Electrospray Ionization-ion Mobility Spectrometry-mass Spectrometry Research Articles

Helical sulfonyl-γ-AApeptides modulating Aβ oligomerization and cytotoxicity by recognizing Aβ helix

In contrast to prevalent strategies which make use of β-sheet mimetics to block Aβ fibrillar growth, in this study, we designed a series of sulfonyl-γ-AApeptide helices that targeted the crucial α-helix domain of Aβ13-26 and stabilized Aβ conformation to avoid forming the neurotoxic Aβ oligomeric β-sheets. Biophysical assays such as amyloid kinetics and TEM demonstrated that the Aβ oligomerization and fibrillation could be greatly prevented and even reversed in the presence of sulfonyl-γ-AApeptides in a sequence-specific and dose-dependent manner. The studies based on circular dichroism, Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) spectra unambiguously suggested that the sulfonyl-γ-AApeptide Ab-6 could bind to the central region of Aβ42 and induce α-helix conformation in Aβ. Additionally, Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) was employed to rule out a colloidal mechanism of inhibitor and clearly supported the capability of Ab-6 for inhibiting the formation of Aβ aggregated forms. Furthermore, Ab-6 could rescue neuroblastoma cells by eradicating Aβ-mediated cytotoxicity even in the presence of pre-formed Aβ aggregates. The confocal microscopy demonstrated that Ab-6 could still specifically bind Aβ42 and colocalize into mitochondria in the cellular environment, suggesting the rescue of cell viability might be due to the protection of mitochondrial function otherwise impaired by Aβ42 aggregation. Taken together, our studies indicated that sulfonyl-γ-AApeptides as helical peptidomimetics could direct Aβ into the off-pathway helical secondary structure, thereby preventing the formation of Aβ oligomerization, fibrillation and rescuing Aβ induced cell cytotoxicity.

Characterization of styrene/methyl methacrylate/n-butyl acrylate terpolymer with Kendrick mass defect analysis in electrospray ionization-ion mobility spectrometry-mass spectrometry.

Synthesis of copolymer is one of the major approaches to establish sophisticated polymers. In copolymer analysis in mass spectrometry (MS), an increase in the number of monomer unit species and/or the polymer molecular weight results in complex mass spectra, and a method is required to solve this problem. In this study, electrospray ionization-ion mobility spectrometry-mass spectrometry was employed to analyze styrene/methyl methacrylate/n-butyl acrylate (St/MMA/nBA) terpolymers via Kendrick mass defect (KMD) analysis. An expanded Fourier transform-based noise reduction method for the copolymer was developed to improve the decrease in the signal/noise ratio observed in the higher m/z region. Low-mass terpolymers (0.7kDa) were identified by comparing the observed and theoretical results using a two-dimensional KMD plot. For the 1.5 kDa terpolymer, the signal overlap, of which KMD value was not matched with the theoretical one, was interpreted by the shift from the theoretical value in the KMD plot. For the 3.0 kDa terpolymers, the compositional candidates were determined by the prediction based on the compositional information of low-mass terpolymers previously analyzed. The 4.5kDa terpolymer was interpreted after the expanded noise filtering. The terpolymers, whose molecular weight was up to 4.5kDa, were successfully characterized at a molecular level. The dependency of the St/MMA/nBA composition on molecular weight was observed; that is, the nBA content decreased with an increase in the molecular weight.

Proton Transfers to DNA in Native Electrospray Ionization Mass Spectrometry: A Quantum Mechanics/Molecular Mechanics Study.

Native electrospray ionization-ion mobility mass spectrometry (N-ESI/IM-MS) is a powerful approach for low-resolution structural studies of DNAs in the free state and in complex with ligands. Solvent vaporization is coupled with proton transfers from ammonium ions to the DNA, resulting in a reduction of the DNA charge. Here we provide insight into these processes by classical molecular dynamics and quantum mechanics/molecular mechanics free energy calculations on the d(GpCpGpApApGpC) heptamer, for which a wealth of experiments is available. Our multiscale simulations, consistent with experimental data, reveal a highly complex scenario. The proton either sits on one of the molecules or is fully delocalized on both, depending on the level of hydration of the analytes and the size of the droplets formed during the electrospray experiments. This work complements our previous study of the intramolecular proton transfer on the same heptamer occurring after the processes studied here, and together, they provide a first molecular view of proton transfer in N-ESI/IM-MS.

