蛋白质组学中质谱分析前的预富集研究进展

回顾了近年来生物质谱分析鉴定蛋白质及多肽前的预富集方法研究进展,其中包括固相微萃取富集方法、靶上富集方法、电洗脱富集方法以及磷酸化蛋白质/肽段的富集方法,总结和归纳了各种方法所具有的优缺点,引用文献59篇。     

Mass spectrometric imaging of synthetic polymers

The analysis of synthetic polymers represents today an important part of polymer science to determine their physical properties and to optimize the performance of polymeric materials for block copolymers as well as blend systems. The characterization can easily and rapidly be performed by mass spectrometry. In particular, the film formation of a synthetic polymer is of interest in material research and quality control, which can be determined by employing mass spectrometric imaging (MSI) using secondary ion mass spectrometry (SIMS) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. MALDI-MSI has been rapidly improved for the analysis of tissue cross-sections due to its soft ionization and accessible m/z range, which both also play an important role in polymer science. On the other hand, SIMS-MSI enables a sub-micrometer molecular spatial resolution, which is limited in MALDI-MSI due to the spatial resolution capabilities of the laser desorption process. The aim of the present contribution is to summarize recent advances in both imaging techniques for the analysis of synthetic polymers and to highlight their capabilities to correlate several imaging modalities in future applications.     

Organic ion imaging of biological tissue with secondary ion mass spectrometry and matrix-assisted laser desorption/ionization

Organic secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be used to produce molecular images of samples. This is achieved through ionization from a clearly identified point on a flat sample, and performing a raster of the sample by moving the point of ionization over the sample surface. The unique analytical capabilities of mass spectrometry for mapping a variety of biological samples at the tissue level are discussed. SIMS provides information on the spatial distribution of the elements and low molecular mass compounds as well as molecular structures on these compounds, while MALDI yields spatial information about higher molecular mass compounds, including their distributions in tissues at very low levels, as well as information on the molecular structures of these compounds. Application of these methods to analytical problems requires appropriate instrumentation, sample preparation methodology, and a data presentation usually in a three-coordinate plot where x and y are physical dimensions of the sample and z is the signal amplitude. The use of imaging mass spectrometry is illustrated with several biological systems.     

二次离子质谱生物成像

二次离子质谱作为目前空间分辨率最高的质谱成像技术,以其免标记、高灵敏、多组分检测优势和亚微米级高空间分辨成像优势为诸多生命科学问题的研究提供了全新的分析手段,在基础细胞生物学、组织生理病理学、生物医药与临床医学等领域的研究中得到了广泛应用.本文综述了二次离子质谱在生物组织、细胞、仿生生物膜等体系中的质谱成像研究进展.     

MALDI imaging directed laser ablation tissue microsampling for data independent acquisition proteomics

A multimodal workflow for mass spectrometry imaging was developed that combines MALDI imaging with protein identification and quantification by liquid chromatography tandem mass spectrometry (LC‐MS/MS). Thin tissue sections were analyzed by MALDI imaging, and the regions of interest (ROI) were identified using a smoothing and edge detection procedure. A midinfrared laser at 3‐μm wavelength was used to remove the ROI from the brain tissue section after MALDI mass spectrometry imaging (MALDI MSI). The captured material was processed using a single‐pot solid‐phase‐enhanced sample preparation (SP3) method and analyzed by LC‐MS/MS using ion mobility (IM) enhanced data independent acquisition (DIA) to identify and quantify proteins; more than 600 proteins were identified. Using a modified database that included isoform and the post‐translational modifications chain, loss of the initial methionine, and acetylation, 14 MALDI MSI peaks were identified. Comparison of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the identified proteins was achieved through an evolutionary relationships classification system.     

The Future of Biological Mass Spectrometry

Biological applications of mass spectrometry have grown exponentially since the discovery of MALDI and electrospray ionization techniques. This growth has been further fueled by the massive volume of DNA sequence information that is now available. An ambitious goal of some of this research is to monitor the level and modification of all proteins and metabolites in a biological sample such as plasma. A major research effort in mass spectrometry and related disciplines has been expended over the past several years toward reaching this and other less ambitious goals, and considerable progress has been made; but the presently available tools are clearly not sufficient for these very difficult tasks. In this “critical insight” discussion we suggest that recent advances in time-of-flight (TOF) technology with MALDI ionization may provide some important new tools for achieving the goals of biological research.     

Mass spectral imaging and profiling of neuropeptides at the organ and cellular domains

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a rapid and sensitive analytical method that is well suited for determining molecular weights of peptides and proteins from complex samples. MALDI-MS can be used to profile the peptides and proteins from single-cell and small tissue samples without the need for extensive sample preparation. Furthermore, the recently developed MALDI imaging technique enables mapping of the spatial distribution of signaling molecules in tissue samples. Several examples of signaling molecule analysis at the single-cell and single-organ levels using MALDI-MS technology are highlighted followed by an outlook of future directions.     

