Electron capture dissociation, electron detachment dissociation, and collision-induced dissociation of polyamidoamine (PAMAM) dendrimer ions with amino, amidoethanol, and sodium carboxylate surface groups.
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ABSTRACT:
SUBMITTER: Kaczorowska MA
PROVIDER: S-EPMC2571976 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The electron capture dissociation (ECD) and collision-induced dissociation (CID) of complexes of polyamidoamine (PAMAM) dendrimers with metal ions Ag(+), Cu(2+), Zn(2+), Fe(2+), and Fe(3+) were determined by Fourier transform ion cyclotron resonance mass spectrometry. Complexes were of the form [PD + M + mH](5+) where PD = generation two PAMAM dendrimer with amidoethanol surface groups, M = metal ion, m = 2-4. Complementary information regarding the site and coordination chemistry of the metal ions can be obtained from the two techniques. The results suggest that complexes of Fe(3+) and Cu(2+) are coordinated via both core tertiary amines, whereas coordination of Ag(+) involves a single core tertiary amine. The Zn(2+) and Fe(2+) complexes do not appear to involve coordination by the dendrimer core.
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Project description:Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6-1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.43 ± 0.20%. Furthermore, we compare our CCS results to previously reported DTIM values, finding strong agreement across similarly configured instrumentation (average RSD of 0.82 ± 0.73%), and systematic differences for DTIM CCS values commonly used to calibrate traveling-wave IM separators (-3% average RSD). Our CIU experiments reveal that the modified DTIM-MS instrument described here achieves enhanced levels of ion activation when compared with any previously reported IM-MS platforms, allowing for comprehensive unfolding of large multiprotein complex ions as well as interplatform CIU comparisons. Using our modified DTIM instrument, we studied two protein complexes. The enhanced CIU capabilities enable us to study the gas phase stability of the GroEL 7-mer and 14-mer complexes. Finally, we report CIU-ECD experiments for the alcohol dehydrogenase tetramer, demonstrating improved sequence coverage by combining ECD fragmentation integrated over multiple CIU intermediates. Further improvements for such native top-down sequencing experiments were possible by leveraging IM separation, which enabled us to separate and analyze CID and ECD fragmentation simultaneously.
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Project description:The structural analysis of sulfated carbohydrates such as glycosaminoglycans (GAGs) has been a long- standing challenge for the field of mass spectrometry. The dissociation of sulfated carbohydrates by collisionally- activated dissociation (CAD) or infrared multiphoton dissociation (IRMPD), which activate ions via vibrational excitation, typically result in few cleavages and abundant SO(3) loss for highly sulfated GAGs such as heparin and heparan sulfate, hampering efforts to determine sites of modification. The recent application of electron activation techniques, specifically electron capture dissociation (ECD) and electron detachment dissociation (EDD), provides a marked improvement for the mass spectrometry characterization of GAGs. In this work, we compare ECD, EDD and IRMPD for the dissociation of the highly sulfated carbohydrate sucrose octasulfate (SOS). Both positive and negative multiply-charged ions are investigated. ECD, EDD and IRMPD of SOS produce abundant and reproducible fragmentation. The product ions produced by ECD are quite different than those produced by IRMPD of SOS positive ions, suggesting different dissociation mechanisms as a result of electronic versus vibrational excitation. The product ions produced by EDD and IRMPD of SOS negative ions also differ from each other. Evidence for SO(3) rearrangement exists in the negative ion IRMPD data, complicating the assignment of product ions.
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Project description:Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissociation, electron-transfer dissociation, and electron-capture dissociation combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.
| S-EPMC9311227 | biostudies-literature
Project description:The effect of site and frequency of phosphorylation on the electron capture dissociation of peptide ions has been investigated. The ECD of a suite of synthetic peptides (APLSFRGSLPKSYVK; one unmodified, three singly-phosphorylated, three-doubly phosphorylated, and one triply-phosphorylated); two tryptic phosphopeptides (YKVPQLEIVPN(p)SAEER, alpha-casein and FQ(p)SEEQQQTEDELQDK, beta-casein) and their unmodified counterparts, were determined over a range of ECD cathode potentials. The results show that, for doubly-charged precursor ions, the presence of phosphorylation has a deleterious effect on ECD sequence coverage. The fragmentation patterns observed suggest that for peptides with multiple basic residues, the phospho-groups exist in their deprotonated form and form salt-bridges with protonated amino acid side chains. The fragmentation observed for the acidic tryptic peptides suggested the presence of noncovalent interactions, which were perturbed on phosphorylation. Increasing the ECD electron energy significantly improves sequence coverage. Alternatively, improved sequence coverage can be achieved by performing ECD on triply-charged precursor ions. The findings are important for the understanding of gas-phase fragmentation of phosphopeptides.
| S-EPMC2570175 | biostudies-literature
Project description:Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. But, once the protein is already oxidized at least once, the spatial distribution of any consecutive oxidation no longer truly reflects initial protein solvent accessibility and residue reactivity, because the site preference of further oxidation may also be influenced by the changes caused by the prior oxidation. Thus, it would be interesting to obtain an assessment of the protein structure based on the FPOP data, by analyzing only singly oxidized protein populations. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is selection of a suitable method of ion isolation, excitation and fragmentation. Here we employ and compare collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-capture dissociation (ECD) combined with multi continuous accumulation of selected ions (CASI). Singly oxidized subpopulation of FPOP labeled ubiquitin was used to optimize the method. The usefulness was then demonstrated further by using it to visualize structural changes induced by cofactor removal from holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in a good agreement, which indicates that monitoring singly FPOP oxidized ion population by top-down is a functional workflow for oxidative protein footprinting.
