Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry

Microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein–protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein–peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a predefined raster of matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium–tin oxide. Furthermore, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI–TOF instruments and has general utility for the label-free analysis of microarray assays.     

Probing neuropeptide signaling at the organ and cellular domains via imaging mass spectrometry

Imaging mass spectrometry (IMS) has evolved to be a promising technology due to its ability to detect a broad mass range of molecular species and create density maps for selected compounds. It is currently one of the most useful techniques to determine the spatial distribution of neuropeptides in cells and tissues. Although IMS is conceptually simple, sample preparation steps, mass analyzers, and software suites are just a few of the factors that contribute to the successful design of a neuropeptide IMS experiment. This review provides a brief overview of IMS sampling protocols, instrumentation, data analysis tools, technological advancements and applications to neuropeptide localization in neurons and endocrine tissues. Future perspectives in this field are also provided, concluding that neuropeptide IMS would greatly facilitate studies of neuronal network and biomarker discovery.     

Imaging mass spectrometry in biomarker discovery and validation

Biomarker discovery and validation involves the consideration of many issues and challenges in order to be effectively used for translation from bench to bedside. Imaging mass spectrometry (IMS) is a new technology to assess spatial molecular arrangements in tissue sections, going far beyond microscopy in providing hundreds of different molecular images from a single scan without the need of target-specific reagents. The possibility to correlate distribution maps of multiple analytes with histological and clinical features makes it an ideal tool to discover diagnostic and prognostic markers of diseases. Some recently published studies that show the usefulness and advantages of this technology in the field of cancer research are highlighted.     

In situ metabolomic mass spectrometry imaging: recent advances and difficulties

MS imaging (MSI) is a remarkable new technology that enables us to determine the distribution of biological molecules present in tissue sections by direct ionization and detection. This technique is now widely used for in situ imaging of endogenous or exogenous molecules such as proteins, lipids, drugs and their metabolites, and it is a potential tool for pathological analysis and the investigation of disease mechanisms. MSI is also thought to be a technique that could be used for biomarker discovery with spatial information. The application of MSI to the study of endogenous metabolites has received considerable attention because metabolites are the result of the interactions of a system's genome with its environment and a total set of these metabolites more closely represents the phenotype of an organism under a given set of conditions. Recent studies have suggested the importance of in situ metabolite imaging in biological discovery and biomedical applications, but several issues regarding the technical application limits of MSI still remained to be resolved. In this review, we describe the capabilities of the latest MSI techniques for the imaging of endogenous metabolites in biological samples, and also discuss the technical problems and new challenges that need to be addressed for effective and widespread application of MSI in both preclinical and clinical settings.     

Imaging mass spectrometry statistical analysis

Imaging mass spectrometry is increasingly used to identify new candidate biomarkers. This clinical application of imaging mass spectrometry is highly multidisciplinary: expertise in mass spectrometry is necessary to acquire high quality data, histology is required to accurately label the origin of each pixel's mass spectrum, disease biology is necessary to understand the potential meaning of the imaging mass spectrometry results, and statistics to assess the confidence of any findings. Imaging mass spectrometry data analysis is further complicated because of the unique nature of the data (within the mass spectrometry field); several of the assumptions implicit in the analysis of LC-MS/profiling datasets are not applicable to imaging. The very large size of imaging datasets and the reporting of many data analysis routines, combined with inadequate training and accessible reviews, have exacerbated this problem. In this paper we provide an accessible review of the nature of imaging data and the different strategies by which the data may be analyzed. Particular attention is paid to the assumptions of the data analysis routines to ensure that the reader is apprised of their correct usage in imaging mass spectrometry research.     

