A high resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is used for characterizing the fragmentation of chlorophyll-a. Three tandem mass spectrometry (MS/MS) techniques, including electron-induced dissociation (EID), collisionally activated dissociation (CAD), and infrared mutiphoton dissociation (IRMPD) are applied to the singly protonated chlorophyll-a. Some previously unpublished fragments are identified unambiguously by utilizing high resolution and accurate mass value provided by the FTICR mass spectrometer. According to this research, the two long aliphatic side chains are shown to be the most labile parts, and favorable cleavage sites are proposed. Even though similar fragmentation patterns are generated by all three methods, there are much more abundant peaks in EID and IRMPD spectra. The similarities and differences are discussed in detail. Comparatively, cleavage leading to odd electron species and H? loss both seem more common in EID experiments. Extensive loss of small side groups (e.g., methyl and ethyl) next to the macrocyclic ring was observed. Coupling the high performance FTICR mass spectrometer with contemporary MS/MS techniques, especially IRMPD and EID, proved to be very promising for the structural characterization of chlorophyll, which is also suitable for the rapid and accurate structural investigation of other singly charged porphyrinic compounds. 相似文献
Tandem mass spectrometry (MS/MS) of intact, noncovalently-bound protein-ligand complexes can yield structural information on the site of ligand binding. Fourier transform ion cyclotron resonance (FT-ICR) top-down MS of the 29 kDa carbonic anhydrase-zinc complex and adenylate kinase bound to adenosine triphosphate (ATP) with collisionally activated dissociation (CAD) and/or electron capture dissociation (ECD) generates product ions that retain the ligand and their identities are consistent with the solution phase structure. Increasing gas phase protein charging from electrospray ionization (ESI) by the addition of supercharging reagents, such as m-nitrobenzyl alcohol and sulfolane, to the protein analyte solution improves the capability of MS/MS to generate holo-product ions. Top-down proteomics for protein sequencing can be enhanced by increasing analyte charging. 相似文献
Over the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)–protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations.
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.
Sphingolipids serve not only as components of cellular membranes but also as bioactive mediators of numerous cellular functions. As the biological activities of these lipids are dependent on their structures, and due to the limitations of conventional ion activation methods employed during tandem mass spectrometry (MS/MS), there is a recognized need for the development of improved structure-specific methods for their comprehensive identification and characterization. Here, positive-ionization mode 193 nm ultraviolet photodissociation (UVPD)-MS/MS has been implemented for the detailed structural characterization of lipid species from a range of sphingolipid classes introduced to the mass spectrometer via electrospray ionization as their lithiated or protonated adducts. These include sphingosine d18:1(4E), dihydrosphingosine (sphinganine) d18:0, sphingadiene d18:2(4E,11Z), the isomeric sphingolipids ceramide d18:1(4E)/18:0 and dihydroceramide d18:0/18:1(9Z), ceramide-1-phosphate d18:1(4Z)/16:0, sphingomyelin d18:1(4E)/18:1(9Z) the glycosphingolipids galactosyl ceramide d18:1(4E)/24:1(15Z) and lactosyl ceramide d18:1(4E)/24:0, and several endogenous lipids present within a porcine brain total lipid extract. In addition to the product ions formed by higher energy collision dissociation (HCD), UVPD is shown to yield a series of novel structurally diagnostic product ions resulting from cleavage of both sphingosine carbon–carbon and acyl chain carbon–carbon double bonds for direct localization of site(s) of unsaturation, as well as via diagnostic cleavages of the sphingosine backbone and N–C amide bond linkages. With activation timescales and dissociation efficiencies similar to those found in conventional MS/MS strategies, this approach is therefore a promising new tool in the arsenal of ion activation techniques toward providing complete structural elucidation in automated, high-throughput lipid analysis workflows.