2023-07-20 | PXD031926 | Pride
Project description:Heparin glycosaminoglycans (GAGs) present the most difficult glycoform for analytical characterization due to high levels of sulfation and structural heterogeneity. Recent contamination of the clinical heparin supply and subsequent fatalities has highlighted the need for sensitive methodologies of analysis. In the last decade, tandem mass spectrometry has been increasingly applied for the analysis of GAGs, but developments in the characterization of highly sulfated compounds have been minimal due to the low number of cross-ring cleavages generated by threshold ion activation by collisional induced dissociation (CID). In the current work, electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) are applied to a series of heparin tetrasaccharides. With both activation methods, abundant glycosidic and cross-ring cleavages are observed. The concept of Ionized Sulfate Criteria (ISC) is presented as a succinct method for describing the charge state, degree of ionization and sodium/proton exchange in the precursor ion. These factors contribute to the propensity for useful fragmentation during MS/MS measurements. Precursors with ISC values of 0 are studied here, and shown to yield adequate structural information from ion activation by EDD or IRMPD.
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Project description:The structural characterization of glycosaminoglycans (GAG) oligosaccharides has been a long-standing challenge in the field of mass spectrometry. In this work, we present the application of electron detachment dissociation (EDD) Fourier transform mass spectrometry to the analysis of dermatan sulfate (DS) oligosaccharides up to 10 residues long. The EDD mass spectra of DS oligosaccharides were compared with their infrared multiphoton dissociation (IRMPD) mass spectra. EDD produces more abundant fragmentation than IRMPD with far less loss of SO3 from labile sulfate modifications. EDD cleaves all glycosidic bonds, yielding both conventional glycosidic bond fragmentation as well as satellite peaks resulting from the additional loss of 1 or 2 hydrogen atoms. EDD also yields more cross-ring fragmentation than IRMPD. For EDD, abundant cross-ring fragmentation in the form of A- and X-ions is observed, with 1,5Xn cleavages occurring for all IdoA residues and many of the GalNAc4S residues, except at the reducing and nonreducing ends. In contrast, IRMPD produces only A-type cross-ring fragmentation for long oligosaccharides (dp6-dp10). As all the structurally informative fragment ions observed by IRMPD appear as a subset of the peaks found in the EDD mass spectrum, EDD shows great potential for the characterization of GAG oligosaccharides using a single tandem mass spectrometry experiment.
| S-EPMC2696562 | biostudies-literature
Project description:Electron transfer dissociation (ETD) and collision-induced dissociation (CID) were used to investigate underivatized, metal-cationized oligosaccharides formed via electrospray ionization (ESI). Reducing and non-reducing sugars were studied including the tetrasaccharides maltotetraose, 3α,4β,3α-galactotetraose, stachyose, nystose, and a heptasaccharide, maltoheptaose. Univalent alkali, divalent alkaline earth, divalent and trivalent transition metal ions, and a boron group trivalent metal ion were adducted to the non-permethylated oligosaccharides. ESI generated [M + Met]+, [M + 2Met]2+, [M + Met]2+, [M + Met - H]+, and [M + Met - 2H]+ most intensely along with low intensity nitrate adducts, depending on the metal and sugar ionized. The ability of these metal ions to produce oligosaccharide adduct ions by ESI had the general trend: Ca(II) > Mg(II) > Ni(II) > Co(II) > Zn(II) > Cu(II) > Na(I) > K(I) > Al(III) ≈ Fe(III) ≈ Cr(III). Although trivalent metals were utilized, no triply charged ions were formed. Metal cations allowed for high ESI signal intensity without permethylation. ETD and CID on [M + Met]2+ produced various glycosidic and cross-ring cleavages, with ETD producing more cross-ring and internal ions, which are useful for structural analysis. Product ion intensities varied based on glycosidic-bond linkage and identity of monosaccharide sub-unit, and metal adducts. ETD and CID showed high fragmentation efficiency, often with complete precursor dissociation, depending on the identity of the adducted metal ion. Loss of water was occasionally observed, but elimination of small neutral molecules was not prevalent. For both ETD and CID, [M + Co]2+ produced the most uniform structurally informative dissociation with all oligosaccharides studied. The ETD and CID spectra were complementary. Graphical Abstract ᅟ.
| S-EPMC5943087 | biostudies-literature
Project description:The use of tumor-specific therapeutic agents is a promising option for efficient and safe nonviral gene transfer in gene therapy. In this study, we describe the efficacy of polyamidoamine (PAMAM)-based nonviral gene delivery carriers, namely, an ornithine conjugated PAMAM (PAMAM-O) dendrimer in delivering apoptin, a tumor-specific killer gene, into human hepatocellular carcinoma (HepG2 cells) and dermal fibroblasts. We analyzed the transfection efficiency by the luciferase assay and assessed cell viability in both cell types. The transfection efficiency of the PAMAM-O dendrimer was found to be higher than that of the PAMAM dendrimer. Moreover, the cytotoxicity of the PAMAM-O dendrimer was very low. We treated both cell types with a polyplex of PAMAM-O dendrimer with apoptin, and analyzed its cellular uptake and localization by confocal microscopy. Cell cycle distribution, tetramethylrhodamine, ethyl ester (TMRE) analysis, and transmission electron microscopy imaging showed that apoptin induced cell death in HepG2 cells. We therefore demonstrated that a PAMAM-O/apoptin polyplex can be used as an effective therapeutic strategy in cancer owing to its effectiveness as a suitable nonviral gene vector for gene therapy.
| S-EPMC6432117 | biostudies-literature