Role of mass spectrometry in the study of interactions between amylin and metal ions.

Amylin (islet amyloid polypeptide [IAPP]) is a neuroendocrine hormone synthesized with insulin in the beta cells of pancreatic islets. The two hormones act in different ways: in fact insulin triggers glucose uptake in muscle and liver cells, removing glucose from the bloodstream and making it available for energy use and storage, while amylin regulates glucose homeostasis. Aside these positive physiological aspects, human amyloid polypeptide (hIAPP) readily forms amyloid in vitro. Amyloids are aggregates of proteins and in the human body amyloids are considered responsible of the development of various diseases. These aspects have been widely described and discussed in literature and to give a view of the highly complexity of this biochemical behavior the different physical, chemical, biological and medical aspects are shortly described in this review. It is strongly affected by the presence on metal ions, responsible for or inhibiting the formation of fibrils. Mass spectrometry resulted (and still results) to be a particularly powerful tool to obtain valid and effective experimental data to describe the hIAPP behavior. Aside classical approaches devoted to investigation on metal ion-hIAPP structures, which reflects on the identification of metal-protein interaction site(s) and of possible metal-induced conformational changes of the protein, interesting results have been obtained by ion mobility mass spectrometry, giving, on the basis of collisional cross-section data, information on both the oligomerization processes and the conformation changes. Laser ablation electrospray ionization-ion mobility spectrometry-mass spectrometry (LAESI-IMS-MS), allowed to obtain information on the binding stoichiometry, complex dissociation constant, and the oxidation state of the copper for the amylin-copper interaction. Alternatively to inorganic ions, small organic molecules have been tested by ESI-IMS-MS as inhibitor of amyloid assembly. Also in this case the obtained data demonstrate the validity of the ESI-IMS-MS approach as a high-throughput screen for inhibitors of amyloid assembly, providing valid information concerning the identity of the interacting species, the nature of binding and the effect of the ligand on protein aggregation. Effects of Cu2+ and Zn2+ ions in the degradation of human and murine IAPP by insulin-degrading enzyme were studied by liquid chromatography/mass spectrometry (LC/MS). The literature data show that mass spectrometry is a highly valid and effective tool in the study of the amylin behavior, so to individuate medical strategies to avoid the undesired formation of amyloids in in vivo conditions.

Validation of ion mobility spectrometry - mass spectrometry as a screening tool to identify type II kinase inhibitors of FGFR1 kinase.

The protein kinase FGFR1 regulates cellular processes in human development. As over-activity of FGFR1 is implicated with cancer, effective inhibitors are in demand. Type I inhibitors, which bind to the active form of FGFR1, are less effective than type II inhibitors, which bind to the inactive form. Screening to distinguish between type I and type II inhibitors is required. X-ray crystallography was used to indicate whether a range of potential inhibitors bind to the active or inactive FGFR1 kinase conformation. The binding affinity of each ligand to FGFR1 was measured using biochemical methods. Electrospray ionisation - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) in conjunction with collision-induced protein unfolding generated a conformational profile of each FGFR1-ligand complex. The results indicate that the protein's conformational profile depends on whether the inhibitor is type I or type II. X-ray crystallography confirmed which of the kinase inhibitors bind to the active or inactive form of FGFR1 kinase. Collision-induced unfolding combined with ESI-IMS-MS showed distinct differences in the FGFR1 folding landscape for type I and type II inhibitors. Biochemical studies indicated a similar range of FGFR1 affinities for both types of inhibitors, thus providing confidence that the conformational variations detected using ESI-IMS-MS can be interpretated unequivocally and that this is an effective screening method. A robust ESI-IMS-MS method has been implemented to distinguish between the binding mode of type I and type II inhibitors by monitoring the conformational unfolding profile of FGFR1. This rapid method requires low sample concentrations and could be used as a high-throughput screening technique for the characterisation of novel kinase inhibitors.

