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1.
Probing the Lipid Annular Belt by Gas‐Phase Dissociation of Membrane Proteins in Nanodiscs
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Dr. Michael T. Marty Kin Kuan Hoi Dr. Joseph Gault Prof. Carol V. Robinson 《Angewandte Chemie (International ed. in English)》2016,55(2):550-554
Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment. 相似文献
2.
Wagner Y Sickmann A Meyer HE Daum G 《Journal of the American Society for Mass Spectrometry》2003,14(9):1003-1011
The analysis of macromolecular protein complexes is an important factor in understanding most cellular processes, e.g., protein transport into cell organells, signal transduction via biological membranes, apoptosis, energy metabolism, directed motion of cells, and cell division. These complexes are not only built of various numbers of different proteins but also of prosthetic groups and RNA molecules. To understand the role each protein plays in a complex, a complete analysis of all protein compounds is necessary. Therefore, several separation steps have to be coupled to mass spectrometry to identify the proteins. In this work, we describe the application of multidimensional liquid chromatography, SCX-RP-LC as well as SAX-RP-LC, coupled to electrospray ion trap mass spectrometry. Tryptic digested ribosomes were separated by ion exchange chromatography manually collected and prepared for reversed phase chromatography to analyze the peptides via nano-ESI mass spectrometry. The total numbers of identified proteins are compared in consideration of the separation method (SCX-RP versus SAX-RP). 相似文献
3.
质谱技术在免疫分子的结构研究中的应用 总被引:1,自引:0,他引:1
质谱技术用于生物大分子的研究具有直接、简单、快速、经济等优点。近十年来 ,基质辅助激光解吸质谱 (MALDI MS)和电喷雾质谱 (ESI MS)在免疫学领域的研究中作出了重要贡献。本文着重对抗原、抗体、抗原 抗体复合物、抗原决定簇等免疫分子结构的质谱研究作一评述。大体分为四方面内容 :免疫分子的分子量、翻译后修饰、异质性、构象变化的分析 ;质谱指纹图的取得和串联质谱测序 ;抗原 抗体复合物的证明 ;B 细胞表位和T 细胞表位序列的测定。这些研究结果对于理解免疫分子的免疫功能、对于疾病的早期诊断、对于发展新药和疫苗具有重要意义 相似文献
4.
Mass spectrometry (MS) with electrospray ionization (ESI) has the capability to measure and detect noncovalent protein-ligand and protein-protein complexes. However, information on the sites of ligand binding is not easily obtained by the ESI-MS methodology. Electron capture dissociation (ECD) favors cleavage of covalent backbone bonds of protein molecules. We show that this characteristic of ECD translates to noncovalent protein-ligand complexes, as covalent backbone bonds of protein complexes are dissociated, but the noncovalent ligand interaction is retained. For the complex formed from 140-residue, 14.5 kDa alpha-synuclein protein, and one molecule of polycationic spermine (202 Da), ECD generates product ions that retain the protein-spermine noncovalent interaction. Spermine binding is localized to residues 106-138; the ECD data are consistent with previous solution NMR studies. Our studies suggest that ECD mass spectrometry can be used to determine directly the sites of ligand binding to protein targets. 相似文献
5.
Collette C Dehareng D De Pauw E Dive G 《Journal of the American Society for Mass Spectrometry》2001,12(3):304-316
The host-guest interactions play a very important role in chemical and biological processes. It is therefore important to be able to characterize these complexes. Electrospray mass spectrometry can be used to characterize the complex formation. It provides information on the mass and the charge of these ionic complexes. In this article, we show that the use of ab initio and semiempirical calculations, in addition to the results obtained by electrospray mass spectrometry, reveal to be a promising tool for the study of these noncovalent complexes. In this article, host-guest complexes formed by macropolycyclic polyammonium host molecules and dicarboxylic acids are studied. 相似文献
6.
Top-down mass spectrometry is an emerging approach for the analysis of intact proteins. The term was coined as a contrast with the better-established, bottom-up strategy for analysis of peptide fragments derived from digestion, either enzymatically or chemically, of intact proteins. Although the term top-down originates from proteomics, it can also be applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein assemblies or complexes. Traditionally, mass spectrometry has usually started with intact molecules, and in this regard, top-down approaches reflect the spirit of mass spectrometry. This article provides an overview of the methodologies in top-down mass spectrometry and then reviews applications covering protein posttranslational modifications, protein biophysics, DNAs/RNAs, and protein assemblies. Finally, challenges and future directions are discussed. 相似文献
7.
