Elemental mass spectrometry for quantitative proteomics

In the last decade mass-spectrometry-based proteomics has become an indispensable analytical tool for molecular biology, cellular biology and, lately, for the emerging systems biology. This review summarises the evolution and great potential of analytical methods based on elemental mass-spectrometric detection for quantitative proteomic analysis.     

A novel mass spectrometry cluster for high-throughput quantitative proteomics

We have developed and implemented a novel mass spectrometry (MS) platform combining the advantages of high mass accuracy and resolving power of Fourier transform ion cyclotron resonance (FTICR) with the economy and speed of multiple ion traps for tandem mass spectrometry. The instruments are integrated using novel algorithms and software and work in concert as one system. Using chromatographic time compression, a single expensive FTICR mass spectrometer can match the throughput of multiple relatively inexpensive ion trap instruments. Liquid chromatography (LC)-mass spectrometry data from the two types of spectrometers are aligned and combined to hybrid datasets, from which peptides are identified using accurate mass from the FTICR data and tandem mass spectra from the ion trap data. In addition, the high resolving power and dynamic range of a 12 tesla FTICR also allows precise label-free quantitation. Using two ion traps in parallel with one LC allows simultaneous MS/MS experiments and optimal application of collision induced dissociation and electrontransfer dissociation throughout the chromatographic separation for increased proteome coverage, characterization of post-translational modifications and/or simultaneous measurement in positive and negative ionization mode. An FTICR-ion trap cluster can achieve similar performance and sample throughput as multiple hybrid ion trap-FTICR instruments, but at a lower cost. We here describe the first such FTICR-ion trap cluster, its performance and the idea of chromatographic compression.     

Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

基于质谱的单细胞蛋白质组学分析方法及应用

细胞是生命体的最小组成单位,遗传及外部环境等因素使单细胞异质性广泛存在于众多生物体中。传统的生物学实验获得的结果多是大量细胞的平均测量值,因此在单细胞层面开展研究对于精确理解细胞的生长发育以及疾病的诊断与治疗至关重要。而作为重要的细胞和生命活动的执行者,蛋白质由于其不具备扩增特性,且种类繁多、丰度低、动态分布范围宽,与核酸等其他生物大分子相比,其单细胞组学研究相对滞后。而在所有的检测手段中,荧光检测以及电化学分析方法具有极高的灵敏度,但是囿于其研究通量有限,以及电化学活性依赖,很难成为普适性的单细胞蛋白质组学研究方法。质谱分析作为传统蛋白质组学中最为核心的研究技术,由于其高灵敏、高通量、结构信息丰富等特点,在单细胞蛋白质组学研究中独树一帜。该文综述了近年来基于质谱的单细胞蛋白质组学研究中的代表性方法,根据质谱分析前蛋白质分离方式的差异,将其分为基于毛细管电泳分离、液相色谱分离和无分离手段的直接检测3类方法,在介绍研究现状的同时对这些方法在细胞通量、蛋白质鉴定数目、灵敏度以及方法应用方面进行了总结与比较。最后,基于目前研究中面临的挑战以及发展趋势对基于质谱的单细胞蛋白质组学的研究前景进行了展望。     

Practical quantitative biomedical applications of MALDI-TOF mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) is used to obtain fast and accurate determinations of molecular mass, but quantitative determinations are generally made by other techniques. In this study we illustrate the practical utility of automated MALDI-TOFMS as a tool for quantifying a diverse array of biomolecules covering an extensive molecular weight range, and present in biological extracts and fluids. Growth hormone was measured in rat pituitary tissue; insulin in human pancreatic tissue; homovanillic acid in human urine; and LVV-hemorphin-7, epinephrine and norepinephrine in human adrenal and pheochromocytoma tissues. Internal standards including compounds of similar molecular weight, structural analogs or isotopomers were incorporated into each analysis. We report on the current practical limitations of quantitative MALDI-TOFMS and highlight some of the potential benefits of this technique as a quantitative tool.     

High-performance separation techniques hyphenated to mass spectrometry for ganglioside analysis

Gangliosides, sialic-acid-containing glycosphingolipids are involved in numerous biological processes and play essential roles in severe pathologies, with predilection in those of the central nervous system. Formerly, ganglioside composition and quantity were assessed exclusively by thin-layer chromatographic (TLC), immunochemical, and immunohistochemical methods, which have limited effectiveness being unable to detect minor components in mixtures of high heterogeneity. Increased awareness of the biological importance of gangliosides stimulated the development of analytical methods that are better amenable to complex ganglioside mixtures. More recently, MS in online conjunction with high-performance separation techniques brought a significant progress to the field. This review highlights the state-of-the-art development and application of separation methods online coupled to MS for ganglioside analysis. Most original and successful protocols based on GC-MS, LC-MS, and CE-MS are presented here together with the special instrumental and sample preparation requirements to be met for effective ganglioside separation, detection, and structural identification. Finally, the advantages and downsides of each methodology as well as the perspectives for simplification, standardization, and upgrading are assessed.     

