Mass‐spectrometry‐based lipidomics

Lipids, which have a core function in energy storage, signalling and biofilm structures, play important roles in a variety of cellular processes because of the great diversity of their structural and physiochemical properties. Lipidomics is the large‐scale profiling and quantification of biogenic lipid molecules, the comprehensive study of their pathways and the interpretation of their physiological significance based on analytical chemistry and statistical analysis. Lipidomics will not only provide insight into the physiological functions of lipid molecules but will also provide an approach to discovering important biomarkers for diagnosis or treatment of human diseases. Mass‐spectrometry‐based analytical techniques are currently the most widely used and most effective tools for lipid profiling and quantification. In this review, the field of mass‐spectrometry‐based lipidomics was discussed. Recent progress in all essential steps in lipidomics was carefully discussed in this review, including lipid extraction strategies, separation techniques and mass‐spectrometry‐based analytical and quantitative methods in lipidomics. We also focused on novel resolution strategies for difficult problems in determining C=C bond positions in lipidomics. Finally, new technologies that were developed in recent years including single‐cell lipidomics, flux‐based lipidomics and multiomics technologies were also reviewed.     

电喷雾离子化接口及其在毛细管电泳／质谱法中的应用

论述了ＥＳＩ的工作原理，结构和ＣＥ－ＥＳＩ－ＭＳ的应用。     

Ultraviolet Photodissociation: Developments towards Applications for Mass‐Spectrometry‐Based Proteomics

Unraveling of all of the information contained in proteomes poses a tremendous chemical challenge, which is balanced by the promise of potentially transformational knowledge. Mass spectrometry offers an unprecedented arsenal of tools for diverse proteomic investigations. Recently, it was demonstrated that ultraviolet light can be utilized to initiate unique and potentially useful fragmentations in peptides and proteins. Either nonspecific dissociation or highly specific dissociation at engineered chromophoric sites is possible following photon absorption. The level of specificity and control over fragmentation in these experiments is greater than with other fragmentation methods. Novel techniques made possible by this technology are poised to make substantial contributions to the field of proteomics.     

MALDI‐TOF Mass‐Spectrometry‐Based Versatile Method for the Characterization of Protein Kinases

We describe a MALDI‐TOF mass‐spectrometry‐based method that is rapid and versatile for the characterization of protein kinases and their inhibitors. We have designed new kinase substrates by the modification of common synthetic peptides, such as kemptide (LRRALS G), CaMKII substrate (KRQQS FDLF), erktide (ATGPLS PGPFGRR), abltide (EAIY AAPFAKKK), srctide (AEEEIY GEFEAKKKK), neurogranin (AAAKIQAS FRGHMARKK), and casein kinase I (CKI) substrate (RRKDLHDDEEDEAMS ITA). There are two fundamental points on which the proposed method is based to improve the mass‐spectrometric response: 1) mass tag technology by N‐derivatization through stable isotope labeling and 2) C‐terminal conjugation with tryptophanylarginine (WR). It was suggested that C‐terminal conjugation with the WR moiety enhances the ionization potency of these new substrates 1.5–13.7 times as much as those of the original peptides. We demonstrated, by using modified abltide (Ac‐EAIY AAPFAKKKWR‐NH₂), that WR conjugation at the C‐terminus in combination with stable‐isotope labeling at the N‐terminus allowed the quantitative assay of recombinant c‐Abl kinase in the presence of adenosine 5′‐triphosphate (ATP; K_M,ATP=18.6 μM and V_max=642 pmol min^?1 μg^?1). The present protocol made a simple and reliable inhibition assay of recombinant c‐Abl kinase by imatinib possible (IC_{50(recombinant)}=291 nM ; STI571, Gleevec; Novartis Pharma). Moreover, it was also demonstrated that this ATP noncompetitive inhibitor differentiates between two conformers of c‐Abl kinases: the phosphorylated active and dephosphorylated inactive forms (IC_{50(active form)}=1049 nM and IC_{50(inactive form)}=54 nM ). The merit of this approach is evident because the present protocol can be applied to the direct monitoring of the activities of living cell kinases by using cancer‐cell lines, such as mouse B16 melanoma cells and human lung cancer K562 cells. A multiple‐kinase assay that uses K562 cell lysate in the presence of seven new synthetic substrates made high‐throughput inhibitor profiling possible. It should be emphasized that this radioactive isotope‐free quantitative kinase assay will greatly accelerate the discovery of a new generation of potential kinase inhibitors that exhibit highly selective or unique inhibitory profiles.     

