Structural investigation of bacterial lipopolysaccharides by mass spectrometry and tandem mass spectrometry

Mass spectrometric studies are now playing a leading role in the elucidation of lipopolysaccharide (LPS) structures through the characterization of antigenic polysaccharides, core oligosaccharides and lipid A components including LPS genetic modifications. The conventional MS and MS/MS analyses together with CID fragmentation provide additional structural information complementary to the previous analytical experiments, and thus contribute to an integrated strategy for the simultaneous characterization and correct sequencing of the carbohydrate moiety. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 29:606–650, 2010     

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。     

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.     

Biological microanalysis by secondary ion mass spectrometry: status and prospects

Secondary ion mass spectrometric (SIMS) analysis of biological problems is an evolving technique. Lateral resolution of currently available commercial instrumentation estimated from actual samples is 0.5 micron, and subcellular organelles can be distinguished. The interrelationship of lateral resolution, elemental concentration and ionizability are, however, important in controlling the actual lateral resolution achievable. Although depth resolutions of 5 nm have been measured in other systems, no test of depth resolution in biological systems has been done, and this parameter is also concentration and ionization dependent. The development of liquid metal ion sources in combination with scanning ion microprobes has a potential lateral resolution of as little as 20 nm, but initial studies with this instrumentation show that tissue preservation at the submicron level becomes an important issue. The current development of a cold-transfer stage for SIMS instruments may obviate the problem of submicron localization of diffusible elements, and initial studies indicate that much more needs to be understood about the ionization process in hydrated samples. Quantitation of diffusible elements using external standards has been achieved over a 30 micron diameter analyzed area. Strategies for analysis of areas limited to 1 micron or less has been suggested using image processing techniques, which take advantage of the lateral resolution inherent in the ion optical system. Matrix effects in biological tissues have been reported and constitute a serious problem for analysis of biologicals which must be addressed for each question. However, development of laser ionization of sputtered particles may both increase the sensitivity of analysis and decrease the importance of ionizability of elements. Chemical analysis of organic molecules is another use of SIMS, but, at present, at the cost of losing localized information. SIMS analysis of biological samples is being systematically evaluated and requires increased accessibility of this instrumentation to the end-user for full development of its role in physiological problems.     

Top‐down mass spectrometry for the analysis of combinatorial post‐translational modifications

Protein post‐translational modifications (PTMs) are critically important in regulating both protein structure and function, often in a rapid and reversible manner. Due to its sensitivity and vast applicability, mass spectrometry (MS) has become the technique of choice for analyzing PTMs. Whilst the “bottom‐up' analytical approach, in which proteins are proteolyzed generating peptides for analysis by MS, is routinely applied and offers some advantages in terms of ease of analysis and lower limit of detection, “top‐down” MS, describing the analysis of intact proteins, yields unique and highly valuable information on the connectivity and therefore combinatorial effect of multiple PTMs in the same polypeptide chain. In this review, the state of the art in top‐down MS will be discussed, covering the main instrumental platforms and ion activation techniques. Moreover, the way that this approach can be used to gain insights on the combinatorial effect of multiple post‐translational modifications and how this information can assist in studying physiologically relevant systems at the molecular level will also be addressed. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:27–42, 2013     

Microorganism characterization by single particle mass spectrometry

In recent years a major effort by several groups has been undertaken to identify bacteria by mass spectrometry at the single cell level. The intent of this review is to highlight the recent progress made in the application of single particle mass spectrometry to the analysis of microorganisms. A large portion of the review highlights improvements in the ionization and mass analysis of bio-aerosols, or particles that contain biologically relevant molecules such as peptides or proteins. While these are not direct applications to bacteria, the results have been central to a progression toward single cell mass spectrometry. Developments in single particle matrix-assisted laser desorption/ionization (MALDI) are summarized. Recent applications of aerosol laser desorption/ionization (LDI) to the analysis of single microorganisms are highlighted. Successful applications of off-line and on-the-fly aerosol MALDI to microorganism detection are discussed. Limitations to current approaches and necessary future achievements are also addressed.     

Characterization of proteins by ambient mass spectrometry

Proteins play important roles in living systems and are topics of many fundamental and applied research projects. With the introduction of electrospray ionization and matrix‐assisted laser desorption/ionization for analysis of biomacromolecules in the late 1980s, mass spectrometry has become an important tool for characterization of proteins. Characterization of proteins in raw samples by these mass spectrometric techniques, however, usually requires extensive sample pretreatment. Ambient ionization techniques are new mass spectrometric techniques that allow direct analysis of samples with no or little sample preparation. Can these techniques facilitate or even eliminate sample preparation for mass spectrometric analysis of proteins? Apart from sample preparation, do these techniques offer any new features for characterization of proteins as compared with conventional ESI or MALDI? Recent advances in characterization of proteins by ambient mass spectrometry are summarized and commented in this article. © 2011 Wiley Periodicals, Inc. Mass Spec Rev 31:437–447, 2012     

