introduced that allows the analysis of peptides and protein mixtures in underivatized fused-silica capillaries without any pretreatment, thereby increasing throughput. The most important characteristic of these polymer additives is that they do not significantly suppress the signals of the proteins and peptides under electrospray ionization, thereby allowing them to be used as an additive to common BGEs that are used for CE-MS analysis of peptide and protein mixtures. In addition, because the fused-silica capillary inner wall is continuously coated with the polymer additive, migration irreproducibility, due to the degradation of the capillary inner wall coating, under CE-MS is minimized. High sensitivity of detection, migration reproducibility, and ease of fabrication allow CE-MS analyses that require long analysis time, such as (CE-MS/MS)n, to be performed with ease. The utility of this background electrolyte has been demonstrated for the analysis of complex protein digests and intact proteins.     

Capillary electrophoresis-mass spectrometry of a new cross-linker with acrylic functionality

Analytical characterization of dimethacrylate-tyrosine-lysine-tyrosine (DMTLT, a new biodegradable acrylic cross-linker synthesized at our laboratory) is carried out using CE-MS. DMTLT is a pseudopeptide composed by tyrosine-lysine-tyrosine amino acids linked through urea bonds with two methacrylic groups, one at each end of the molecule, making this compound an excellent cross-linker for polymerization reactions and for obtaining new biodegradable materials. A new CE-MS method is developed for the characterization of DMTLT and its products of degradation after basic hydrolysis. In order to carry out an exhaustive examination of such degradation products methods based on CE coupled to IT and TOF-MS are employed. Based on CE-IT-MS results and the elemental composition of the degradation products obtained by CE-TOF-MS, conclusions on the mechanism and kinetic of hydrolysis of DMTLT are obtained confirming both the usefulness of CE-MS to characterize new biomaterials and the applicability of DMTLT for preparing new biodegradable polymers. These results are corroborated through the CE-MS detection of the identified products of degradation in a dimethyl acrylamide polymer cross-linked with DMTLT.     

Sensitivity enhancement of CE and CE-MS for the analysis of peptides by a dynamic pH junction

An analytical method of CE-MS and CE with an online preconcentration technique induced by a dynamic pH junction, addition of organic solvent and large volume injection was developed for sensitive determination of peptides in biological samples. Leucine enkephalin, methionine enkephalin, dynorphin A, β-endorphin and angiotensin II were used as model peptides. The optimal online preconcentration conditions were obtained at a sample matrix consisting of 100?mM borate buffer (pH 10.0) with 50% v/v acetonitrile and a BGE containing 1?M formic acid at pH 2.0, along with a 25-cm injection length. Under the optimized conditions, a 4.0×10(3)-1.1×10(4)-fold increase in peak intensity was achieved without degrading the peak shape. This online preconcentration method was applied to analyze the intracellular angiotensin II within the peptides extracted from HL1 cells and approximately increase of 1×10(4)-fold sensitivity was achieved compared to normal condition. Thus, the developed method could be applied to the analysis of various peptides for peptidomics study in biological samples.     

Capillary electrophoresis-mass spectrometry methods for tryptic peptide mapping of therapeutic antibodies

Tryptic peptide mapping is routinely used in the biotech industry to confirm primary sequence, cell line stability, and to analyze posttranslational modifications. Peptide analysis is generally done by reverse phase liquid chromatography with UV or mass spectrometric detection. This method provides excellent resolution and sequence coverage. However, traditional methods are slow, and generally cannot detect small, hydrophilic peptides due to coelution with the column void volume. In this work, complementary CE-MS peptide analysis methods have been developed. The analyses are performed on a traditional CE-MS instrument with a sheath interface, and also on a novel sheathless interface that promises improved resolution and limit of detection. The methods were performed on a tryptic digest of a therapeutic monoclonal antibody for which LC-MS detects 97% sequence coverage. The 3% not covered consists of 11 peptides containing three amino acids or fewer, including two in the critical complementarity binding domain. Without further processing, the same tryptic digest was analyzed by CE-MS. Separation and detection of the 11 small peptides was achieved on CE-MS systems with both interfaces. The sheathless system produced better peak capacity and gave mass spectra with significantly less noise, while the sheath system proved to have better repeatability.     

Peptide correlation: a means to identify high quality quantitative information in large-scale proteomic studies

A major challenge of proteomic studies is the accurate quantitation of proteins. LC-MS/MS-based methods are especially suited for profiling proteins in large sample sets. In this setup, the measurement of relative protein abundance relies on the correct quantitation of tryptic peptides. However, peptide intensities often do not unequivocally reflect the abundance of the native proteins in the sample. In this study, we show that peptides that accurately reflect relative protein abundances in large-scale sample sets can be selected based on the correlation to each other. This strategy was tested in a well-controlled experiment using a set of spiked serum samples as well as 55 clinical serum samples from schizophrenia patients and healthy volunteers. The peptide correlation analysis we present here provides an intuitive and simple procedure to obtain a high quality quantitative information from proteomics data.     

Sensitive CE-MS method for monitoring of riociguat and desmethylriociguat levels in human serum

Riociguat is novel antihypertensive drug for treatment of pulmonary hypertension. As such, it is still being tested in many clinical and pharmacokinetic trials. Existing methods that determine serum riociguat and desmethylriociguat (DMR) are based solely on liquid chromatography with mass spectrometry. Therefore, we present a novel capillary electrophoresis with mass spectrometry method (CE-MS) for their determination in human serum as alternative method for ongoing trials. Complete resolution of both analytes was achieved by means of pH optimization of ammonium formate background electrolytes that are fully compatible with ESI/MS detection. Simple liquid-liquid extraction was used as sample pretreatment. The calibration dependence of the method was linear (in the range of 10–1000 ng/mL), with adequate accuracy (90.1–114.9%) and precision (13.4%). LOD and LOQ were arbitrarily set at 10 ng/mL for both analytes. Clinical applicability was validated using serum samples from patients treated with riociguat in pharmacokinetic study and the results corresponded with reference HPLC-MS/MS values. Capillary electrophoresis proved to be sensitive and selective tool for the analysis of riociguat and DMR.