An Integrated Microfluidic Probe for Mass Spectrometry Imaging of Biological Samples**

Ambient ionization based on liquid extraction is widely used in mass spectrometry imaging (MSI) of molecules in biological samples. The development of nanospray desorption electrospray ionization (nano-DESI) has enabled the robust imaging of tissue sections with high spatial resolution. However, the fabrication of the nano-DESI probe is challenging, which limits its dissemination to the broader scientific community. Herein, we describe the design and performance of an integrated microfluidic probe (iMFP) for nano-DESI MSI. The glass iMFP, fabricated using photolithography, wet etching, and polishing, shows comparable performance to the capillary-based nano-DESI MSI in terms of stability and sensitivity; a spatial resolution of better than 25 μm was obtained in these first proof-of-principle experiments. The iMFP is easy to operate and align in front of a mass spectrometer, which will facilitate broader use of liquid-extraction-based MSI in biological research, drug discovery, and clinical studies.     

Applications of solid‐phase micro‐extraction with mass spectrometry in pesticide analysis

Pesticides, widely applied in agriculture, can produce a variety of transformation products and their continuous use causes deleterious effects to ecosystem. Efficient and sensitive analytical techniques for enrichment and analysis of pesticides samples are highly required. Compared with other extraction methods, solid‐phase micro extraction is a solvent free, cost effective, robust, versatile, and high throughput sample preparation technique, especially for the analysis of pesticides from complicated matrices. Coupling of solid‐phase micro extraction with gas chromatography and mass spectrometry and liquid chromatography–mass spectrometry has been extensively applied in pesticide analysis. On the other hand, in recent years, combination of fast separation using solid‐phase micro extraction and rapid detection using ambient mass spectrometry is providing highly efficient pesticide screening. This article summarizes the applications of solid‐phase micro extraction coupled to mass spectrometry for pesticides analysis.     

毛细管电泳-电感耦合等离子质谱联用的接口设计

描述了毛细管电泳电感耦合等离子体质谱(CE-ICP-MS)联用技术的单T型接口,自行设计了双T型接口,并对两接口的分析性能作了比较。解决了接口中的常见问题,使用节流阀减小自吸作用并降低了CE分离物的稀释倍数,排气阀使提升量保持稳定。经考察得知,采用自吸作用提升液流流量稳定,其重现性RSD小于5％;双T型接口较单T型接口对CE分离更有利。采用双T型接口联用时,CE分离La、Ce、Nd混合离子迁移时间RSD小于2％,MS信号RSD小于15％,且不同浓度样品经CE分离后其MS信号基本呈线性关系。     

毛细管电泳及其质谱联用技术分析重组人促红细胞生成素

用高效毛细管电泳测定了重组人促红细胞生成素（ｒｈＥＰＯ）的微多相性、肽图，同时采用毛细管电泳质谱联用技术鉴定了部分非糖基化胰酶消解片段和Ｏ－１２６糖基化肽的结构。方法简便、快速，适用于ｒｈＥＰＯ半成品的质量控制。     

Analysis of Sulfonamides by Liquid Chromatography Mass Spectrometry and Capillary Electrophoresis Combing with Voltage‐Assisted Liquid‐Phase Microextraction

The method of liquid‐phase microextraction assisted with voltage was developed and applied on determination of sulfonamides in water samples. Four analytes, such as sulfamethazine, sulfathiazole, sulfadimethoxine, and sulfamethoxazole were extracted from a sample solution at pH 4.5 through a polypropylene membrane of immobilized with 2‐octanone, and then into 25 μL of the acceptor phase of 10 mM sodium hydroxide, and applied voltage of 100 V. Subsequently, the acceptor solution was directly subjected to analysis by LC‐MS or capillary zone electrophoresis. Linearity was obtained in the range of 1.0–25.0 ng mL^?1 with R² > 0.992 in LC‐MS, and 50–1000 ng mL^?1 with R² > 0.995 in capillary zone electrophoresis. The development of VA‐LPME was also applied in analysis of sulfonamides in water samples to evaluate its practical applicability.     

Over 10 000 Peptide Identifications from the HeLa Proteome by Using Single‐Shot Capillary Zone Electrophoresis Combined with Tandem Mass Spectrometry

Capillary zone electrophoresis (CZE)–tandem mass spectrometry (MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has so far fallen far below that of reversed‐phase liquid chromatography (RPLC)–MS/MS. The use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (ca. 300) is reported. This method was coupled to an Orbitrap Fusion mass spectrometer through an electrokinetically pumped sheath‐flow interface for the analysis of complex proteome digests. Single‐shot CZE–MS/MS lead to the identification of over 10 000 peptides and 2100 proteins from a HeLa cell proteome digest in approximately 100 min. This performance is nearly an order of magnitude better than earlier CZE studies and is within a factor of two to four of the state‐of‐the‐art nano ultrahigh‐pressure LC system.     

