Electrospray interfacing of polymer microfluidics to MALDI-MS

The off-line coupling of polymer microfluidics to MALDI-MS is presented using electrospray deposition. Using polycarbonate microfluidic chips with integrated hydrophobic membrane electrospray tips, peptides and proteins are deposited onto a stainless steel target followed by MALDI-MS analysis. Microchip electrospray deposition is found to yield excellent spatial control and homogeneity of deposited peptide spots, and significantly improved MALDI-MS spectral reproducibility compared to traditional target preparation methods. A detection limit of 3.5 fmol is demonstrated for angiotensin. Furthermore, multiple electrospray tips on a single chip provide the ability to simultaneously elute parallel sample streams onto a MALDI target for high-throughput multiplexed analysis. Using a three-element electrospray tip array with 150 microm spacing, the simultaneous deposition of bradykinin, fibrinopeptide, and angiotensin is achieved with no cross talk between deposited samples. In addition, in-line proteolytic digestion of intact proteins is successfully achieved during the electrospray process by binding trypsin within the electrospray membrane, eliminating the need for on-probe digestion prior to MALDI-MS. The technology offers promise for a range of microfluidic platforms designed for high-throughput multiplexed proteomic analyses in which simultaneous on-chip separations require an effective interface to MS.     

Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein identification

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be performed. The sensitivity of nano-ESI/MS, however, is lower than that of MALDI-MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI-MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI-directed nano-ESI-MS/MS, makes use of information obtained from the more sensitive MALDI-MS experiments in order to direct subsequent nano-ESI-MS/MS experiments. Peptide molecular ions found in the MALDI-MS analysis are then selected, as their (+2) precursor ions, for nano-ESI-MS/MS sequencing, even though these ions cannot be detected in the ESI-MS spectra. This method, originally proposed by Tempst et al. (Anal. Chem. 2000, 72: 777-790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time-of-flight instruments equipped with commercially available nano-ESI sources, have allowed the unambiguous identification of in-gel digested proteins at levels below their ESI-MS detection limits, even in the presence of salts and detergents.     

生物质谱

质谱已成为生物和生物化学研究的一个重要的分析工具，特别是在蛋白质组学研究的作用更显突出，它的分析速度、准确性和灵敏度都是传统分析技术所不可比拟的。主要介绍了两种近年来发展最为迅速、应用最为广泛的软离子化质谱技术：即基体辅助激光解吸离子化质谱（MALDI－MS）和电喷了子化质谱（ESI－MS）的原理、技术的最新进展，并简单介绍了它们在蛋白质和多肽分析中的应用。     

Recent advances in the MS analysis of glycoproteins: Capillary and microfluidic workflows

Recent developments in bioanalytical instrumentation, MS detection, and computational data analysis approaches have provided researchers with capabilities for interrogating the complex cellular glycoproteome, to help gain a better insight into the cellular and physiological processes that are associated with a disease and to facilitate the efforts centered on identifying disease-specific biomarkers. This review describes the progress achieved in the characterization of protein glycosylation by using advanced capillary and microfluidic MS technologies. The major steps involved in large-scale glycoproteomic analysis approaches are discussed, with special emphasis given to workflows that have evolved around complex MS detection functions. In addition, quantitative analysis strategies are assessed, and the bioinformatics aspects of glycoproteomic data processing are summarized. The developments in commercial and custom fabricated microfluidic front-end platforms to ESI- and MALDI-MS instrumentation, for addressing major challenges in carbohydrate analysis such as sensitivity, throughput, and ability to perform structural characterization, are further evaluated and illustrated with relevant examples.     

New advances in microfluidic flow cytometry

In recent years, researchers are paying the increasing attention to the development of portable microfluidic diagnostic devices including microfluidic flow cytometry for the point‐of‐care testing. Microfluidic flow cytometry, where microfluidics and flow cytometry work together to realize novel functionalities on the microchip, provides a powerful tool for measuring the multiple characteristics of biological samples. The development of a portable, low‐cost, and compact flow cytometer can benefit the health care in underserved areas such as Africa or Asia. In this article, we review recent advancements of microfluidics including sample pumping, focusing and sorting, novel detection approaches, and data analysis in the field of flow cytometry. The challenge of microfluidic flow cytometry is also examined briefly.     