High resolution mass spectrometry (HRMS) was successfully applied to elucidate the structure of a polyfluorinated polyether (PFPE)-based formulation. The mass spectrum generated from direct injection into the MS was examined by identifying the different repeating units manually and with the aid of an instrument data processor. Highly accurate mass spectral data enabled the calculation of higher-order mass defects. The different plots of MW and the nth-order mass defects (up to n = 3) could aid in assessing the structure of the different repeating units and estimating their absolute and relative number per molecule. The three major repeating units were -C2H4O-, -C2F4O-, and -CF2O-. Tandem MS was used to identify the end groups that appeared to be phosphates, as well as the possible distribution of the repeating units. Reversed-phase HPLC separated of the polymer molecules on the basis of number of nonpolar repeating units. The elucidated structure resembles the structure in the published manufacturer technical data. This analytical approach to the characterization of a PFPE-based formulation can serve as a guide in analyzing not just other PFPE-based formulations but also other fluorinated and non-fluorinated polymers. The information from MS is essential in studying the physico-chemical properties of PFPEs and can help in assessing the risks they pose to the environment and to human health.
The study of protein kinetics requires an accurate measurement of isotopic ratios of peptides. Although Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometers yield accurate mass measurements of analytes, the isotopologue ratios are consistently lower than predicted. Recently, we demonstrated that the magnitude of the spectral error in the FT-ICR mass spectrometer is proportional to the scan duration of ions. Here, we present a novel isotopic ratio extrapolation (IRE) method for obtaining accurate isotopic ratio measurements. Accuracy is achieved by performing scans with different duration and extrapolation of the data to the initial moment of the ion rotation; IRE minimizes the absolute isotopic ratio error to ≤1 %. We demonstrate the application of IRE in protein turnover studies using 2H2O-metabolic labeling. Overall, this technique allows accurate measurements of the isotopic ratios of proteolytic peptides, a critical step for enabling routine studies of proteome dynamics. 相似文献
We report identification of the binding sites for an organometallic ruthenium anticancer complex [(η6-biphenyl)Ru(en)Cl][PF6] (1; en = ethylenediamine) on the 15-mer single-stranded oligodeoxynucleotides (ODNs), 5′-CTCTCTX7G8Y9CTTCTC-3′ [X = Y = T (I); X = C and Y = A (II); X = A and Y = T (III); X = T and Y = A (IV)] by electrospray ionization mass spectrometry (ESI-MS) in conjunction with enzymatic digestion or tandem mass spectrometry (top-down MS). ESI-MS combined with enzymatic digestion (termed MS-based ladder-sequencing), is effective for identification of the thermodynamically-favored G-binding sites, but not applicable to determine the thermodynamically unstable T-binding sites because the T-bound adducts dissociate during enzymatic digestion. In contrast, top-down MS is efficient for localization of the T binding sites, but not suitable for mapping ruthenated G bases, due to the facile fragmentation of G bases from ODN backbones prior to the dissociation of the phosphodiester bonds. The combination of the two MS approaches reveals that G8 in each ODN is the preferred binding site for 1, and that the T binding sites of 1 are either T7 or T11 on I and IV, and either T6 or T11 on II and III, respectively. These findings not only demonstrate for the first time that T-bases in single-stranded oligonucleotides are kinetically competitive with guanine for such organoruthenium complexes, but also illustrate the relative merits of the combination of ladder-sequencing and top-down MS approaches to elucidate the interactions of metal anticancer complexes with DNA. 相似文献
Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has the ability to provide an enormous amount of information on the abundances and spatial distributions of molecules within biological tissues. The rapid progress in the development of this technology significantly improves our ability to analyze smaller and smaller areas and features within tissues. The mammalian eye has evolved over millions of years to become an essential asset for survival, providing important sensory input of an organism’s surroundings. The highly complex sensory retina of the eye is comprised of numerous cell types organized into specific layers with varying dimensions, the thinnest of which is the 10 μm retinal pigment epithelium (RPE). This single cell layer and the photoreceptor layer contain the complex biochemical machinery required to convert photons of light into electrical signals that are transported to the brain by axons of retinal ganglion cells. Diseases of the retina, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, occur when the functions of these cells are interrupted by molecular processes that are not fully understood. In this report, we demonstrate the use of high spatial resolution MALDI IMS and FT-ICR tandem mass spectrometry in the Abca4–/– knockout mouse model of Stargardt disease, a juvenile onset form of macular degeneration. The spatial distributions and identity of lipid and retinoid metabolites are shown to be unique to specific retinal cell layers.