Protein disulfide bond determination by mass spectrometry

The determination of disulfide bonds is an important aspect of gaining a comprehensive understanding of the chemical structure of a protein. The basic strategy for obtaining this information involves the identification of disulfide-linked peptides in digests of proteins and the characterization of their half-cystinyl peptide constituents. Tools for disulfide bond analysis have improved dramatically in the past two decades, especially in terms of speed and sensitivity. This improvement is largely due to the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), and complementary analyzers with high resolution and accuracy. The process of pairing half-cystinyl peptides is now generally achieved by comparing masses of non-reduced and reduced aliquots of a digest of a protein that was proteolyzed with intact disulfide bonds. Pepsin has favorable properties for generating disulfide-linked peptides, including its acidic pH optimum, at which disulfide bond rearrangement is precluded and protein conformations are likely to be unfolded and accessible to cleavage, and broad substrate specificity. These properties potentiate cleavage between all half-cystine residues of the substrate protein. However, pepsin produces complex digests that contain overlapping peptides due to ragged cleavage. This complexity can produce very complex spectra and/or hamper the ionization of some constituent peptides. It may also be more difficult to compute which half-cystinyl sequences of the protein of interest are disulfide-linked in non-reduced peptic digests. This ambiguity is offset to some extent by sequence tags that may arise from ragged cleavages and aid sequence assignments. Problems associated with pepsin cleavage can be minimized by digestion in solvents that contain 50% H(2) (18)O. Resultant disulfide-linked peptides have distinct isotope profiles (combinations of isotope ratios and average mass increases) compared to the same peptides with only (16)O in their terminal carboxylates. Thus, it is possible to identify disulfide-linked peptides in digests and chromatographic fractions, using these mass-specific markers, and to rationalize mass changes upon reduction in terms of half-cystinyl sequences of the protein of interest. Some peptides may require additional cleavages due to their multiple disulfide bond contents and/or tandem mass spectrometry (MS/MS) to determine linkages. Interpretation of the MS/MS spectra of peptides with multiple disulfides in supplementary digests is also facilitated by the presence of (18)O in their terminal carboxylates.     

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.     

Proteome analysis of tissues by mass spectrometry

Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh‐frozen and formalin‐fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue‐sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom‐up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys‐C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue‐specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm² obtained with laser capture microdissection to much larger amounts that weight several milligrams.     

Genotyping single nucleotide polymorphisms by mass spectrometry

In the last decade, the demand for high-throughput DNA analysis methods has dramatically increased, mainly due to the advent of the human genome sequencing project that is now nearing completion. Even though mass spectrometry did not contribute to that project, it is clear that it will have an important role in the post-genome sequencing era, in genomics and proteomics. In genomics, mainly matrix-assisted laser desorption/ionization (MALDI) mass spectrometry will contribute to large-scale single nucleotide polymorphism (SNP) genotyping projects. Here, the development and history of DNA analysis by mass spectrometry is reviewed and put into the context with the requirements of genomics. All major contributions to the field and their status and limitations are described in detail.     

Depth profiling by secondary ion mass spectrometry

The principles and applications of depth profiling by secondary ion mass spectrometry (SIMS) are reviewed. Discussed are the basic physical processes and instrumental factors which influence the shape of depth profiles and which have to be understood or controlled for successful experimental measurements. Microroughness caused by sputtering, atomic mixing by primary beam knock-on, and sample consumption limit the depth resolution which can be achieved while the chemical effect of ion yield enhancement by reactive species, matrix effects, and preferential sputtering can strongly affect the secondary ion signal. Instrumental effects to be controlled include beam uniformity, sample charging, and beam, and residual gas contamination. High depth resolution and sensitivity are the reasons for a wide variety of applications for SIMS depth profiling. Reviewed are measurements of the range distribution of ions implanted into semiconductors and their redistribution by subsequent annealing, studies of thin films and of oxide layers, diffusion measurements in metals, semiconductors, and minerals, measurements of elemental surface enhancements in airborne particles, and lunar glass spherules, and the search for solar wind implanted ions in lunar crystals.     

Membrane introduction mass spectrometry: trends and applications

Recent advances in membrane introduction mass spectrometry (MIMS) are reviewed. On-line monitoring is treated by focusing on critical variables, including the nature and dimensions of the membrane, and the analyte vapor pressure, diffusivity, and solubility in the membrane barrier. Sample introduction by MIMS is applied in (i) on-line monitoring of chemical and biological reactors, (ii) analysis of volatile organic compounds in environmental matrices, including air, water and soil, and (iii) in more fundamental studies, such as measurements of thermochemical properties, reaction mechanisms, and kinetics. New semipermeable membranes are discussed, including those consisting of thin polymers, low vapor pressure liquids, and zeolites. These membranes have been used to monitor polar compounds, selectively differentiate compounds through affinity-binding, and provide isomer differentiation based on molecular size. Measurements at high spatial resolution, for example, using silicone-capped hypodermic needle inlets, are also covered, as is electrically driven sampling through microporous membranes. Other variations on the basic MIMS experiment include analyte preconcentration through cryotrapping (CT-MIMS) or trapping in the membrane (trap-and-release), as well as differential thermal release methods and reverse phase (i.e., organic solvent) MIMS. Method limitations center on semivolatile compounds and complex mixture analysis, and novel solutions are discussed. Semivolatile compounds have been monitored with thermally assisted desorption, ultrathin membranes and derivatization techniques. Taking advantage of the differences in time of membrane permeation, mixtures of structurally similar compounds have been differentiated by using sample modulation techniques and by temperature-programmed desorption from a membrane interface. Selective ionization techniques that increase instrument sensitivity towards polar compounds are also described, and comparisons are made with other direct sampling (nonchromatographic) methods that are useful in mixture analysis.