基于微流控芯片-质谱联用的细胞分析研究进展

微流控芯片与质谱联用为细胞研究提供了一个很好的研究平台.质谱的高灵敏度和对化合物独特的鉴别能力可以从复杂的化学信息背景中筛选识别出微量目标物,是细胞分析理想的检测手段.本文重点综述了近年来基于微流控芯片-质谱联用技术的细胞研究进展,从芯片-电喷雾质谱(ESI-MS)接口技术、集成化的样品前处理技术、细胞的药物代谢和细胞相互作用研究及基质辅助激光解吸电离质谱(MALDI-MS)的细胞分析应用等方面总结了最新的方法和技术发展.并展望了芯片-质谱联用新技术应用于细胞分析的可能性.     

Optical and electrochemical detection techniques for cell-based microfluidic systems

The ability to fabricate microfluidic systems with complex structures and with compatible dimensions between the microfluidics and biological cells have attracted significant attention in the development of microchips for analyzing the biophysical and biochemical functions of cells. Just as cell-based microfluidics have become a versatile tool for biosensing, diagnostics, drug screening and biological research, detector modules for cell-based microfluidics have also undergone major development over the past decade. This review focuses on detection methods commonly used in cell-based microfluidic systems, and provides a general survey and an in-depth look at recent developments in optical and electrochemical detection methods for microfluidic applications for biological systems, particularly cell analysis. Selected examples are used to illustrate applications of these detection systems and their advantages and weaknesses.     

Microfluidic electronics

Microfluidics, a field that has been well-established for several decades, has seen extensive applications in the areas of biology, chemistry, and medicine. However, it might be very hard to imagine how such soft microfluidic devices would be used in other areas, such as electronics, in which stiff, solid metals, insulators, and semiconductors have previously dominated. Very recently, things have radically changed. Taking advantage of native properties of microfluidics, advances in microfluidics-based electronics have shown great potential in numerous new appealing applications, e.g. bio-inspired devices, body-worn healthcare and medical sensing systems, and ergonomic units, in which conventional rigid, bulky electronics are facing insurmountable obstacles to fulfil the demand on comfortable user experience. Not only would the birth of microfluidic electronics contribute to both the microfluidics and electronics fields, but it may also shape the future of our daily life. Nevertheless, microfluidic electronics are still at a very early stage, and significant efforts in research and development are needed to advance this emerging field. The intention of this article is to review recent research outcomes in the field of microfluidic electronics, and address current technical challenges and issues. The outlook of future development in microfluidic electronic devices and systems, as well as new fabrication techniques, is also discussed. Moreover, the authors would like to inspire both the microfluidics and electronics communities to further exploit this newly-established field.     

Microfluidic platforms for lab-on-a-chip applications

We review microfluidic platforms that enable the miniaturization, integration and automation of biochemical assays. Nowadays nearly an unmanageable variety of alternative approaches exists that can do this in principle. Here we focus on those kinds of platforms only that allow performance of a set of microfluidic functions--defined as microfluidic unit operations-which can be easily combined within a well defined and consistent fabrication technology to implement application specific biochemical assays in an easy, flexible and ideally monolithically way. The microfluidic platforms discussed in the following are capillary test strips, also known as lateral flow assays, the "microfluidic large scale integration" approach, centrifugal microfluidics, the electrokinetic platform, pressure driven droplet based microfluidics, electrowetting based microfluidics, SAW driven microfluidics and, last but not least, "free scalable non-contact dispensing". The microfluidic unit operations discussed within those platforms are fluid transport, metering, mixing, switching, incubation, separation, droplet formation, droplet splitting, nL and pL dispensing, and detection.     