Laser mass spectrometry: an overview of techniques,instruments and applications

Lasers have been used to produce intact molecular ions and structurally informative fragment ions of otherwise intractable biomolecules for examination by mass spectrometry. While laser microprobes have been used for this purpose, this paper focusses on other instrumental configurations, which require less focussed laser beams and which record ions that are produced by mechanisms which can be described thermodynamically. The time-of-flight and Fourier-transform ion-cyclotron resonance mass spectrometers emerge as the most suitable for use with pulsed laser desorption. Classes of compounds which are amenable to the technique include industrial polymers and carbohydrates.     

Interpretation of mass spectrometry data for high-throughput proteomics

Recent developments in proteomics have revealed a bottleneck in bioinformatics: high-quality interpretation of acquired MS data. The ability to generate thousands of MS spectra per day, and the demand for this, makes manual methods inadequate for analysis and underlines the need to transfer the advanced capabilities of an expert human user into sophisticated MS interpretation algorithms. The identification rate in current high-throughput proteomics studies is not only a matter of instrumentation. We present software for high-throughput PMF identification, which enables robust and confident protein identification at higher rates. This has been achieved by automated calibration, peak rejection, and use of a meta search approach which employs various PMF search engines. The automatic calibration consists of a dynamic, spectral information-dependent algorithm, which combines various known calibration methods and iteratively establishes an optimised calibration. The peak rejection algorithm filters signals that are unrelated to the analysed protein by use of automatically generated and dataset-dependent exclusion lists. In the "meta search" several known PMF search engines are triggered and their results are merged by use of a meta score. The significance of the meta score was assessed by simulation of PMF identification with 10,000 artificial spectra resembling a data situation close to the measured dataset. By means of this simulation the meta score is linked to expectation values as a statistical measure. The presented software is part of the proteome database ProteinScape which links the information derived from MS data to other relevant proteomics data. We demonstrate the performance of the presented system with MS data from 1891 PMF spectra. As a result of automatic calibration and peak rejection the identification rate increased from 6% to 44%.Abbreviations 2-DE Two-dimensional gel electrophoresis - MALDI Matrix-assisted laser desorption ionisation - PMF Peptide mass fingerprinting - MS Mass spectrometry - TOF Time of flight     

Electrospray mass spectrometry: a tool for elemental speciation

Recent progress in the development of electrospray mass spectrometry (ESMS) as a tool for elemental speciation is reviewed. Reports wherein ESMS is used to qualitatively determine the presence of metal ions (inorganic, organometallic and complexed) and non-metallic inorganic species have grown exponentially over the last decade. In addition to elemental speciation, impact in other areas such as gas-phase chemistry, inorganic–organometallic chemistry and biological mass spectrometry has been prolific. The review is structured to cover each of the areas listed above, and also includes a brief introduction, discussion of the electrospray process, discussion of instrumentation and other relevant application areas. An overview of the types of species/complexes studied is given in each section along with a brief discussion of the application objectives and analytical aspects. Analytical considerations for the development of ESMS as a tool for elemental speciation are also raised, including, application, quantitation, sensitivity, limitations and future directions. The impact of speciation strategies involving stand-alone ESMS, ESMS coupled with on-line separation techniques and the inclusion of ESMS in dual (multiple) technique strategies are presented. High backgrounds due to chemical noise and signal suppression (matrix effects) appear to be two important factors limiting sensitive detection of most analytes. The use of sample pre-treatment, pre-concentration or separation techniques is necessary to alleviate these problems. Although ESMS currently suffers from a number of limitations, continued instrumentation and methods development will improve its capability and diversify the impact of ESMS as a tool for elemental speciation.     

