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.     

Advances in proteomics data analysis and display using an accurate mass and time tag approach

Proteomics has recently demonstrated utility for increasing the understanding of cellular processes on the molecular level as a component of systems biology approaches and for identifying potential biomarkers of various disease states. The large amount of data generated by utilizing high efficiency (e.g., chromatographic) separations coupled with high mass accuracy mass spectrometry for high-throughput proteomics analyses presents challenges related to data processing, analysis, and display. This review focuses on recent advances in nanoLC-FTICR-MS-based proteomics approaches and the accompanying data processing tools that have been developed to display and interpret the large volumes of data being produced.     

A History of Molecular Level Analysis of Natural Organic Matter by FTICR Mass Spectrometry and The Paradigm Shift in Organic Geochemistry

Natural organic matter (NOM) is a complex mixture of biogenic molecules resulting from the deposition and transformation of plant and animal matter. It has long been recognized that NOM plays an important role in many geological, geochemical, and environmental processes. Of particular concern is the fate of NOM in response to a warming climate in environments that have historically sequestered carbon (e.g., peatlands and swamps) but may transition to net carbon emitters. In this review, we will highlight developments in the application of high-field Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) in identifying the individual components of complex NOM mixtures, focusing primarily on the fraction that is dissolved in natural waters (dissolved organic matter or DOM). We will first provide some historical perspective on developments in FTICR technology that made molecular-level characterizations of DOM possible. A variety of applications of the technique will then be described, followed by our view of the future of high-field FTICR MS in carbon cycling research, including a particularly exciting metabolomic approach.     

Seasonal changes predominant over manure application in driving dissolved organic matter shifts in agricultural runoff

Non-point source nutrient pollution can cause eutrophication, hypoxic events, and harmful algal blooms in surface waters. Surface water eutrophication is frequently attributed to agricultural runoff, a known source of inorganic and organic nutrients to surface and subsurface waters. Analysis of dissolved organic matter (DOM) using ultrahigh resolution mass spectrometry is an effective tool for characterizing diverse organic nitrogen and phosphorus components of DOM that may be mineralized and/or assimilated and ultimately contribute to eutrophication. An anion exchange resin was applied to extract DOM from surface and subsurface tile runoff from agricultural plots located within the Maumee River watershed (Ohio, USA) that received raw liquid manure, polymer-treated dewatered manure, or no manure (control). DOM extracts were analyzed using Fourier transform ion cyclotron resonance mass spectrometry prior to manure application and during three runoff events after manure application. Organic nitrogen compounds were predominant, making up 38–48% of the total assigned formulas. Principle coordinates analysis indicated clustering predominately by sample month and secondarily by treatment. These findings show that a large amount of variability in DOM in runoff was observed regardless of manure application, indicating the important role played by natural processes in driving transformation of the DOM pool during the growing season. Molecular ions unique to runoff from manure-applied plots were also identified that may be useful in source-tracking of non-point source nutrient pollution in waters draining to Lake Erie.     

傅里叶变换离子回旋共振质谱仪表征VGO馏分油中噻吩类含硫化合物

在平均质量分辨率为220000,m/z检测范围150～1200的条件下,采用大气压光致电离源(APPI)的9.4 T傅里叶变换离子回旋共振质谱仪(FTICR MS),建立了VGO馏分油中噻吩类硫化物的表征方法。在所研究的VGO中,主要包括2种(含1个硫原子S₁和含2个硫原子S₂),共计29类噻吩类硫化物,碳数分布范围为15～50。对VGO依沸点分布的窄馏分进行含硫化合物种类分布考察结果表明,在4个窄馏分中,均,通以S₁硫化物为主, S₂硫化物含量较少,而且随着馏分沸点的增加,其中的烷基取代基的碳数增加,多环芳烃含硫化合物含量增加,S₂硫化物含量增加。     

Peptide mapping of proteins in human body fluids using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Human body fluids have been rediscovered in the post-genomic era as great sources of biological markers and perhaps particularly as sources of potential protein biomarkers of disease. Analytical tools that allow rapid screening, low sample consumption, and accurate protein identification are of great importance in studies of complex biological samples and clinical diagnosis. Mass spectrometry is today one of the most important analytical tools with applications in a wide variety of fields. One of the fastest growing applications is in proteomics, or the study of protein expression in an organism. Mass spectrometry has been used to find post-translational modifications and to identify key functions of proteins in the human body. In this study, we review the use of human body fluids as sources for clinical markers and present new data that show the ability of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to identify and characterize proteins in four human body fluids: plasma, cerebrospinal fluid (CSF), saliva, and urine. The body fluids were tryptically digested without any prior separation, purification, or selection, and the digest was introduced into a 9.4 T FTICR mass spectrometer by direct-infusion electrospray ionization (ESI). Even though these samples represent complex biological mixtures, the described method provides information that is comparable with traditional 2D-PAGE data. The sample consumption is extremely low, a few microliters, and the analysis time is only a few minutes. It is, however, evident that the separation of proteins and/or peptides must be included in the methodology, in order to detect low-abundance proteins and other proteins of biological relevance.     

Analysis of natural organic matter via fourier transform ion cyclotron resonance mass spectrometry: an overview of recent non-petroleum applications

Among the different techniques for mass analysis, ultra-high-resolution Fourier transform ion cyclotron resonance (FTICR) is the method of choice for highly complex samples, as it offers unrivaled mass accuracy and resolving power, combined with a high degree of flexibility in hybrid instruments as well as for ion activation techniques. FTICR instruments are readily embraced by the biological and biomedical research communities and applied over a wide range of applications for the analysis of biomolecules such as carbohydrates, lipids, nucleic acids, and proteins. In the field of natural organic matter (NOM) analysis, petroleum-related studies currently dominate FTICR-MS applications. Recently, however, there is a growing interest in developing high-performance MS methods for the characterization of NOM samples from natural aquatic and terrestrial environments. Here, we present an overview of FTICR-MS techniques for complex, non-petroleum NOM samples, including data analysis and novel tandem mass spectrometry (MS/MS) methods for structural classifications. © 2020 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.     

Towards Lipidomics of Low-Abundant Species for Exploring Tumor Heterogeneity Guided by High-Resolution Mass Spectrometry Imaging

Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization of specific low-abundant lipid species involved in cancer biology are still challenging for both fundamental studies and lipid marker discovery. In this paper, we report the identification and the localization of specific isobaric minor phospholipids in human breast cancer xenografts by FTICR MALDI imaging supported by histochemistry. These potential candidates can be further confirmed by liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS) after extraction from the region of interest defined by MALDI imaging. Finally, this study highlights the importance of characterizing the heterogeneous distribution of low-abundant lipid species, relevant in complex histological samples for biological purposes.     

另辟蹊径——串联质谱肽段断裂的

一种新型的串联质谱断裂方式——电子捕获解离,以其完全不同于其他串联质谱肽段断裂的机理,提供了全新的肽段序列标签。它集众多优点于一身:对蛋白质和多肽的主链断裂无偏好;高断裂覆盖度;优先断裂二硫键;保留翻译后修饰基团;中性丢失少、可区分亮氨酸和异亮氨酸等。这些特点使电子捕获解离充满魅力,特别在Top Down技术和翻译后修饰中具有广阔的应用前景。