Micellar electrokinetic chromatography of proteins 总被引:1,自引:0,他引:1
Micellar electrokinetic capillary chromatography (MECC) of proteins is a high resolution capillary electrophoretic (CE) analysis method that utilizes the hydrophobic and electrostatic interaction of protein analytes with surfactant micelles present in the buffer medium to facilitate separation. Through the manipulation of the protein-micelle interaction by the adjustment of variables such as surfactant concentration, solution pH, ionic strength, the presence of an organic modifier and the use of coated capillaries, MECC analyses of a wide variety of proteins have been optimized. MECC has been demonstrated to provide resolution of mixtures consisting of proteins with minor structural variations and also has provided the successful quantitative analysis of protein present in complex matrices. The adoption of protein MECC as a routine analytical technique may be dependent upon the successful interface of MECC with detection methodology, such as mass spectrometry, which can provide analyte characterization information. 相似文献
8.
McCammon MG Hernández H Sobott F Robinson CV 《Journal of the American Chemical Society》2004,126(19):5950-5951
We have used tandem mass spectrometry to examine the stoichiometry and binding sites of trp molecules in various assemblies of the protein complex TRAP. The results show that TRAP forms oligomers containing 11 and 12 subunits. MS/MS experiments show that up to 11 trp molecules bind to the 12-mer but that during gas-phase dissociation 5 then 6 trp molecules are released reflecting the different gas-phase stabilities of the partially ligated forms. At high trp concentrations, the protein assembles to form a double ring structure. Tandem mass spectrometry reveals that it is composed of 24 subunits with up to 22 molecules of trp. Dissociation of the complex reveals the same dissociation pathway as for the single ring structure, allowing us to propose a model for the assembly of the TRAP 24-mer based on the different environments of trp molecules. More generally, these results demonstrate the power of tandem mass spectrometry for defining the stoichiometry and quaternary structural arrangement of subunits and ligands within a 46-component multiprotein multiligand complex. 相似文献
9.
Jani Reddy Bolla Robin A. Corey Cagla Sahin Joseph Gault Alissa Hummer Jonathan T. S. Hopper David P. Lane David Drew Timothy M. Allison Phillip J. Stansfeld Carol V. Robinson Michael Landreh 《Angewandte Chemie (International ed. in English)》2020,59(9):3523-3528
Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non‐specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid‐binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent‐resistant lipids bound at the dimer interface in the leucine transporter show decreased koff rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid‐II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non‐annular lipids based on their exchange rates in solution. 相似文献
10.
Morgner N Kleinschroth T Barth HD Ludwig B Brutschy B 《Journal of the American Society for Mass Spectrometry》2007,18(8):1429-1438
A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices. 相似文献
11.
Wang SC Politis A Di Bartolo N Bavro VN Tucker SJ Booth PJ Barrera NP Robinson CV 《Journal of the American Chemical Society》2010,132(44):15468-15470
Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for the ion channel are in accord with a compact configuration of subunits, suggesting that the native-like structure can be preserved under the harsh activation conditions required to release it from the detergent micelle into the gas phase. We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable once stripped of detergents and bulk water. These results highlight the potential of ion mobility mass spectrometry for characterizing the overall topologies of membrane protein complexes and the structural changes associated with nucleotide, lipid, and drug binding. 相似文献
12.
A recent major advance in the field of mass spectrometry in the biomolecular sciences is represented by the study of the supramolecular interactions among two or more partners in the gas phase. A great deal of chemistry and most of biochemistry concerns molecular interactions taking place in solution. The electrospray technique, which allows direct sampling from solution, and soft ionization of the solute without deposition into the analyte of large amounts of energy, guarantees in many cases the survival of noncovalent bondings and, hence, the direct analysis of the supramolecular complexes present in the condensed phase. The proper preparation of the solution to be studied and also the expert and accurate setting and use of the instrumental parameters are the prerequisites for gaining results as to the specific interactions between, for instance, a protein conformationally modified by its specific metal ion, eventually, and a ligand molecule. The analysis of the charge state of the protein itself and of the modifications of the complex integrity by activating collisions are also methods for studying the biomolecule-molecule interactions. Accordingly, this new mass spectrometric approach to the supramolecular chemistry, which could be also defined as 'supramolecular mass spectrometry', allows the study of ion-protein, protein-protein, protein-ligand and DNA-drug interactions. Chiral recognition can also be performed in the gas phase, studying by electrospray mass spectrometry the fragmentation of diastereomeric complex ions. Not the least, a deep insight can also be obtained into the formation and nature of inclusion complexes like those formed with crown ethers, cyclodextrins and calixarenes as host molecules. All these topics are treated to a certain extent in this special feature article. 相似文献
13.
《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2017,129(27):8020-8023
Nonvolatile salts are essential for the structures and functions of many proteins and protein complexes but can severely degrade performance of native mass spectrometry by adducting to protein and protein complex ions, thereby reducing sensitivity and mass measuring accuracy. Small nanoelectrospray emitters are used to form protein and protein complex ions directly from high‐ionic‐strength (>150 mm ) nonvolatile buffers with salts that mimic the extracellular environment. Charge‐state distributions are not obtained for proteins and protein complexes from six commonly used nonvolatile buffers and ≥150 mm Na+ with conventionally sized nanoelectrospray emitter tips but are resolved with 0.5 μm tips. This method enables mass measurements of proteins and protein complexes directly from a variety of commonly used buffers with high concentrations of nonvolatile salts and eliminates the need to buffer exchange into volatile ammonium buffers traditionally used in native mass spectrometry. 相似文献
14.