双步激光解析/激光电离质谱法及其应用的新进展

激光光电离技术已广泛应用于质谱领域.基于单束激光的基质辅助激光解析(MALDI)质谱分析方法,已成为质谱分析生物大分子的标准方法之一.本文介绍的是另一种新的激光质谱分析方法:双步激光解析/激光电离质谱法(L2MS),与MALDI相比,该方法不需要加入与样品形成共结晶的基质,同时可通过独立地改变两束激光的光强和波长达到优...     

Measuring salty samples without adducts with MALDI MS

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been used for the discovery of hundreds of novel cell to cell signaling peptides. Beyond its advantages of sensitivity and minimal sample preparation requirements, MALDI MS is attractive for biological analyses as high quality mass spectra may be obtained directly from specific locations within prepared tissue sections. However, due to the large quantity of salts present in physiological tissues, these mass spectra often contain many adducts of cationic salts such as sodium and potassium, in addition to the molecular ion [M + H]⁺. To reduce the presence of cation adducts in MALDI mass spectra obtained directly from tissues, we present a methodology that uses a slow condensation procedure to enable the formation of distinct regions of matrix/analyte crystals and cation (salt) crystals. Secondary ion mass spectrometric imaging suggests that the salts and MALDI matrix undergo a mutually exclusive crystallization process that results in the separation of the salts and matrix in the sample.     

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix‐assisted laser desorption ionization (MALDI) is recognized as the method‐of‐choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix‐free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI‐MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227‐fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295‐fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na⁺. Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49‐fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19‐fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.     

In situ isobaric lipid mapping by MALDI–ion mobility separation–mass spectrometry imaging

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high‐resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.     

Comparision of FAB and MALDI Mass Spectrometry of Ginsenosides

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Subcellular imaging mass spectrometry of brain tissue

Imaging mass spectrometry provides both chemical information and the spatial distribution of each analyte detected. Here it is demonstrated how imaging mass spectrometry of tissue at subcellular resolution can be achieved by combining the high spatial resolution of secondary ion mass spectrometry (SIMS) with the sample preparation protocols of matrix-assisted laser desorption/ionization (MALDI). Despite mechanistic differences and sampling 10(5) times less material, matrix-enhanced (ME)-SIMS of tissue samples yields similar results to MALDI (up to m/z 2500), in agreement with previous studies on standard compounds. In this regard ME-SIMS represents an attractive alternative to polyatomic primary ions for increasing the molecular ion yield. ME-SIMS of whole organs and thin sections of the cerebral ganglia of Lymnaea stagnalis demonstrate the advantages of ME-SIMS for chemical imaging mass spectrometry. Subcellular distributions of cellular analytes are clearly obtained, and the matrix provides an in situ height map of the tissue, allowing the user to identify rapidly regions prone to topographical artifacts and to deconvolute topographical losses in mass resolution and signal-to-noise ratio.     

Spatially Resolved Quantification of Gadolinium(III)‐Based Magnetic Resonance Agents in Tissue by MALDI Imaging Mass Spectrometry after In Vivo MRI

Gadolinium(III)‐based contrast agents improve the sensitivity and specificity of magnetic resonance imaging (MRI), especially when targeted contrast agents are applied. Because of nonlinear correlation between the contrast agent concentration in tissue and the MRI signal obtained in vivo, quantification of certain biological or pathophysiological processes by MRI remains a challenge. Up to now, no technology has been able to provide a spatially resolved quantification of MRI agents directly within the tissue, which would allow a more precise verification of in vivo imaging results. MALDI imaging mass spectrometry for spatially resolved in situ quantification of gadolinium(III) agents, in correlation to in vivo MRI, were evaluated. Enhanced kinetics of Gadofluorine M were determined dynamically over time in a mouse model of myocardial infarction. MALDI imaging was able to corroborate the in vivo imaging MRI signals and enabled in situ quantification of the gadolinium probe with high spatial resolution.     

Data Processing for 3D Mass Spectrometry Imaging

Data processing for three dimensional mass spectrometry (3D-MS) imaging was investigated, starting with a consideration of the challenges in its practical implementation using a series of sections of a tissue volume. The technical issues related to data reduction, 2D imaging data alignment, 3D visualization, and statistical data analysis were identified. Software solutions for these tasks were developed using functions in MATLAB. Peak detection and peak alignment were applied to reduce the data size, while retaining the mass accuracy. The main morphologic features of tissue sections were extracted using a classification method for data alignment. Data insertion was performed to construct a 3D data set with spectral information that can be used for generating 3D views and for data analysis. The imaging data previously obtained for a mouse brain using desorption electrospray ionization mass spectrometry (DESI-MS) imaging have been used to test and demonstrate the new methodology.     

Utilization of matrix-assisted laser desorption/ionization imaging mass spectrometry to search for cannabis in herb mixtures

Herb mixtures including cannabis among the other herbs have recently appeared. When cannabinoids from herb extracts are detected by chemical examinations such as gas chromatography/mass spectrometry, forensic analysts have to determine whether cannabis is actually in the mixture or the cannabinoids are spiked. Morphological examinations are time-consuming, since it is difficult to find several pieces of cannabis among a large number of herb pieces using a microscope. Here, we propose a procedure for efficiently searching for cannabis in herb mixtures using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI/IMS). Pieces of herb mixtures were spread on double-sided adhesive tape attached to a stainless steel plate. The pieces were then covered with a conductive sheet and pressed. After a solution containing a matrix reagent was sprayed, the distribution of cannabinoids in the sample was visualized by MALDI/IMS. Then, just the pieces with cannabinoids could be picked up selectively with tweezers and decolorized. Cystolith hairs and trichomes, which are characteristic of cannabis, were observed in most of these pieces using a biological microscope. This MALDI/IMS procedure enables cannabis to be found in herb mixtures without inefficient random sampling and microscopic morphological examination.     