ESI-IM-MS reveals the metal binding of three analog methanobactin peptides with different numbers of free Cys at physiological pH

Three analog methanobactin (amb7) peptides containing the primary structure acetyl-Leu1-His2-Cys3-Gly4-Ser5-Cys6-Tyr7-Pro8-His9-Cys10-Ser11-Cys12-Met13 but differing by the number of disulfide bridges, have been studied for their reactivity with Fe(III), Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Ag(I), and Pb(II) using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) and fluorescence quenching. The Pb(II) bound only with the amb7-0 (0 disulfide bonds), Zn(II) only to amb7-0 and amb7-1 (1 disulfide bond), Ni(II) only to amb7-1 and amb7-2 (2 disulfide bonds), and Cu(II) to amb7-2, concomitant with the oxidation of amb7-0 and amb7-1. The reduction of Cu(II) to Cu(I) resulted with 1-4Cu(I) binding to amb7-1 and amb7-0, while Ag(I) also exhibited 1-6 Ag(I) ions binding through a mechanism indicative of labile proton exchange. The binding of the oxidized Co(III/V) and Mn(III/V) were observed at pH 9 and 11, while Fe(III) did not exhibit significant binding. The observed negatively charged complexes were consistent with the deprotonation of the metal sites of Cys, His, carboxyl terminus and indicative of ion-ion interactions with the metal at pH 7, 9, and 11. The positively charged complexes were indicative of ion-dipole metal binding but at pH 9 and pH 11 gave results similar to those of the negative ion analyses. Fluorescence quenching also qualitatively agreed with the ESI-IM-MS results providing evidence that they revealed the solution-phase metal binding behavior of amb7.

Formation of Co(II), Ni(II), Zn(II) complexes of alternative metal binding heptapeptides and nitrilotriacetic acid: Discovering new potential affinity tags

Using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) a new series of alternative metal binding-5 (amb5) heptapeptides that provide various combinations of His, Cys and Asp as potential metal binding sites are compared for their relative formation of complexes with the metal ions Co(II), Ni(II) and Zn(II). These amb5 peptides have the general primary structure acetyl-Aa1-Aa2-Gly3-Pro4-Tyr5-Aa6-Aa7, where Aa1,2,6,7 are the four amino acids that include the His, Cys, and/or Asp potential metal binding sites. The study includes two polyhistidine heptapeptides; the His-tag and His-tag with Pro4, because they represent the widely applied His affinity tag used in expression cloning and purification of recombinant proteins with immobilized metal affinity chromatography (IMAC). We compare these amb5 heptapeptides for their formation of the amb5+M(II) and amb5+M(II)+NTA complexes, where M = Co, Ni, or Zn, and NTA is nitrilotriacetic acid as a model for the binding of amb5 in a IMAC column. Overall, the results show that the inclusion of Asp at positions 1, 2, and 7; i.e., ac-Asp1-Asp2-Gly3-Pro4-Tyr5-Aa6-Asp7, generally favors the formation of amb5+Ni(II) and amb5+Ni(II)+NTA complexes, while the inclusion of Cys at positions 2 and 6; i.e., ac-Aa1-Cys2-Gly3-Pro4-Tyr5-Aa6-Cys7, with Aa1 = His, Cys or Asp, favors the formation of amb5+Zn(II) complexes. The comparison with the His-tag and competition between Ni(II) and Zn(II) for forming the amb5+M(II)+NTA complexes, showed the structure ac-Asp1-Asp2-Gly3-Pro4-Tyr5-His6-Aa7, with Aa7 = Cys or Asp, were the most selective for Ni(II) and forming the amb5+Ni(II)+NTA complex. Collision-induced dissociation of the single negatively-charged [amb5+Ni(II)+NTA]− shows it competitively dissociates into [amb5−3H+Ni(II)]− or [NTA−3H+Ni(II)]− while the doubly-charged [amb5+Ni(II)+NTA]2− complex dissociates into [NTA−3H+Ni(II)]− and [amb5J−H]−, indicating these complexes are realistic mimics for the complex inside an IMAC column.

Structural insights into peptide self-assembly using photo-induced crosslinking experiments and discontinuous molecular dynamics.

Determining the structure of the (oligomeric) intermediates that form during the self‐assembly of amyloidogenic peptides is challenging because of their heterogeneous and dynamic nature. Thus, there is need for methodology to analyze the underlying molecular structure of these transient species. In this work, a combination of fluorescence quenching, photo‐induced crosslinking (PIC) and molecular dynamics simulation was used to study the assembly of a synthetic amyloid‐forming peptide, Aβ16‐22. A PIC amino acid containing a trifluormethyldiazirine (TFMD) group—Fmoc(TFMD)Phe—was incorporated into the sequence (Aβ*16–22). Electrospray ionization ion‐mobility spectrometry mass‐spectrometry (ESI‐IMS‐MS) analysis of the PIC products confirmed that Aβ*16–22 forms assemblies with the monomers arranged as anti‐parallel, in‐register β‐strands at all time points during the aggregation assay. The assembly process was also monitored separately using fluorescence quenching to profile the fibril assembly reaction. The molecular picture resulting from discontinuous molecule dynamics simulations showed that Aβ16‐22 assembles through a single‐step nucleation into a β‐sheet fibril in agreement with these experimental observations. This study provides detailed structural insights into the Aβ16‐22 self‐assembly processes, paving the way to explore the self‐assembly mechanism of larger, more complex peptides, including those whose aggregation is responsible for human disease.