Going forward: Increasing the accessibility of imaging mass spectrometry

The driving force behind the high and increasing popularity of imaging mass spectrometry is its demonstrated potential for the determination of new diagnostic/prognostic biomarkers and its ability to simultaneously trace the distributions of pharmaceuticals and their metabolites in tissues without the need to develop expensive radioactively-labeled analogues. Both of these applications would benefit from standardized methods, for the development of novel MS-based molecular histology tests and governmental-approved MS-based assays for pharmaceutical development. In addition, the broader scientific community would benefit from the increased accessibility of the technique. Currently imaging MS studies are individual endeavors, utilizing the individual expertise and infrastructure of a single laboratory and their immediate collaborators. A wide array of tissue preparation, data acquisition and data analysis techniques has been developed but lacks an international collaborative structure and data sharing capabilities. Such a collaborative framework would enable methodological exchange and detailed comparisons of analytical capabilities, to explore synergies between the different methods and result in the development of robust standardized methods. Here we describe the activities of a new European imaging MS network that will explicitly compare and contrast existing methods to provide best practice guidelines for the entire healthcare research community.     

Imaging mass spectrometry: principle and application

Imaging mass spectrometry (IMS) is two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules, and enables us to monitor not only the identification of unknown molecules but also the localization of numerous molecules simultaneously. Among the ionization techniques, matrix assisted laser desorption/ionization (MALDI) is one of the most generally used for IMS, which allows the analysis of numerous biomolecules ranging over wide molecular weights. Proper selection and preparation of matrix is essential for successful imaging using IMS. Tandem mass spectrometry, which is referred to MSⁿ, enables the structural analysis of a molecule detected by the first step of IMS. Applications of IMS were initially developed for studying proteins or peptides. At present, however, targets of IMS research have expanded to the imaging of small endogenous metabolites such as lipids, exogenous drug pharmacokinetics, exploring new disease markers, and other new scientific fields. We hope that this new technology will open a new era for biophysics.     

Determination of oligonucleotide molecular masses by MALDI mass spectrometry

MALDI mass spectrometry (MS) of 14- to 42-mer homogeneous oligonucleotides and their mixtures was carried out using a Vision 2000 instrument (Thermo BioAnalysis, Finnigan, United States). Conditions for the determination of oligonucleotide molecular masses were optimized by applying various matrices and operation modes. The most reproducible results with minimal uncontrolled decomposition of the oligonucleotides including their apurinization during the MALDI MS registration were obtained using 2,4,6-trihydroxyacetophenone as a matrix instead of 3-hydroxypicolinic acid usually employed in the mass spectrometry of oligonucleotides. Our approach allows the determination of molecular masses of oligonucleotides obtained by chemical synthesis and the evaluation of their component composition and purity. It was applied to the mass spectrometric analysis of oligonucleotides containing a 3-(methyl-C-phosphonate) group or a modified 1,N ⁶-ethenodeoxyadenosine unit.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 151–158.Original Russian Text Copyright © 2005 by Streletskii, Kozlova, Esipov, Kayushin, Korosteleva, Esipov.     

Advances in mass spectrometry-based cancer research and analysis: from cancer proteomics to clinical diagnostics

Introduction: The last 20 years have seen significant improvements in the analytical capabilities of biological mass spectrometry (MS). Studies using advanced MS have resulted in new insights into cell biology and the etiology of diseases as well as its use in clinical applications.

Areas covered: This review discusses recent developments in MS-based technologies and their cancer-related applications with a focus on proteomics. It also discusses the issues around translating the research findings to the clinic and provides an outline of where the field is moving.

Expert commentary: Proteomics has been problematic to adapt for the clinical setting. However, MS-based techniques continue to demonstrate potential in novel clinical uses beyond classical cancer proteomics.     