Chemical and Topographical Single‐Cell Imaging by Near‐Field Desorption Mass Spectrometry

Simultaneously acquiring chemical and topographical information within a single cell at nanoscale resolutions is vital to cellular biology, yet it remains a great challenge due to limited lateral resolutions and detection sensitivities. Herein, the development of near‐field desorption mass spectrometry for correlated chemical and topographical imaging is reported, thereby bridging the gap between laser‐based mass spectrometry (MS) methods and multimodal single‐cell imaging. Using this integrated platform, an imaging resolution of 250 nm and 3D topographically reconstructed chemical single‐cell imaging were achieved. This technique offers more in‐depth cellular information than micrometer‐range laser‐based MS imaging methods. Considering the simplicity and compact size of the near‐field device, this technique can be introduced to MALDI‐MS, expanding the multimodal abilities of MS at nanoscale resolutions.     

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the most widely used techniques in proteomics to achieve structural identification and characterization of proteins and peptides, including their variety of proteoforms due to post-translational modifications (PTMs) or protein–protein interactions (PPIs). MALDI-MS and MALDI tandem mass spectrometry (MS/MS) have been developed as analytical techniques to study small and large molecules, offering picomole to femtomole sensitivity and enabling the direct analysis of biological samples, such as biofluids, solid tissues, tissue/cell homogenates, and cell culture lysates, with a minimized procedure of sample preparation. In the last decades, structural identification of peptides and proteins achieved by MALDI-MS/MS helped researchers and clinicians to decipher molecular function, biological process, cellular component, and related pathways of the gene products as well as their involvement in pathogenesis of diseases. In this review, we highlight the applications of MALDI ionization source and tandem approaches for MS for analyzing biomedical relevant peptides and proteins. Furthermore, one of the most relevant applications of MALDI-MS/MS is to provide “molecular pictures”, which offer in situ information about molecular weight proteins without labeling of potential targets. Histology-directed MALDI-mass spectrometry imaging (MSI) uses MALDI-ToF/ToF or other MALDI tandem mass spectrometers for accurate sequence analysis of peptide biomarkers and biological active compounds directly in tissues, to assure complementary and essential spatial data compared with those obtained by LC-ESI-MS/MS technique.     

Quantification of pharmaceutical compounds in tissue and plasma samples using selective ion accumulation with multiple mass isolation windows

Quantification of pharmaceutical compounds using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is an alternative to traditional liquid chromatography (LC)-MS techniques. Benefits of MALDI-based approaches include rapid analysis times for liquid samples and imaging mass spectrometry capabilities for tissue samples. As in most quantification experiments, the use of internal standards can compensate for spot-to-spot and shot-to-shot variability associated with MALDI sampling. However, the lack of chromatographic separation in traditional MALDI analyses results in diminished peak capacity due to the chemical noise background, which can be detrimental to the dynamic range and limit of detection of these approaches. These issues can be mitigated by using a hybrid mass spectrometer equipped with a quadrupole mass filter (QMF) that can be used to fractionate ions based on their mass-to-charge ratios. When the masses of the analytes and internal standards are sufficiently disparate in mass, it can be beneficial to effect multiple narrow mass isolation windows using the QMF, as opposed to a single wide mass isolation window, to minimize chemical noise while allowing for internal standard normalization. Herein, we demonstrate a MALDI MS quantification workflow incorporating multiple sequential mass isolation windows enabled on a QMF, which divides the total number of MALDI laser shots into multiple segments (i.e., one segment for each mass isolation window). This approach is illustrated through the quantitative analysis of the pharmaceutical compound enalapril in human plasma samples as well as the simultaneous quantification of three pharmaceutical compounds (enalapril, ramipril, and verapamil). Results show a decrease in the limit of detection, relative standard deviations below 10%, and accuracy above 85% for drug quantification using multiple mass isolation windows. This approach has also been applied to the quantification of enalapril in brain tissue from a rat dosed in vitro. The average concentration of enalapril determined by imaging mass spectrometry is in agreement with the concentration determined by LC–MS, giving an accuracy of 104%.     

有机硼化合物的质谱分析

对有机硼化合物进行质谱分析,考察不同种类仪器对样品分析的影响,确定使用电喷雾质谱仪效果最好。进一步考察了流动相、正负离子检测模式、离子化试剂及其浓度等因素的影响。实验发现:样品浓度为1×10-4mol/L,使用V(甲醇):V(H2O)=1:1的混合溶剂为流动相,正离子检测,0.1mol/LNa 为离子化试剂条件下可得到满意的谱图。