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Analytical Chemistry Jun 2019Native liquid extraction surface analysis (LESA) mass spectrometry allows direct analysis of folded proteins and protein complexes from biological substrates, such as...
Native liquid extraction surface analysis (LESA) mass spectrometry allows direct analysis of folded proteins and protein complexes from biological substrates, such as dried blood spots and thin tissue sections, by use of native-like extraction/ionization solvents. Previously, we have demonstrated native LESA mass spectrometry of folded proteins up to 16 kDa as well as the 64 kDa hemoglobin tetramer, from mouse tissues. With denaturing LESA solvents, the highest mass protein detected in tissue to date is ∼37 kDa. Here, we demonstrate native LESA mass spectrometry by use of a Q Exactive UHMR Hybrid Quadrupole-Orbitrap (QE-UHMR) mass spectrometer, pushing the upper mass limit of proteins detected in tissue to >70 kDa. Moreover, a protein trimer of 42 kDa was detected and its stoichiometry confirmed by higher energy collision dissociation (HCD). The benefits of inclusion of detergents in the LESA sampling solvent are also demonstrated.
Topics: Animals; Brain Chemistry; Detergents; Kidney; Male; Mass Spectrometry; Multiprotein Complexes; Protein Denaturation; Protein Multimerization; Proteins; Rats, Wistar
PubMed: 31062957
DOI: 10.1021/acs.analchem.9b00971 -
Methods (San Diego, Calif.) Oct 2018The interaction between microbial communities and their environment, such as the human gastrointestinal tract, has been an area of microbiology rapidly advanced, by... (Review)
Review
The interaction between microbial communities and their environment, such as the human gastrointestinal tract, has been an area of microbiology rapidly advanced, by developments in sequencing technology. However, these techniques are largely limited to the detection of the taxonomic composition of a microbial community and/or its genetic functional capacity. Here, we discuss a range of mass spectrometry-based approaches which researchers can employ to explore the host-microbiome interactions at the metabolic level. Traditional approaches to mass spectrometry are detailed, alongside new developments in the field, namely ambient ionisation mass spectrometry and imaging mass spectrometry, which we believe will prove to be important to future work in this field. We further discuss considerations for experimental workflows, data analysis options and propose a methodology for the establishment of causal relationships between functional host-microbiome interactions with regards to health and disease in the human gastrointestinal tract.
Topics: Gastrointestinal Microbiome; Gastrointestinal Tract; Humans; Mass Spectrometry; Metabolome; Metabolomics; Microbiota; Tandem Mass Spectrometry
PubMed: 29704664
DOI: 10.1016/j.ymeth.2018.04.027 -
The Tohoku Journal of Experimental... Mar 2021Despite ongoing concerns regarding its clinical application, mass spectrometry (MS)-based steroid assay represents a promising tool in endocrine research. Recent studies...
Despite ongoing concerns regarding its clinical application, mass spectrometry (MS)-based steroid assay represents a promising tool in endocrine research. Recent studies indicate that monitoring the blood levels of individual sterols provides improved diagnostic insight into hyperlipidemia compared with immunoassays routinely used in clinical practice. Hypercortisolism and hyperaldosteronism can also be easily evaluated along with successful subtyping of adrenal diseases using MS-based methods, while metabolic signatures of sex steroids provide experimental evidence of abnormal puberty and male infertility. Many MS-based biological and clinical studies are based on liquid chromatography-mass spectrometry (LC-MS) coupled to electrospray ionization and tandem MS scan modes. However, gas chromatography-mass spectrometry (GC-MS) provides better chromatographic separation. Improved chromatographic resolution enables large-scale steroid profiling to allow a bird-eye view and increase the chances of identifying potent biomarkers in endocrine research. In addition to the technical advantages of MS-based assays over immunoassays, minimizing the sample amounts with acceptable analytical sensitivity and standardization of surrogate materials provides cutting-edge tools for precision and personalized medicine.
Topics: Adrenal Glands; Animals; Endocrinology; Gas Chromatography-Mass Spectrometry; Humans; Mass Spectrometry; Steroids
PubMed: 33716273
DOI: 10.1620/tjem.253.171 -
Journal of the American Society For... Feb 2021Native mass spectrometry (native MS) has emerged as a powerful technique to study the structure and stoichiometry of large protein complexes. Traditionally, native MS...
Native mass spectrometry (native MS) has emerged as a powerful technique to study the structure and stoichiometry of large protein complexes. Traditionally, native MS has been performed on modified time-of-flight (TOF) systems combined with detectors that do not provide information on the arrival coordinates of each ion at the detector. In this study, we describe the implementation of a Timepix (TPX) pixelated detector on a modified orthogonal TOF (O-TOF) mass spectrometer for the analysis and imaging of native protein complexes. In this unique experimental setup, we have used the impact positions of the ions at the detector to visualize the effects of various ion optical parameters on the flight path of ions. We also demonstrate the ability to unambiguously detect and image individual ion events, providing the first report of single-ion imaging of protein complexes in native MS. Furthermore, the simultaneous space- and time-sensitive nature of the TPX detector was critical in the identification of the origin of an unexpected TOF signal. A signal that could easily be mistaken as a fragment of the protein complex was explicitly identified as a secondary electron signal arising from ion-surface collisions inside the TOF housing. This work significantly extends the mass range previously detected with the TPX and exemplifies the value of simultaneous space- and time-resolved detection in the study of ion optical processes and ion trajectories in TOF mass spectrometers.