基质辅助激光解吸电离质谱和电喷雾电离质谱在辣根过氧化物酶糖肽结构分析中的应用

糖链结构的质谱解析是今后糖蛋白分析中的重要研究内容,其中完整糖肽的分析,由于可以同时获得糖基化位点和对应糖链的结构信息,更具有重要意义和研究前景。本工作对质谱软电离技术在完整糖肽分析中的应用进行了研究,其中包括了基质辅助激光解吸电离(matrix-assisted laser desorption ionization, MALDI)和电喷雾电离(electrospray ionization, ESI)技术。通过平行使用两种串联质谱(tandem mass spectrometry, MS/MS)分析策略: MALDI-MS/MS和ESI-MS/MS对目标糖蛋白——辣根过氧化物酶进行分析,并讨论了其互补性。结果表明,MALDI和ESI技术各有优劣,结合串联质谱分析,可获得糖肽的糖链结构信息;两条路线互补使用,在揭示蛋白质糖基化修饰(位点和结构)的研究中十分必要。     

Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads

A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor protein.     

Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry

The structural characterization of Glycosyl-Inositol-Phospho-Ceramides (GIPCs), which are the main sphingolipids of plant tissues, is a critical step towards the understanding of their physiological function. After optimization of their extraction, numerous plant GIPCs have been characterized by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) full scan analysis of negative ions provides a quick overview of GIPC distribution. Clear differences were observed for the two plant models studied: six GIPC series bearing from two to seven saccharide units were detected in tobacco BY-2 cell extracts, whereas GIPCs extracted from A. thaliana cell cultures and leaves were less diverse, with a dominance of species containing only two saccharide units. The number of GIPC species was around 50 in A. thaliana and 120 in tobacco BY-2 cells. MALDI-MS/MS spectra gave access to detailed structural information relative to the ceramide moiety, the polar head, as well as the number and types of saccharide units. Once released from GIPCs, fatty acid chains and long-chain bases were analyzed by GC/MS to verify that all GIPC series were taken into account by the MALDI-MS/MS approach. ESI-MS/MS provided complementary information for the identification of isobaric species and fatty acid chains. Such a methodology, mostly relying on MALDI-MS/MS, should open new avenues to determine structure-function relationships between glycosphingolipids and membrane organization.     

Recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices

AC electrokinetics is a generic term that refers to an induced motion of particles and fluids under nonuniform AC electric fields. The AC electric fields are formed by application of AC voltages to microelectrodes, which can be easily integrated into microfluidic devices by standard microfabrication techniques. Moreover, the magnitude of the motion is large enough to control the mass transfer on the devices. These advantages are attractive for biomolecular analysis on the microfluidic devices, in which the characteristics of small space and microfluidics have been mainly employed. In this review, I describe recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices. The applications include fluid pumping and mixing by AC electrokinetic flow, and manipulation of biomolecules such as DNA and proteins by various AC electrokinetic techniques. Future prospects for highly functional biomolecular analysis on microfluidic devices with the aid of AC electrokinetics are also discussed.     

Determination of the fatty acyl profiles of phosphatidylethanolamines by tandem mass spectrometry of sodium adducts

Phosphatidylethanolamines (PEs) are one of the major constituents of cellular membranes, and, along with other phospholipid classes, have an essential role in the physiology of cells. Profiling of phospholipids in biological samples is currently done using mass spectrometry (MS). In this work we describe the MS fragmentation of sodium adducts of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and 2-linoleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine (PLPE). This study was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) using three different instruments and also by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). All MS/MS spectra show product ions related to the polar head fragmentation and product ions related to the loss of acyl chains. In ESI-MS/MS spectra, the product ions [M+Na-R1COOH-43]+ and [M+Na-R2COOH-43]+ show different relative abundance, as well as [M+Na-R1COOH]+ and [M+Na-R2COOH]+ product ions, allowing identification of both fatty acyl residues of PEs, and their specific location. MALDI-MS/MS shows the same product ions reported before and other ions generated by charge-remote fragmentation of the C3-C4 bond (gamma-cleavage) of fatty acyl residues combined with loss of 163 Da. These fragment ions, [M+Na-(R2-C2H3)-163]+ and [M+Na-(R1-C2H3)-163]+, show different relative abundances, and the product ion formed by the gamma-cleavage of sn-2 is the most abundant. Overall, differences noted that are important for identification and location of fatty acyl residues in the glycerol backbone are: relative abundance between the product ions [M+Na-R1COOH-43]+ > [M+Na-R2COOH-43]+ in ESI-MS/MS spectra; and relative abundance between the product ions [M+Na-(R2-C2H3)-163]+ > [M+Na-(R1-C2H3)-163]+ in MALDI-MS/MS spectra.     