生物质谱与蛋白质组学

蛋白质组学是后基因组学时代最受关注的研究领域之一,其核心的鉴定技术——生物质谱近年来在仪器设计以及鉴定通量、分辨率和灵敏度等各方面均有质的飞跃,促进了蛋白质表达谱作图、定量蛋白质组分析、亚细胞器蛋白质组作图、蛋白质翻译后修饰以及蛋白质相互作用等蛋白质组研究各个领域的飞速发展。本综述了生物质谱技术的最新进展,及其在蛋白质组学研究中的应用。     

微生物学研究中的软电离质谱技术

包括基质辅助激光解吸电离（ＭＡＬＤＩ）和电喷雾（ＥＳＩ）在内的软电离质谱是最近发展起来的质谱技术,由于这些电离方式对样品的破坏性小,质量测定范围大,分子量测定准确,样品纯度要求不高很适合分析成分复杂的微生物样品,ＭＡＬＩＤＩ－ＴＯＦ－ＭＳ结合高分辨率的二维ＳＤＳ－ＰＡＧＥ可以分析１０＾－１２摩尔水平的蛋白,是细菌蛋白质研究过程中必不可少的工具。最近的研究工作表明,通过ＭＡＩＤＩ－ＴＯＦ－ＭＳ或ＨＰ     

The MALDI mass spectrometry in the identification of new proteins in snake venoms

By MALDI MS, we searched cobra venoms for new low-content polypeptides. A number of new proteins with molecular masses 7–25 kDa, characteristic of the known snake protein toxins, were identified, with the content of one of them less than 0.02%.     

Mass spectrometry of nucleotides and oligonucleotides

Recent advances in the use of mass spectrometry for the determination of the molecular weight and sequencing of oligonucleotides are disscussed. Matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) mass spectrometry have been shown to be especially important techniques for both molecular weight assignment and sequencing of oligonucleotides, and are the focus of this article which covers the literature through early 1996.     

Bioinformatics in mass spectrometry data analysis for proteomics studies

Mass spectrometry is a technique widely employed for the identification and characterization of proteins. The role of bioinformatics is fundamental for the elaboration of mass spectrometry data due to the amount of data that this technique can produce. To process data efficiently, new software packages and algorithms are continuously being developed to improve protein identification and characterization in terms of high-throughput and statistical accuracy. However, many limitations exist concerning bioinformatics spectral data elaboration. This review aims to critically cover the recent and future developments of new bioinformatics approaches in mass spectrometry data analysis for proteomics studies.     

Towards GAG glycomics: analysis of highly sulfated heparins by MALDI-TOF mass spectrometry

Glycomics is a developing field that provides structural information on complex populations of glycans isolated from tissues, cells and organs. Strategies employing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are central to glycomic analysis. Current MALDI-based glycomic strategies are capable of efficiently analyzing glycoprotein and glycosphingolipid glycomes but little attention has been paid to devising glycomic methodologies suited to the analysis of glycosaminoglycan (GAG) polysaccharides which pose special problems for MALDI analysis because of their high level of sulfation and large size. In this paper, we describe MALDI strategies that have been optimized for the analysis of highly sulfated GAG-derived oligosaccharides. A crystalline matrix norharmane, as well as an ionic liquid 1-methylimidazolium alpha-cyano-4-hydroxycinnamate (ImCHCA), have been used for the analysis of heparin di-, tetra-, hexa- and decasaccharides carrying from 2 to 13 sulfate groups. Information about the maximum number of sulfate groups is obtained using the ionic liquid whereas MALDI-TOF/TOF MS/MS experiments using norharmane allowed the determination of the nature of the glycosidic backbone, and more precise information about the presence and the position in the sequence of N-acetylated residues.     

Convenient and rapid analysis of linkage isomers of fucose-containing oligosaccharides by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was used to analyze three pyridylamino (PA)-fucosyloligosaccharides isolated from human milk: lacto-N-fucopentaose (LNFP) I [Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-PA], LNFP II [Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-PA], and LNFP III [Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-PA]. These oligosaccharides are linkage isomers. MALDI-QIT-TOF MS provides MSⁿ spectra, which we used to characterize these PA-oligosaccharides. MS/MS/MS analysis of the non-reducing end tri-saccharide ions generated by MS/MS was able to distinguish these oligosaccharide isomers. The MALDI-QIT-TOF MS is a very convenient and rapid method, therefore, it would be useful for high throughput structural analyses of various types of pyridylaminated oligosaccharide isomers.     