Topics: Electrons; Equipment Design; Ions; Mass Spectrometry; Molecular Imaging; Molecular Weight; Multiprotein Complexes; Proteins; Spectrometry, Mass, Electrospray Ionization
PubMed: 33439014
DOI: 10.1021/jasms.0c00412 -
The Analyst Mar 2015Ion Mobility Spectrometry (IMS) is a widely used and 'well-known' technique of ion separation in the gaseous phase based on the differences in ion mobilities under an... (Review)
Review
Ion Mobility Spectrometry (IMS) is a widely used and 'well-known' technique of ion separation in the gaseous phase based on the differences in ion mobilities under an electric field. All IMS instruments operate with an electric field that provides space separation, but some IMS instruments also operate with a drift gas flow that provides also a temporal separation. In this review we will summarize the current IMS instrumentation. IMS techniques have received an increased interest as new instrumentation and have become available to be coupled with mass spectrometry (MS). For each of the eight types of IMS instruments reviewed it is mentioned whether they can be hyphenated with MS and whether they are commercially available. Finally, out of the described devices, the six most-consolidated ones are compared. The current review article is followed by a companion review article which details the IMS hyphenated techniques (mainly gas chromatography and mass spectrometry) and the factors that make the data from an IMS device change as a function of device parameters and sampling conditions. These reviews will provide the reader with an insightful view of the main characteristics and aspects of the IMS technique.
Topics: Humans; Ions; Mass Spectrometry
PubMed: 25465076
DOI: 10.1039/c4an01100g -
Journal of Visualized Experiments : JoVE Nov 2017Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore...
Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement. In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.
Topics: Mass Spectrometry; Models, Molecular; Peptides; Protein Subunits
PubMed: 29286378
DOI: 10.3791/56747 -
Analytical and Bioanalytical Chemistry Mar 2011In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated... (Review)
Review
In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets-pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches.
Topics: Animals; Biological Assay; Complex Mixtures; Humans; Mass Spectrometry; Proteins
PubMed: 21107824
DOI: 10.1007/s00216-010-4406-0 -
Journal of Chromatography. A Apr 2021Mass spectrometry is now in widespread use for the detection of the analytes separated by chromatography. Electron ionization is the most frequently used method in mass... (Review)
Review
Mass spectrometry is now in widespread use for the detection of the analytes separated by chromatography. Electron ionization is the most frequently used method in mass spectrometry. However, this ionization technique sometimes suffers from extensive fragmentation of analytes, which makes identification difficult. A photoionization technique has been developed for suppressing this fragmentation and for subsequently observing a molecular ion. A variety of lasers have been employed for the sensitive and selective ionization of organic compounds. A femtosecond laser has a high peak power and is preferential for efficient ionization as well as for suppressing fragmentation, providing valuable information concerning molecular weight and chemical structure as well. In this review, we report on applications of femtosecond ionization mass spectrometry combined with gas chromatography.
Topics: Chromatography; Gas Chromatography-Mass Spectrometry; Mass Spectrometry; Polycyclic Aromatic Hydrocarbons; Spectrum Analysis, Raman; Thermodynamics
PubMed: 33714081
DOI: 10.1016/j.chroma.2021.462023 -
Journal of Molecular Biology Oct 2012That membrane protein complexes could survive in the gas phase had always seemed impossible. The lack of chargeable residues, high hydrophobicity, and poor solubility... (Review)
Review
That membrane protein complexes could survive in the gas phase had always seemed impossible. The lack of chargeable residues, high hydrophobicity, and poor solubility and the vast excess of detergent contributed to the view that it would not be possible to obtain mass spectra of intact membrane complexes. With the recent success in recording mass spectra of these complexes, first from recombinant sources and later from the cellular environment, many surprising properties of these gas phase membrane complexes have been revealed. The first of these was that the interactions between membrane and soluble subunits could survive in vacuum, without detergent molecules adhering to the complex. The second unexpected feature was that their hydrophobicity and, consequently, lower charge state did not preclude ionization. The final surprising finding was that these gas phase membrane complexes carry with them lipids, bound specifically in subunit interfaces. This provides us with an opportunity to distinguish annular lipids that surround the membrane complexes, from structural lipids that have a role in maintaining structure and subunit interactions. In this perspective, we track these developments and suggest explanations for the various discoveries made during this research.
Topics: Detergents; Gases; Hydrophobic and Hydrophilic Interactions; Lipids; Mass Spectrometry; Membrane Proteins; Models, Molecular; Protein Conformation
PubMed: 22750574
DOI: 10.1016/j.jmb.2012.06.033 -
Current Opinion in Chemical Biology Feb 2018Ion mobility spectrometry-mass spectrometry (IMS-MS) provides information about the structures of gas-phase ions in the form of a collision cross section (CCS) with a... (Review)
Review
Ion mobility spectrometry-mass spectrometry (IMS-MS) provides information about the structures of gas-phase ions in the form of a collision cross section (CCS) with a neutral buffer gas. Indicating relative ion size, a CCS value alone is of limited utility. Although such information can be used to propose different conformer types, finer details of structure are not captured. The increased accessibility of IMS-MS measurements with commercial instrumentation in recent years has ballooned its usage in combination with separate measurements to provide enhanced data from which greater structural inferences can be drawn. This short review presents recent outstanding developments in scientific research that employs complementary measurements that when combined with IMS-MS data are used to characterize the structures of a wide range of compounds.
Topics: Biochemical Phenomena; Ion Mobility Spectrometry; Mass Spectrometry; Molecular Structure
PubMed: 29241076
DOI: 10.1016/j.cbpa.2017.11.013