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used to determine the structures of anhydroicaritin glycosides by the MS/MS experiments of anhydroicaritin glycosides and their methylated derivatives,With high accuracy FT-ICR-MS provides much information about the structures of compounds ,FT-ICR-MS shows the great potential application in the structural characterization of unknown compounds. 相似文献
Steroid conjugates, which often occur as metabolites, are challenging to characterize. One application is female-mouse urine, where steroid conjugates serve as important ligands for the pheromone-sensing neurons. Although the two with the highest abundance in mouse urine were previously characterized with mass spectrometry (MS) and NMR to be sulfated steroids, many more exist but remain structurally unresolved. Given that their physical and chemical properties are similar, they are likely to have a sulfated steroid ring structure. Because these compounds occur in trace amounts in mouse urine and elsewhere, their characterization by NMR will be difficult. Thus, MS methods become the primary approach for determining structure. Here, we show that a combination of MS tools is effective for determining the structures of sulfated steroids. Using 4-pregnene analogs, we explored high-resolving power MS (HR-MS) to determine chemical formulae; HD exchange MS (HDX-MS) to determine number of active, exchangeable hydrogens (e.g., OH groups); methoxyamine hydrochloride (MOX) derivatization MS, or reactive desorption electrospray ionization with hydroxylamine to determine the number of carbonyl groups; and tandem MS (MSn), high-resolution tandem MS (HRMS/MS), and GC-MS to obtain structural details of the steroid ring. From the fragmentation studies, we deduced three major fragmentation rules for this class of sulfated steroids. We also show that a combined MS approach is effective for determining structure of steroid metabolites, with important implications for targeted metabolomics in general and for the study of mouse social communication in particular.
Correct and rapid identification of microorganisms is the key to the success of many important applications in health and safety, including, but not limited to, infection treatment, food safety, and biodefense. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is challenging correct microbial identification because of the large number of choices present. To properly disentangle candidate microbes, one needs to go beyond apparent morphology or simple ‘fingerprinting’; to correctly prioritize the candidate microbes, one needs to have accurate statistical significance in microbial identification. We meet these challenges by using peptidome profiles of microbes to better separate them and by designing an analysis method that yields accurate statistical significance. Here, we present an analysis pipeline that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using MS/MS data of 81 samples, each composed of a single known microorganism, that the proposed pipeline can correctly identify microorganisms at least at the genus and species levels. We have also shown that the proposed pipeline computes accurate statistical significances, i.e., E-values for identified peptides and unified E-values for identified microorganisms. The proposed analysis pipeline has been implemented in MiCId, a freely available software for Microorganism Classification and Identification. MiCId is available for download at http://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html.
High-resolution mass spectrometry (HRMS) continues to play an important role in the compositional characterization of larger organic molecules. In the field of polymer characterization, however, the application of HRMS has made only slow progress because of lower compatibility between matrix-assisted laser desorption/ionization (MALDI) and ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS). In this study, a newly developed type of MALDI high-resolution time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS) was applied to the structural and compositional characterization of polymers. To create a graphical distribution of polymer components on a two-dimensional plot converted from complex mass spectra, we adopted a slightly modified Kendrick mass defect (KMD) analysis based on accurate masses determined using spiral-TOFMS. By setting the Kendrick mass scale based on the mass of the repeating units of a given polymer, components with common repeat units lined up in the horizontal direction on the KMD plot, whereas those components with different structures were shifted vertically. This combination of MALDI spiral-TOFMS measurement and KMD analysis enabled the successful discrimination of the polymer components in a blend of poly(alkylene oxide)s, the compositional analysis of poly(ethylene oxide)/poly(propylene oxide) block copolymers, and profiling of the end-group distribution of poly(ε-caprolactone)s synthesized under different conditions.