Hydrogen/deuterium exchange mass spectrometry of actin in various biochemical contexts

Hydrogen/deuterium exchange mass spectrometry (H/D MS) of monomeric actin (G-actin), polymeric actin (F-actin), phalloidin-bound F-actin and G-actin complexed with DNase I provides new insights into the architecture of F-actin and the effects of phalloidin and DNase I binding. Although the overall pattern of deuteration change supports the gross features of the Holmes F-actin model, two important differences were observed. Most significantly, no change in deuteration was observed in the critical "hydrophobic plug" region, suggesting this feature may not be present. Polymerization also produced deuteration increases for peptide fragments containing the ATP phosphate-binding loops, suggesting G-actin transitions to a more "open" conformation upon polymerization. However, polymerization produced decreases in deuteration mainly localized to the "inner", filament-axis side as predicted by the Holmes model. Mapping the phalloidin-induced decreases in F-actin deuteration onto the Lorenz binding site produced a single common patch straddling two monomers across the 1-start helix contact, again consistent with the Holmes architecture. Finally, both DNase I and phalloidin were able to alter the deuteration of regions distal to their respective binding sites. These results highlight the great opportunities for H/D MS to exploit high-resolution structures for detailed studies of the organization and dynamics of complex molecular assemblies.     

Peptide profiling of cerebrospinal fluid by mass spectrometry

The search for biomarkers is driven by the increasing clinical importance of early diagnosis. Reliable biomarkers can also assist in directing therapy, monitoring disease activity and the efficacy of treatment. In addition, the discovery of novel biomarkers might provide clues to the pathogenesis of a disease. The dynamic range of protein concentrations in body fluids exceeds 10 orders of magnitude. These huge differences in concentrations complicate the detection of proteins with low expression levels. Since all classical biomarkers have low expression levels (e.g., prostate-specific antigen: 2–4 µg/l; and CA125: 20–35 U/ml), new developments with respect to identification and validation techniques of the low-abundance proteins are required. This review will discuss the current status of profiling cerebrospinal fluid using mass spectrometry-based techniques, and new developments in this area.     

Detection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling

Although colorectal cancer is one of the best-characterized tumors with regard to the multistep progression, it remains one of the most frequent and deadly neoplasms. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, changes in protein expression between microdissected normal and tumorous colonic epithelium were analyzed. Cryostat sections from colorectal tumors, adenoma tissue, and adjacent normal mucosa were laser-microdissected and analyzed using ProteinChip Arrays. The derived MS profiles exhibited numerous statistical differences. One peak showing significantly high expression in the tumor was purified by reverse-phase chromatography and SDS-PAGE. The protein band of interest was passively eluted from the gel and identified as heat shock protein 10 (HSP 10) by tryptic digestion, peptide mapping, and MS/MS analysis. This tumor marker was further characterized by immunohistochemistry. Analysis of HSP 10-positive tissue by ProteinChip technology confirmed the identity of this protein. This work demonstrates that biomarker in colorectal cancer can be detected, identified, and assessed by a proteomic approach comprising tissue microdissection, protein profiling, and immunological techniques. In our experience, histological defined microdissected tissue areas should be used to identify proteins that might be responsible for tumorigenesis.     

Quantitative mass spectrometry imaging of propranolol and olanzapine using tissue extinction calculation as normalization factor

In order to quantify small molecules at the early stage of drug discovery, we developed a quantitation approach based on mass spectrometry imaging (MSI) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) without the use of a labeled compound. We describe a method intended to respond to the main challenges encountered in quantification through MALDI imaging dedicated to whole-body or single heterogeneous organ samples (brain, eye, liver). These include the high dependence of the detected signal on the matrix deposition, the MALDI ionization yield of specific target molecules, and lastly, the ion suppression effect on the tissue. To address these challenges, we based our approach on the use of a normalization factor called the TEC (Tissue Extinction Coefficient). This factor takes into account the ion suppression effect that is both tissue- and drug-specific. Through this protocol, the amount of drug per gram of tissue was determined, which in turn, was compared with other analytical techniques such as Liquid Chromatography-Mass spectrometry (LC-MS/MS).