Crocus-derived compounds alter the aggregation pathway of Alzheimer\u2019s Disease - associated beta amyloid protein

Natural products have played a dominant role in the discovery of lead compounds for the development of drugs aimed at the treatment of human diseases. This electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)—based study demonstrates that dietary antioxidants, isolated components from the stigmas of saffron (Crocus sativus L.) may be effective in inhibiting Aβ fibrillogenesis, a neuropathological hallmark of Alzheimer’s Disease (AD). This study reveals a substantial alteration in the monomer/oligomer distribution of Aβ1-40, concomitant with re-direction of fibril formation, induced by the natural product interaction. These alterations on the Aβ1-40 aggregation pathway are most prominent for trans-crocin-4 (TC4). Use of ESI-IMS-MS, electron microscopy alongside Thioflavin-T kinetics, and the interpretation of 3-dimensional Driftscope plots indicate a correlation of these monomer/oligomer distribution changes with alterations to Aβ1-40 amyloid formation. The latter could prove instrumental in the development of novel aggregation inhibitors for the prevention, or treatment of AD.

Studies on the cross-interaction between hIAPP and Aβ25-35 and the aggregation process in binary mixture by electrospray ionization-ion mobility-mass spectrometry.

A wealth of epidemiological evidence indicates a strong link between type 2 diabetes (T2D) and Alzheimer's disease (AD). The fiber deposition with cross-β-sheet structure formed by self-aggregation and misfolding of amyloidogenic peptides is a common hallmark of both diseases. For the patients with T2D, the fibrils are mainly found in the islets of Langerhans that results from the accumulation of human islet amyloid polypeptide (hIAPP). The major component of aggregates located in the brain of AD patients is amyloid-β (Aβ). Many biophysical and physiological properties are shared by hIAPP and Aβ, and both peptides show similar cytotoxic mechanisms. Therefore, it is meaningful to investigate the possible cross-interactions of hIAPP and Aβ in both diseases. In this article, the segment 25-35 of Aβ was selected because Aβ25-35 was a core region in the process of amyloid formation and showed similar aggregation tendency and toxicity with full-length Aβ. The electrospray ionization-ion mobility-mass spectrometry analysis and thioflavin T fluorescence kinetic analysis combined with transmission electron microscopy were used to explore the effects of the coexistence of Aβ25-35 and hIAPP on the self-aggregation of both peptides and whether there was co-assembly in fibrillation. The results indicated that the aggregation of hIAPP and Aβ25-35 had two nucleation stages in the binary mixtures. hIAPP and Aβ25-35 had a high binding affinity and a series of hetero-oligomers formed in the mixtures of hIAPP and Aβ25-35 in the early stage. The cross-reaction between hIAPP monomers and Aβ25-35 monomers as well as a little of oligomers during primary nucleation stage could accelerate the aggregation of Aβ25-35 . However, owing to the obvious difference in aggregation ability between hIAPP and Aβ25-35 , this cross-interaction had no significant impact on the self-assembly of hIAPP. Our study may offer a better understanding for exploring the molecular mechanism of the association between AD and T2D observed in clinical and epidemiological studies and developing therapeutic strategies against amyloid diseases.