High‐Throughput Reaction Optimisation and Activity Screening of Ferrocene‐Based Lewis Acid–Catalyst Complexes by Using Continuous‐Flow Reaction Detection Mass Spectrometry

Optimising synthetic conversions and assessing catalyst performance is a tedious and laborious endeavour. Herein, we present an automated alternative to the commonly applied sequential approaches that are used to increase catalyst discovery process efficiencies by increasing the number of entities that can be tested. This new approach combines conversion of the reactants and determination of product formation into a single comprehensive reaction detection system that can be operated with minimal catalyst and reactant consumption. With this approach, rudimentary reaction conditions can be quickly optimised and the same system can then be used to screen for the optimal homogenous catalyst in a selected solution‐phase synthetic conversion. The system, which is composed of standard HPLC components, can be used to screen catalyst libraries at a repetition rate of five minutes and can be run unsupervised. The sensitive mass spectrometric detection that is implemented in the reaction detection methodology can be used for the simultaneous monitoring of reactants, catalysts and product ions. In the experiments, the three‐component reaction that gives a substituted 2‐imidazoline was optimised. Afterwards, the same method was used to assess a library of ferrocene‐based Lewis acid catalysts for performance in the aforementioned conversion in six different solvents. We demonstrate the feasibility of using this methodology to directly compare the performance results obtained in different solvents by calibrating the solvent‐specific MS responses.     

Integrated Micro Flow Synthesis Based on Sequential Br–Li Exchange Reactions of p‐, m‐, and o‐Dibromobenzenes

A micro flow system consisting of micromixers and microtube reactors provides an effective method for the introduction of two electrophiles onto p‐, m‐, and o‐dibromobenzenes. The Br–Li exchange reaction of p‐dibromobenzene with nBuLi can be conducted by using the micro flow system at 20 °C, although much lower temperatures (p‐bromophenyllithium was allowed to react with an electrophile in the micro flow system at 20 °C. The p‐substituted bromobenzene thus obtained was subjected to a second Br–Li exchange reaction followed by reaction with a second electrophile at 20 °C in one flow. A similar transformation can be carried out with m‐dibromobenzene by using the micro flow system. However, the Br–Li exchange reaction of o‐dibromobenzene followed by reaction with an electrophile should be conducted at ?78 °C to avoid benzyne formation. The second Br–Li exchange reaction followed by reaction with an electrophile can be carried out at 0 °C. By using the present method, a variety of p‐, m‐, and o‐disubstituted benzenes were synthesized in one flow at much higher temperatures than are required for conventional batch reactions.     

Integrated electrokinetically driven microfluidic devices with pH‐mediated solid‐phase extraction coupled to microchip electrophoresis for preterm birth biomarkers

Integration in microfluidics is important for achieving automation. Sample preconcentration integrated with separation in a microfluidic setup can have a substantial impact on rapid analysis of low‐abundance disease biomarkers. Here, we have developed a microfluidic device that uses pH‐mediated solid‐phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). A reversed‐phase octyl methacrylate monolith was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH‐mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. Nearly 50‐fold enrichment was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility (<7% RSD). The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with ∼15‐fold enrichment. This device shows important progress towards an integrated electrokinetically operated platform for preconcentration and separation of biomarkers.     

A Mass‐Spectrometry‐Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non‐specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid‐binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent‐resistant lipids bound at the dimer interface in the leucine transporter show decreased k_off rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid‐II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non‐annular lipids based on their exchange rates in solution.     

Direct determination of pregabalin in human urine by nonaqueous CE‐TOF‐MS

Determination of pregabalin in urine samples was carried out by nonaqueous CE with TOF‐MS via ESI, with a mixture of 10 mM ammonium formate and 0.05% acetic acid in methanol. By using TOF‐MS, accurate mass information was obtained, thus causing a great improvement in qualitative ability. In order to avoid ionic suppression, urine samples dilution 1:10 was used. This was the only treatment to urine samples before the injection. Despite this dilution, the detection limit was as low as 0.03 μg/mL for pregabalin. The method was validated with respect to accuracy, precision, and linearity, LOD, and LOQ. This method was applied to the analysis of urine samples from seven different cancer patients undergoing treatment with pregabalin. The developed method may find wide application for the routine determination of pregabalin in biological samples in order to establish a more efficient and safe dosage.     

Coupling Microdroplet Microreactors with Mass Spectrometry: Reading the Contents of Single Droplets Online

Fully integrated : Mass spectrometry has been integrated into a detection scheme for microdroplets that are created within microfluidic channels (see picture, scale bar 200 μm). This technique allows droplets to be identified based on the compounds they contain, and combines fluorescence screening with MS analysis. These experiments indicate how similar approaches can be applied to the ambitious goals of on‐chip protein evolution and chemical synthesis.