Two-dimensional nano-liquid chromatography-mass spectrometry system for applications in proteomics

Transient isotachophoresis-capillary zone electrophoresis with artificial seawater as the background electrolyte (BGE) was improved to further lower the limit of detection (LOD) for determination of nitrite and nitrate in seawater. By lowering the pH of BGE, the difference between effective mobility of nitrite and that of nitrate increased, thereby permitting increased sample volumes to be tolerated and their LOD values to decrease. Artificial seawater with pH adjusted to 3.0 using phosphate buffer was adopted as the BGE. To reverse electroosmotic flow (EOF), a capillary was flushed with 0.1 mM dilauryldimethylammonium bromide for 3 min before the capillary was filled with the BGE. Limits of detection (LODs) for nitrite and nitrate were 2.7 and 3.0 microg/l (as nitrogen), respectively. The LODs were obtained at a signal-to-noise ratio of 3. Values of the relative standard deviation (RSD) of peak area for these ions were 2.0 and 0.75%, respectively, when nitrite concentration was 0.05 mg/l and that of nitrate was 0.5 mg/l. The RSDs of peak height were 4.4 and 2.3%. The RSD values of migration time for these ions were 0.19 and 0.17%. The proposed method was applied to determination of nitrite and nitrate in a proposed certified reference material for nutrients in seawater, MOOS-1, distributed by the National Research Council of Canada. Results agreed with the assigned tolerance interval. This method was also applied to determination of these ions in seawater collected around Osaka Bay. Results nearly agreed with those obtained by a conventional spectrophotometric method.     

比较纳升反相色谱-串联质谱与毛细管区带电泳-串联质谱用于自顶向下蛋白质组学

自顶向下蛋白质组学的一个重要难题是缺乏与质谱可以在线连用并且可以提供高效蛋白质分离的液相分离技术。毛细管区带电泳与纳升反相色谱都可以与质谱在线连用,并且在复杂蛋白质样品分析方面也都有了显著的提升。在这里,我们首次比较了先进的纳升反相色谱-串联质谱与毛细管区带电泳-串联质谱平台用于自顶向下蛋白质组学分析。相对于纳升反相色谱-质谱而言,毛细管区带电泳-质谱可以将标准蛋白质样品的消耗量降低10倍,而且保持与纳升反相色谱-质谱相当的蛋白质信号强度。有意思的是,与毛细管区带电泳-质谱相比,纳升反相色谱-质谱可以获得更高的蛋白质分子的气相价态。这个现象可能是由于反相流动相中的高浓度乙腈使得蛋白质变性的更加充分。从1微克的大肠杆菌蛋白质样品中,毛细管区带电泳-串联质谱可以鉴定到159个蛋白质和513个蛋白质变体,而纳升反相色谱-串联质谱仅鉴定到105个蛋白质和277个蛋白质变体。当将大肠杆菌蛋白质的上样量提高到8微克时,纳升反相色谱-串联质谱可以鉴定到245个蛋白质和1004个蛋白质变体。由于纳升反相色谱-串联质谱具有比毛细管区带电泳-串联质谱更高的上样量与更宽的分离窗口,当蛋白质样品量不受限制时,纳升反相色谱-串联质谱具有明显的优势。但是,在痕量样品分析方面,毛细管区带电泳-串联质谱具有更大的潜力。     

Plasma-based mass spectrometry for simultaneous acquisition of elemental and molecular information

Chemical speciation studies are commonly accomplished by resorting to hyphenated analytical techniques, consisting of a powerful chromatographic separation technique coupled to a highly sensitive elemental spectrometric detector. However, in addition to this element-selective information, complementary molecular spectrometric tools are often required for a complete identification of macromolecules. Therefore, there is an increased research effort focused towards the development of integrated instruments to carry out the complete chemical speciation within a sample using a single instrument. An outline of recent developments in plasma-based mass spectrometric instrumentation for such comprehensive chemical speciation studies is here presented and their pros and cons detailed. In this context, the use of complementary techniques operating in parallel after splitting to a single chromatographic separation (dual sources) providing simultaneously elemental and molecular information is critically reviewed. Also, instrumental developments involving the use of stationary plasma sources operated in non-traditional modes (e.g. low pressure and low power) are also discussed. Moreover, the capabilities of tunable plasma-based ionization sources (allowing different ionization processes and, so, quasi-simultaneously providing elemental and molecular information with a single instrument) as a relatively simple and cheap approach are revised.     

Advances in analytical methodology for bioinorganic speciation analysis: metallomics, metalloproteomics and heteroatom-tagged proteomics and metabolomics

The recent developments in analytical techniques capable of providing information on the identity and quantity of heteroatom-containing biomolecules are critically discussed. Particular attention is paid to the emerging areas of bioinorganic analysis including: (i) a comprehensive analysis of the entirety of metal and metalloid species within a cell or tissue type (metallomics), (ii) the study of the part of the metallome involving the protein ligands (metalloproteomics), and (iii) the use of a heteroelement, naturally present in a protein or introduced in a tag added by means of derivatisation, for the spotting and quantification of proteins (heteroatom-tagged proteomics). Inductively coupled plasma mass spectrometry (ICP MS), used as detector in chromatography and electrophoresis, and supported by electrospray and MALDI MS, appears as the linchpin analytical technique for these emerging areas. This review focuses on the recent advances in ICP MS in biological speciation analysis including sensitive detection of non-metals, especially of sulfur and phosphorus, couplings to capillary and nanoflow HPLC and capillary electrophoresis, laser ablation ICP MS detection of proteins in gel electrophoresis, and isotope dilution quantification of biomolecules. The paper can be considered as a followup of a previous review by the author on a similar topic (J. Szpunar, Analyst, 2000, 125, 963).     

Recent directions of electrospray mass spectrometry for elemental speciation analysis

A brief survey is given of the last 2 years’ literature on electrospray mass spectrometry (ESI-MS) for speciation analysis. As observed for many years, the main recent applications in this field concern arsenic and selenium species, especially in studies encompassing combined use of molecular and element mass spectrometry. A further application field is the stoichiometric characterization of metal complexes by ESI-MS, which in some studies was assisted by nuclear magnetic resonance spectroscopy. A few examples are presented to illustrate arsenic species involved in metabolic pathways, sulfur species in oils and bitumen, and aluminum complexes. On the basis of this review, we also give an outlook of expected future developments and trends in this research field.     

磁性离子印迹技术用于元素形态分离分析

元素的形态决定了其在环境和生物过程中的不同行为,形态分析正在被分析化学、环境化学、地球化学、生态学、农学和生物医学等众多学科所关注。环境和生物样品基质复杂、化学形态多样、含量低且易转化是元素形态分析面临的挑战,因此对元素形态的甄别、定量、生态毒性评价和生理功能研究需要对原生形态进行高选择性识别和高效率分离。固相萃取是一种有效应对以上难题的方法,但现有材料和方法远不能满足要求。离子印迹聚合物可与印迹金属离子特异性结合,具有准确、灵敏、可靠的特点,近年来在元素形态分离富集和分析检测方面得到了较为广泛的应用。鉴于非磁性吸附剂在固相萃取操作时,需要将分散在样品溶液中的吸附材料经过离心或过滤分离,操作比较繁琐费时,而磁性材料易被外部磁场快速分离,因此操作简便快速的磁固相萃取正成为元素形态分离富集中一种极具潜力的方法。这篇综述系统总结了离子印迹技术的最新进展,包括离子印迹技术的原理、离子印迹聚合物的制备方法,并根据元素形态分析中离子印迹磁固相萃取的发展现状,分析了离子印迹技术所面临的挑战,最后对元素形态分析中离子印迹技术的未来发展方向和策略提出了建议,提出开发基于有机-无机杂化聚合的多功能磁性离子印迹纳米复合物用于样品的前处理是建立识别选择性高、分离能力强、吸附容量大、形态稳定性好的形态分析方法的一种重要举措。     

Hyphenating liquid phase separation techniques with mass spectrometry: on-line or off-line

The advantages of hyphenating analytical separation techniques to mass spectrometers have been recognised since the 1970s. The first attempts to couple liquid phase separations to mass spectrometry(MS) were problematic, but the development of electrospray ionisation (ES) in 1984 facilitated the production of routinely used, commercial interfaces. This review considers the most recent major developments in hyphenating liquid chromatographic and electrophoretic separations to mass spectrometry, and considers the role the post genomic sciences have played in driving them. It also considers why, despite all of the advances in hyphenation, the future appears to hold a major role for off-line analysis.     

Evolution of esi–mass spectrometry and fourier transform ion cyclotron resonance for proteomics and other biological applications

A perspective is presented centered on the author’s contributions to developments involving electrospray ionization–mass spectrometry, capillary electrophoresis–mass spectrometry, and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and their applications to biological systems, with a special emphasis on the study of noncovalent complexes and proteomics.     

Progress of laser ionization mass spectrometry for elemental analysis — A review of the past decade

Mass spectrometry using a laser ionization source has played a significant role in elemental analysis. Three types of techniques are widely used: high irradiance laser ionization mass spectrometry is capable of rapid determination of elements in solids; single particle mass spectrometry is a powerful tool for single particle characterization; and resonance ionization mass spectrometry is applied for isotope ratio measurements with high sensitivity and selectivity. In this review, the main features of the laser ablation process and plasma characterization by mass spectrometry are summarized. Applications of these three techniques for elemental analysis are discussed.     

Fundamental and applied investigations in plasma-source mass spectrometry for elemental analysis

The current status of plasma source-mass spectrometry (PS-MS) is reviewed. An overview of interference effects that exist, alternative plasma sources available, and mass spectrometer interface studies is provided. A discussion of current and future development areas in plasma source mass spectrometry is also included.