Simultaneous Analysis of Fatty Alcohols, Fatty Aldehydes, and Sterols in Thyroid Tissues by Electrospray Ionization-Ion Mobility-Mass Spectrometry Based on Charge Derivatization

In this work, we developed a rapid and high-sensitivity method for simultaneous analyses of fatty alcohols, fatty aldehydes, and sterols by combining the optimized derivatization reaction with electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS). Pyridine and thionyl chloride were used as derivatization reagents as they were easily removed after the derivatization reaction and could generate permanently charged tags on different functional groups including hydroxyls and aldehydes. Through this one-step derivatization reaction, the sensitivity of detection for fatty alcohols, fatty aldehydes, and sterols was significantly increased. Moreover, the introduction of ion mobility spectrometry (IMS), offering an additional resolution power, ensured more sensitive and accurate detection of derivative products without increasing analytical time. Being connected with high-performance liquid chromatography, more than 15 kinds of compounds were analyzed within 4 min. Relative quantification using peak intensity ratios between d0-/d5-labeled ions were subsequently applied for analyzing these 15 kinds of compounds in human thyroid carcinoma and para-carcinoma tissues. The results showed significant differences in content of some analytes between these two kinds of tissues (p < 0.05). The correlations between most of the analytes in thyroid carcinoma tissues are better than the correlations in para-carcinoma tissues.

Physicochemical and structural properties of lunasin revealed by spectroscopic, chromatographic and molecular dynamics approaches

Lunasin is a 43-amino acid peptide from seeds and grains with bioavailability in humans and potent chemotherapeutic action against several cancer cell lines. Here, we investigate new information about the physicochemical and structural properties of lunasin using circular dichroism (CD), fluorescence spectroscopy, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), size exclusion chromatography (SEC), molecular dynamics (MD), and bioinformatics. CD analysis and disorder prediction obtained by PONDR indicate that lunasin has a mostly unordered structure. Double wavelength [θ]222nm x [θ]200nm plot data suggests that lunasin is an intrinsically disordered peptide (IDP) in a pre-molten globule-like (PMG-like) state, while CD spectrum deconvolution and MD simulation indicate small β-strand content. The presence of residual structure was supported by loss of CD signal at 222 nm after treatment with urea and by increasing fluorescence emission upon bis-ANS binding. Lunasin also demonstrated stability to heating up to the temperature of 100 °C, as verified by CD. MD and CD analyses in the presence of TFE and MoRFpred prediction indicated the helix propensity of lunasin. ESI-IMS-MS data revealed that lunasin shows a propensity to form disulfide bonds at the conditions used. MD data also indicated that disulfide bond formation affects the adopted structure, showing a possible role of aspartyl-end in structure stabilization and compaction. In conclusion, our data support a characterization of lunasin as a peptide with an intrinsic disorder in a PMG-like state and reveal new aspects about its structural stability and plasticity, as well as the effects of disulfide bond formation and electrostatic attractions.

Gas‐phase conformations of intrinsically disordered proteins and their complexes with ligands: Kinetically trapped states during transfer from solution to the gas phase

Flexible structures of intrinsically disordered proteins (IDPs) are crucial for versatile functions in living organisms, which involve interaction with diverse partners. Electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) has been widely applied for structural characterization of apo-state and ligand-associated IDPs via two-dimensional separation in the gas phase. Gas-phase IDP structures have been regarded as kinetically trapped states originated from conformational features in solution. However, an implication of the states remains elusive in the structural characterization of IDPs, because it is unclear what structural property of IDPs is preserved. Recent studies have indicated that the conformational features of IDPs in solution are not fully reproduced in the gas phase. Nevertheless, the molecular interactions captured in the gas phase amplify the structural differences between IDP conformers. Therefore, an IDP conformational change that is not observed in solution is observable in the gas-phase structures obtained by ESI-IM-MS. Herein, we have presented up-to-date researches on the key implications of kinetically trapped states in the gas phase with a brief summary of the structural dynamics of IDPs in ESI-IM-MS.

Effects of aprotic solvents on the stability of metal-free superoxide dismutase probed by native electrospray ionization-ion mobility-mass spectrometry.

Considering that aprotic solvents are often used as cosolvents in investigating the interactions between small molecules and proteins, we assessed the effects of five aprotic solvents represented by dimethylformamide (DMF) on the structure stabilities of metal-free SOD1 (apo-SOD1) by native electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS). These aprotic solvents include DMF, 1,3-dimethyl-2-imidazolidinone (DMI), dimethyl sulfoxide (DMSO), acetonitrile (ACN), and tetrahydrofuran (THF). Results indicated that DMI, DMSO, and DMF at low percentage concentration could reduce the average charge and the dimer dissociation of apo-SOD1. By contrast, ACN and THF at low concentration have no similar effect. DMF was selected as a representative solvent to further investigate the detailed effects on the structure stability of apo-SOD1 by using collision-induced dissociation and unfolding. The results reveal that the addition of minimal DMF to an aqueous protein solution can protect against the unfolding and dissociation of dimer, even under destabilizing conditions (such as low pH or high cone voltage). When the different percentage concentrations of DMF were added, the average collision cross section of apo-SOD1 showed that apo-SOD1 became compacted when the DMF concentration increased from 0% to 1% and eventually started extending when increased from 1% to 20%. The results indicated that DMF has similar effects to DMSO in native mass spectrometry (MS) and it can also be used as a cosolvent besides DMSO in investigating the stabilities of proteins and the interactions between small molecules and proteins.