Immunoassays in microfluidic systems

Immunoassays have greatly benefited from miniaturization in microfluidic systems. This review, which summarizes developments in microfluidics-based immunoassays since 2000, includes four sections, focusing on the configurations of immunoassays that have been implemented in microfluidics, the main fluid handling modalities that have been used for microfluidic immunoassays, multiplexed immunoassays in microfluidic platforms, and the emergence of label-free detection techniques. The field of microfluidic immunoassays is continuously improving and has great promise for the future.     

A matrix-assisted laser desorption/ionization tandem mass spectrometry method for direct screening of small molecule mixtures against an aminoglycoside kinase

Aminoglycoside phosphotransferase 3′IIIa (APH3′IIIa) is a bacterial enzyme involved in antibiotic resistance through phosphorylation of aminoglycosides, which can potentially be overcome by co-administration of an APH3′IIIa inhibitor with the antibiotic. Current assay methods for discovery of APH3′IIIa inhibitors suffer from low specificity and high false positive/negative hit rates. Here, we describe a method for screening APH3′IIIa inhibitors based on direct detection of kanamycin A phosphorylation using MALDI-MS/MS, which is more rapid than conventional assays and does not require secondary assays or sample cleanup. The MALDI-MS/MS assay operates at an ionic strength of 45 mM and co-factors can be utilized at near-physiological levels for optimal enzyme activity. Detection via MALDI-MS/MS allowed for improved reproducibility when compared to ESI-MS/MS. Furthermore, the use of MS/MS provided better signal-to-noise ratios relative to MS alone on the MALDI instrument. The assay was validated via generation of Z′-factors, with values of 0.78 and 0.56 in the absence and presence of 0.2% DMSO, respectively. The assay was used to screen a kinase directed library of >200 compounds, assayed as 21 mixtures of 10 compounds each. Five novel synthetic inhibitors were identified following mixture deconvolution. Inhibition constants were obtained for the aforementioned inhibitors using the MALDI-MS/MS assay, revealing several low to mid micromolar “hits”, and highlighting the quantitative nature of the assay.     

微流控细胞芯片生命分析应用多元化

微流控芯片是现代生命科学研究领域的重要分析工具.结合研究者近年来开展的研究工作和取得的相关进展,本文主要介绍了微流控细胞芯片的功能特征,同时从动物细胞、植物细胞以及微生物细胞三方面系统阐述了微流控芯片生命分析多元化的发展现状,并对其应用前景进行了展望.     

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140DeltaCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis.     

Chip-based nano-LC-MS/MS identification of proteins in complex biological samples using a novel polymer microfluidic device

Although the proteome of each organism is unambiguously coded in its genome, the proteome shows the real biology in action in each particular organism. New powerful tools are being developed for biochemists and biologists to analyze complex biological samples for studying the complete protein supplement of the genome, i. e., the proteome. There are several methods available for proteome analysis including 2-DE and several forms of MS. In recent years, technologies such as microfluidics and array-based systems have appeared in the field of analysis, identification, and quantification of proteins. These novel approaches might help in solving current technical challenges in proteomics. This paper presents a practical application of the first commercially available microfluidic nano-ESI device coupled with nano-LC (i. e., HPLC-chip) for the analysis of samples of some biological protein mixtures.     

Microfluidics for cell-based high throughput screening platforms—A review

In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery.