Automated protein identification by tandem mass spectrometry: issues and strategies

Protein identification by tandem mass spectrometry (MS/MS) is key to most proteomics projects and has been widely explored in bioinformatics research. Obtaining good and trustful identification results has important implications for biological and clinical work. Although well matured, automated software identification of proteins from MS/MS data still faces a number of obstacles due to the complexity of the proteome or procedural issues of mass spectrometry data acquisition. Expected or unexpected modifications of the peptide sequences, polymorphisms, errors in databases, missed or non-specific cleavages, unusual fragmentation patterns, and single MS/MS spectra of multiple peptides of the same m/z are so many pitfalls for identification algorithms. A lot of research work has been carried out in recent years that yielded new strategies to handle a number of these issues. Multiple MS/MS identification algorithms are now available or have been theoretically described. The difficulty resides in choosing the most adapted method for each type of spectra being identified. This review presents an overview of the state-of-the-art bioinformatics approaches to the identification of proteins by MS/MS to help the reader doing the spade work of finding the right tools among the many possibilities offered.     

Chip‐mass spectrometry for glycomic studies

The introduction of micro‐ and nanochip front end technologies for electrospray mass spectrometry addressed a major challenge in carbohydrate analysis: high sensitivity structural determination and heterogeneity assessment in high dynamic range mixtures of biological origin. Chip‐enhanced electrospray ionization was demonstrated to provide reproducible performance irrespective of the type of carbohydrate, while the amenability of chip systems for coupling with different mass spectrometers greatly advance the chip/MS technique as a versatile key tool in glycomic studies. A more accurate representation of the glycan repertoire to include novel biologically‐relevant information was achieved in different biological sources, asserting this technique as a valuable tool in glycan biomarker discovery and monitoring. Additionally, the integration of various analytical functions onto chip devices and direct hyphenation to MS proved its potential for glycan analysis during the recent years, whereby a new analytical tool is on the verge of maturation: lab‐on‐chip MS glycomics. The achievements until early beginning of 2007 on the implementation of chip‐ and functional integrated chip/MS in systems glycobiology studies are reviewed here. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:223–253, 2009     

Elemental and molecular imaging of human hair using secondary ion mass spectrometry.

Secondary ion mass spectrometry (SIMS) is used to image the spatial distribution of elemental and molecular species on the surface and in cross sections of doped human hair using a magnetic sector SIMS instrument operated as an ion microprobe. Analysis of electrically insulating, non-planar hair samples requires one of two different methods of charge compensation to be used depending on the polarity of the sputtered secondary ions. For detection of positive secondary ions, the hair is imaged using a approximately 0.5 micron diameter, 19.5 keV impact energy, O- microbeam with no auxiliary electron bombardment. For detection of negative secondary ions, a approximately 0.2 micron diameter, 14.5 keV impact energy Cs+ microbeam is used in conjunction with normal incidence, low-energy electron bombardment. Both of these methods allow submicrometer spatial resolution elemental and molecular secondary ion images to be obtained from hair samples without metallic coating of the sample surface prior to analysis. Several examples are presented that reflect potential application areas for these analytical methods.     

质谱检测技术及其一些最新进展

质谱作为一种重要的分析技术,被广泛应用于物理、化学、材料学、生物学、医学等领域.质谱技术包括：离子化技术、离子的质量分析技术、离子检测技术.相对于离子化技术和质量分析技术,科研工作者对离子检测技术关注较少,本文拟对离子检测技术近年来取得的进展进行评述,以便引起相关工作者对离子检测技术的重视.     

Microfabricated devices: A new sample introduction approach to mass spectrometry

Instrument miniaturization is one way of addressing the issues of sensitivity, speed, throughput, and cost of analysis in DNA diagnostics, proteomics, and related biotechnology areas. Microfluidics is of special interest for handling very small sample amounts, with minimal concerns related to sample loss and cross-contamination, problems typical for standard fluidic manipulations. Furthermore, the small footprint of these microfabricated structures leads to instrument designs suitable for high-density, parallel sample processing, and high-throughput analyses. In addition to miniaturized systems designed with optical or electrochemical detection, microfluidic devices interfaced to mass spectrometry have also been demonstrated. Instruments for automated sample infusion analysis are now commercially available, and microdevices utilizing chromatographic or capillary electrophoresis separation techniques are under development. This review aims at documenting the technologies and applications of microfluidic mass spectrometry for the analysis of proteomic samples.     

Femtosecond laser ablation elemental mass spectrometry

Laser ablation mass spectrometry (LA-MS) has always been an interesting method for the elemental analysis of solid samples. Chemical analysis with a laser requires small amounts of material. Depending on the analytical detection system, subpicogram quantities may be sufficient. In addition, a focused laser beam permits the spatial characterization of heterogeneity in solid samples typically with micrometer resolution in terms of lateral and depth dimensions. With the advent of high-energy, ultra-short pulse lasers, new possibilities arise. The task of this review is to discuss the principle differences between the ablation process of short (>1 ps) and ultra-short (<1 ps) pulses. Based on the timescales and the energy balance of the process that underlies an ablation event, it will be shown that ultra-short pulses are less thermal and cause less collateral damages than longer pulses. The confinement of the pulse energy to the focal region guarantees a better spatial resolution in all dimensions and improves the analytical figures of merit (e.g., fractionation). Applications that demonstrate these features and that will be presented are in-depth profiling of multi-layer samples and the elemental analysis of biological materials.     

Preparation of cells cultured on silicon wafers for mass spectrometry analysis

The distribution of specific atoms and molecules within living cells is of high interest in bio-medical research. Laser secondary neutral mass spectrometry (laser-SNMS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) detect atoms with high sensitivity and spatial resolution. The application of these methods to cultured cells requires special preparation techniques preserving morphological and chemical integrity of the living cells. The cells should, therefore, be grown on a conducting material preventing charging of the sample during ion bombardment. Silicon is currently used as the preferred support material for non-biological samples in mass spectrometry. This study investigates (1) the influence of silicon surfaces on cell growth and (2) the suitability of a sandwiched, rapid freezing method to analyse transmembrane ion gradients. Human melanoma cells were grown on silicon with polished or etched surfaces. Growth kinetics were studied using the Sulforhodamine-B assay. Number, shape, and morphology of the cells were assessed by epifluorescence microscopy of calcein AM- and DAPI-stained cells. Cells were subjected to rapid freezing, freeze-fracturing, and freeze-drying prior to analysis by TOF-SIMS and laser-SNMS. While cell numbers and morphology on the rough silicon wafers were impaired, morphology and growth kinetics of cells on polished silicon were identical to control cells on cell culture tested polystyrene. TOF-SIMS and laser-SNMS resulted in high-resolution elemental images and mass spectra. Measurement of the intracellular Na+ and K+ concentrations revealed a ratio as observed in living cells. In conclusion, culturing cells on polished silicon wafers followed by sandwiched, rapid freezing is an adequate preparation method to study intracellular ion distribution with mass spectrometry.     

Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions

Closely related to studying the function of a protein is the analysis of its three-dimensional structure and the identification of interaction sites with its binding partners. An alternative approach to the high-resolution methods for three-dimensional protein structure analysis, such as X-ray crystallography and NMR spectroscopy, consists of covalently connecting two functional groups of the protein(s) under investigation. The location of the created cross-links imposes a distance constraint on the location of the respective side chains and allows one to draw conclusions on the three-dimensional structure of the protein or a protein complex. Recently, chemical cross-linking of proteins has been combined with a mass spectrometric analysis of the created cross-linked products. This review article describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the different available strategies that employ chemical cross-linking and different mass spectrometric techniques. The challenges for mass spectrometry caused by the enormous complexity of the cross-linking reaction mixtures are emphasized. The various approaches described in the literature to facilitate the mass spectrometric detection of cross-linked products as well as computer software for data analyses are reviewed.     

Proteomic tools for quantitation by mass spectrometry

Techniques for the quantitation of proteins and peptides by mass spectrometry (MS) are reviewed. A range of labeling processes is discussed, including metabolic, enzymatic, and chemical labeling, and techniques that can be employed for comparative and absolute quantitation are presented. Advantages and drawbacks of the techniques are discussed, and suggestions for the appropriate uses of the methodologies are explained. Overall, the metabolic incorporation of isotopic labels provides the most accurate labeling strategy, and is most useful when an internal standard for comparative quantitation is needed. However, that technique is limited to research that uses cultured cells.     

Environmental and food applications of LC-tandem mass spectrometry in pesticide-residue analysis: an overview

An overview is given on pesticide-residue determination in environmental and food samples by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Pesticides comprise a large number of substances that belong to many completely different chemical groups, the only common characteristic is that they are effective against pests. They still constitute a challenge in MS because there is no collective pathway for fragmentation. A brief introduction to the theory of tandem MS permits a discussion of which parameters influence the ionization efficiency when the ions are subjected to different actions. Emphasis is placed on the different tandem MS instruments: triple and ion-trap quadrupoles, and hybrid quadrupole time-of-flight (Q-TOF), including advantages and drawbacks, typical detection limits, and ion signals at low concentrations. The instrumental setup, as well as LC and mass spectrometric experimental conditions, must be carefully selected to increase the performance of the analytical system. The capacity of each instrument to provide useful data for the identification of pesticides, and the possibility to obtain structural information for the identification of target and non-target compounds, are discussed. Finally, sample preparation techniques and examples of applications are debated to reveal the potential of the current state-of-the-art technology, and to further promote the usefulness of tandem MS.     

Low-vacuum cylindrical ion trap mass spectrometry

Low-vacuum mass spectrometry is desirable because it reduces the size, weight, cost, and power of the instrument by reducing the workload of the pumping system. To investigate low-vacuum mass spectrometry methods, a cylindrical ion trap (CIT) instrument was built. The platform used an electron impact source as the ionization source, a custom CIT as the mass analyzer, and an electron multiplier as the ion detector. The dimensions of the CIT were r₀?=?10?mm and z₀?=?8.98?mm. Aiming at low-vacuum conditions, its working parameters were optimized. By increasing the frequency of the radio frequency (RF) voltage, optimizing the electron impact source, using a higher voltage on the electron multiplier, and improving the current preamplifier, the mass spectrometry of methyl salicylate was successively performed at helium buffer gas pressures up to 2?Pa, which was dozens of times higher than the upper pressure limit of ion trap mass spectrometers. More importantly, this pressure can be obtained using a single pump, avoiding the use of a bulky turbo pump. In addition, we measured and analyzed the mass deviation of methyl salicylate with the changes in the background gas pressure and RF voltage frequency. The results experimentally verified the theory that the stability regions expand with increasing pressure for the first time. The methods we explored could be used to develop next generation hand-portable instruments and bring new applications to mass spectrometry.     

液相色谱-串联质谱法快速测定人血浆中紫杉醇的含量

建立测定人血浆中紫杉醇含量的LC-MS/MS方法。取正常人血浆300μL,加入50ng/mL多西他赛溶液100μL,再加入1.2mL无水乙醚涡旋震荡提取2min后,离心(12000转,15min),取上清液吹干,然后用80%乙腈300μL溶解,取10μL进行LC-MS/MS测定。LC条件:采用ASB C_(18)柱(2.1×50mm,5μm),流动相:乙腈-4mmol/L醋酸铵(80:20,V/V),流速为0.3mL/min。质谱条件:ESI电离源,正离子模式,多反应监测(MRM)方式,用于定量分析的离子对分别为m/z 876.5→m/z 308.1(紫杉醇)和m/z 830.6→m/z 549.2(内标,多西他赛)。该方法紫杉醇的线性范围为0.2～1000ng/mL,最低检测限为0.2ng/mL。本方法操作简便、快速、结果准确、可用于该药物的含量测定,同时也可为临床药代动力学研究提供参考。     

Mass spectrometry in the biosynthetic and structural investigation of lignins

Lignin, a resistant cell-wall constituent of all vascular plants that consists of ether and carbon-linked methoxyphenols, is still far from being structurally described in detail. The main problem in its structural elucidation is the difficulty of isolating lignin from other wood components without damaging lignin itself. Furthermore, the high number and variegated forms of linkages that occur between the monomeric units and the chemical resistance of certain ether bonds limit the extent to which analytical and degradation procedures can be used to elucidate the lignin structure. Most of our present knowledge about the molecular structure of lignin is based on the analysis of monomers, dimers or, at the most, tetramers of degraded isolated lignins. Mass spectrometry (MS), which offers advantages in terms of speed, specificity, and sensitivity, has revealed to be a very powerful technique in the structural elucidation of lignins, in combination with the great number of chemical and thermal degradation methods available in the study of lignin. Moreover, the recent development of new ionization techniques in MS-electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS-has provided new possibilities to also analyze the undegraded lignin macromolecule.     

Investigation of intact protein complexes by mass spectrometry

Mass spectrometry has grown in recent years to a well-accepted and increasingly important complementary technique in structural biology. Especially electrospray ionization mass spectrometry is well suited for the detection of non-covalent protein complexes and their interactions with DNA, RNA, ligands, and cofactors. Over the last decade, significant advances have been made in the ionization and mass analysis techniques, which makes the investigation of even larger and more heterogeneous intact assemblies feasible. These technological developments have paved the way to study intact non-covalent protein-protein interactions, assembly and disassembly in real time, subunit exchange, cooperativity effects, and effects of cofactors, allowing us a better understanding of proteins in cellular processes. In this review, we describe some of the latest developments and several highlights.     

Optical detection methods for mass spectrometry of macroions

Detection of macroions has been a challenge in the field of mass spectrometry. Conventional ionization-based detectors, relying on production and multiplication of secondary electrons, are restricted to detection for charged particles of m/z < 1 x 10(6). While both energy-sensitive and charge-sensitive detectors have been developed recently to overcome the limitation, they are not yet in common use. Photon-sensitive detectors are suggested to be an alternative, with which detection of macroions (or charged particles) by either elastic light scattering (ELS) or laser-induced fluorescence (LIF) has been possible. In this article, we provide a critical review on the developments of novel optical detection methods for mass spectrometry of macroions, including both micron-sized and nano-sized synthetic polymers as well as high-mass biomolecules. Design and development of new spectrometers making possible observations of the mass spectra of macroions with sizes in the range of 10-10(3) nm or masses in the range of 1-10(6) MDa are illustrated. The potential and promise of this optical approach toward macroion detection with high efficiency are discussed in practical aspects.     

Tandem mass spectrometry in the study of fatty acids,bile acids,and steroids

Over the last 50 years, the mass spectrometry of lipids has evolved to become one of the most mature techniques in biomolecule analysis. Many volatile and non-polar lipids are directly amenable to analysis by gas-chromatography-mass spectrometry (GC-MS), a technique that combines the unsurpassed separation properties of gas-chromatography with the sensitivity and selectivity of electron ionization mass spectrometry. Less volatile and/or thermally labile lipids can be analyzed by GC-MS, following appropriate sample derivatization. However, many complex lipids are not readily analyzed by GC-MS, and it is these molecules that are the subject of the current review. Since the early 1970s, there have been three outstanding developments in mass spectrometry that are particularly appropriate in lipid analysis; i.e., the introduction of (i) fast atom bombardment (FAB); (ii) electrospray (ES); and (iii) tandem mass spectrometry (MS/MS). The FAB and ES ionization techniques will be discussed in relation to MS/MS, and examples of their application in biochemical studies will be presented. The review will concentrate on the analysis of fatty acids, bile acids, steroid conjugates, and neutral steroids.