Interaction of peptide backbones and transition metal ions: 1. an IM-MS and DFT study of the binding pattern, structure and fragmentation of Pd(II)/Ni(II)-Polyalanine complexes

The interaction between metal ions and peptides have attracted interests because of the important roles of metal ions in many biological processes and the potential application in different fields. The binding patterns of Pd(II)/Ni(II) with polyalanine and the consequent conformational changes as well as the effects on fragmentation have been studied by electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) experiments and density functional theory (DFT). The collision cross section (CCS) values that were measured showed that such complexes tend to adopt compact globular conformations in the case of polyalanines with more than 6 residues, while conformational change was detected in shorter polyalanines with 4 or 5 residues. Collision induced dissociation (CID) experiments revealed a characteristic fragmentation pattern for Pd(II)/Ni(II)-polyalanine complexes of length n in which neutral part Ala4–H2O + M–2H (M = Pd or Ni) and fragment ions [b4+M–2 H]+, [a4+M–2 H]+ were formed in much higher abundance than any other product ions. Density functional theory (DFT) calculations indicated that Pd(II) and Ni(II) preferred to coordinating to the N atoms of the four residues at the N-terminus. Such a binding pattern accelerates fragmentation involving a structure moiety containing four residues and a metal. This analysis suggested a strong structural correlation between Pd(II) and Ni(II) and the four residues located at the N-terminus of polyalanines.

Oligomeric transition and dynamics of RNA binding by the HuR RRM1 domain in solution.

Human antigen R (HuR) functions as a major post-transcriptional regulator of gene expression through its RNA-binding activity. HuR is composed by three RNA recognition motifs, namely RRM1, RRM2, and RRM3. The two N-terminal RRM domains are disposed in tandem and contribute mostly to HuR interaction with adenine and uracil-rich elements (ARE) in mRNA. Here, we used a combination of NMR and electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to characterize the structure, dynamics, RNA recognition, and dimerization of HuR RRM1. Our solution structure reveals a canonical RRM fold containing a 19-residue, intrinsically disordered N-terminal extension, which is not involved in RNA binding. NMR titration results confirm the primary RNA-binding site to the two central β-strands, β1 and β3, for a cyclooxygenase 2 (Cox2) ARE I-derived, 7-nucleotide RNA ligand. We show by 15N relaxation that, in addition to the N- and C-termini, the β2-β3 loop undergoes fast backbone dynamics (ps-ns) both in the free and RNA-bound state, indicating that no structural ordering happens upon RNA interaction. ESI-IMS-MS reveals that HuR RRM1 dimerizes, however dimer population represents a minority. Dimerization occurs via the α-helical surface, which is oppositely orientated to the RNA-binding β-sheet. By using a DNA analog of the Cox2 ARE I, we show that DNA binding stabilizes HuR RRM1 monomer and shifts the monomer-dimer equilibrium toward the monomeric species. Altogether, our results deepen the current understanding of the mechanism of RNA recognition employed by HuR.

Analysis of Multiply Charged Poly(ethylene oxide-co-propylene oxide) Using Electrospray Ionization-Ion Mobility Spectrometry-Mass Spectrometry.

Poly(ethylene oxide), poly(propylene oxide), and their copolymer (poly(EO-co-PO)) were analyzed by electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS). ESI produced multiply charged analytes of 2 to 5 Na+ additions, and they were separately observed in a 2D map of m/z value vs. drift time. The collision cross-section of the analyte polymers was almost linearly proportional to (molecular weight)0.644, except for the analytes with 2Na+ addition; a nonlinear relation called "folding" was significantly observed for the analytes with 2Na+ addition. An increase in electrostatic repulsion, because of the increase in Na+ addition, suppressed the folding of the polymer. Analyses of poly(EO-co-PO) with different EO compositions revealed that the copolymer with high EO composition tended to show folding. The separation of highly multiply charged poly(EO-co-PO)s with different EO contents by ESI-IMS-MS was successfully demonstrated.

Physico-chemical properties of co-formulated fast-acting insulin with pramlintide

Since the discovery of amylin its use has been discouraged by the inadequacy of the protocol involving multiple injections in addition to insulin. We aimed here to develop a combined fixed-dose formulation of pramlintide with fast-acting insulin. We have investigated the compatibility of regular and fast-acting insulin analogues (Aspart, AspB28, and LisPro, LysB28ProB29) with the amylin analogue pramlintide by using electrospray ionization - ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), kinetic aggregation assays monitored by thioflavin T, and transmission electron microscopy (TEM) in the evaluation of the aggregation product. Insulin interacts with pramlintide, forming heterodimers as probed by ESI-IMS-MS. While their interaction is likely to delay the amyloid aggregation of pramlintide in phosphate-buffered solution pH 7.0, they do not prevent aggregation at this condition. At acidic sodium acetate solution pH 5.0, combination of pramlintide and the fast-acting insulin analogues become stable against amyloid aggregation. The co-formulated product at high concentration of both pramlintide (600 μg/mL,150 μM) and LisPro insulin (50 IU/mL, 300 μM) showed also stability against amyloid aggregation. These data indicate the physico-chemical short-term stability of the co-formulated preparation of LisPro insulin with pramlintide, which could bring benefits for the combined therapy.

Mass Spectrometry of Bacterial Outer Membrane Enzymes within the Native Lipid Environment

Lipid complexity of bacterial membranes and the dynamic interaction of lipophilic substrates with membrane‐embedded enzymes reflect a demand for a robust detection approach that unravels the local lipids and hydrophobic substrates/products either bound or within the milieu of membrane enzymes. In this study, we used styrene maleic acid lipid particle (SMALP) nanodiscs and electrospray ionization‐ion mobility spectrometry–mass spectrometry (ESI‐IMS) mass spectrometry to address the stability of lipid‐bound states of two bacterial β‐barrel enzymes.We overexpressed and purified different oligomeric states of two model β‐barrel enzymes with their co‐associated lipids (i.e. enzyme substrates/products) from the outer membrane of E.coli using various polymer formulations. We further examined the stability of the intact lipid‐protein nanodiscs in solution as well as in the gas phase. Our results, thus far, has demonstrated the higher efficacy of the native nanodiscs over conventional detergents in isolating lipid populations bound to the enzymes, also an improved thermal stability of the resulting β‐barrel complexes in solution. We have, interestingly, observed that some polymers are more compatible with the required conditions in mass spectrometry hence overcoming limitations (e.g. low pH which is known as destabilizing factor for the original SMALP nanodiscs) in the structural analysis of lipid‐bound states of membrane proteins in the gas phase.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A chemometric approach for characterization of serum transthyretin in familial amyloidotic polyneuropathy type I (FAP-I) by electrospray ionization-ion mobility mass spectrometry

In this study, we describe a chemometric data analysis approach to assist in the interpretation of the complex datasets from the analysis of high-molecular mass oligomeric proteins by ion mobility mass spectrometry (IM-MS). The homotetrameric protein transthyretin (TTR) is involved in familial amyloidotic polyneuropathy type I (FAP-I). FAP-I is associated with a specific TTR mutant variant (TTR(Met30)) that can be easily detected analyzing the monomeric forms of the mutant protein. However, the mechanism of protein misfolding and aggregation onset, which could be triggered by structural changes in the native tetrameric protein, remains under investigation. Serum TTR from healthy controls and FAP-I patients was purified under non-denaturing conditions by conventional immunoprecipitation in solution and analyzed by IM-MS. IM-MS allowed separation and characterization of several tetrameric, trimeric and dimeric TTR gas ions due to their differential drift time. After an appropriate data pre-processing, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the complex datasets. A group of seven independent components being characterized by their ion mobility profiles and mass spectra were resolved to explain the observed data variance in control and patient samples. Then, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were considered for exploration and classification. Only four out of the seven resolved components were enough for an accurate differentiation. Furthermore, the specific TTR ions identified in the mass spectra of these components and the resolved ion mobility profiles provided a straightforward insight into the most relevant oligomeric TTR proteoforms for the disease.