Francis Canon Franck Paté Emmanuelle Meudec Thérèse Marlin Véronique Cheynier Alexandre Giuliani Pascale Sarni-Manchado 《Analytical and bioanalytical chemistry》2009,395(8):2535-2545
Numerous protein–polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins,
which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein–ligand complexes are readily
detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly,
the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited,
whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry
has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich
protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID)
measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments.
Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild
ESI interface conditions allowed us to observe intact noncovalent PRP–tannin complexes with stoichiometries ranging from 1:1
to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP–tannin interactions, (2) the determination of stoichiometry,
and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall
subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5·EgCG complexes
are maintained intact in the gas phase.
相似文献
15.
Bornschein RE Hyung SJ Ruotolo BT 《Journal of the American Society for Mass Spectrometry》2011,22(10):1690-1698
Characterizing intact multiprotein complexes in terms of both their mass and size by ion mobility-mass spectrometry is becoming
an increasingly important tool for structural biology. Furthermore, the charge states of intact protein complexes can dramatically
influence the information content of gas-phase measurements performed. Specifically, protein complex charge state has a demonstrated
influence upon the conformation, mass resolution, ion mobility resolution, and dissociation properties of protein assemblies
upon collisional activation. Here we present the first comparison of charge-reduced multiprotein complexes generated by solution
additives and gas-phase ion-neutral reaction chemistry. While the charge reduction mechanism for both methods is undoubtedly
similar, significant gas-phase activation of the complex is required to reduce the charge of the assemblies generated using
the solution additive strategy employed here. This activation step can act to unfold intact protein complexes, making the
data difficult to correlate with solution-phase structures and topologies. We use ion mobility-mass spectrometry to chart
such conformational effects for a range of multi-protein complexes, and demonstrate that approaches to reduce charge based
on ion-neutral reaction chemistry in the gas-phase consistently produce protein assemblies having compact, ‘native-like’ geometries
while the same molecules added in solution generate significantly unfolded gas-phase complexes having identical charge states. 相似文献
16.
Dr. Jani Reddy Bolla Dr. Robin A. Corey Dr. Cagla Sahin Dr. Joseph Gault Alissa Hummer Dr. Jonathan T. S. Hopper Prof. David P. Lane Prof. David Drew Dr. Timothy M. Allison Dr. Phillip J. Stansfeld Prof. Carol V. Robinson Dr. Michael Landreh 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2020,132(9):3551-3556
Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non-specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid-binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent-resistant lipids bound at the dimer interface in the leucine transporter show decreased koff rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid-II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution. 相似文献
17.
Pim G. A. Janssen Joost L. J. van Dongen E. W. Meijer Prof. Dr. Albertus P. H. J. Schenning Dr. 《Chemistry (Weinheim an der Bergstrasse, Germany)》2009,15(2):352-360
Supramolecular complexes consisting of a single‐stranded oligothymine ( dTn ) as the host template and an array of guest molecules equipped with a complementary diaminotriazine hydrogen‐bonding unit have been studied with electrospray‐ionization mass spectrometry (ESI‐MS). In this hybrid construct, a supramolecular stack of guest molecules is hydrogen bonded to dTn . By changing the hydrogen‐bonding motif of the DNA host template or the guest molecules, selective hydrogen bonding was proven. We were able to detect single‐stranded‐DNA (ssDNA)–guest complexes for strands with lengths of up to 20 bases, in which the highest complex mass detected was 15 kDa; these complexes constitute 20‐component self‐assembled objects. Gas‐phase breakdown experiments on single‐ and multiple‐guest–DNA assemblies gave qualitative information on the fragmentation pathways and the relative complex stabilities. We found that the guest molecules are removed from the template one by one in a highly controlled way. The stabilities of the complexes depend mainly on the molecular weight of the guest molecules, a fact suggesting that the complexes collapse in the gas phase. By mixing two different guests with the ssDNA template, a multicomponent dynamic library can be created. Our results demonstrate that ESI‐MS is a powerful tool to analyze supramolecular ssDNA complexes in great detail. 相似文献
18.
Claudia Bich Samuel Baer Matthias C. Jecklin Renato Zenobi 《Journal of the American Society for Mass Spectrometry》2010,21(2):286-289
The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role
of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer
into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc.,
is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass
spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease
S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray
mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with
chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were
observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at
very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to
the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates
that the noncovalently bound complexes are conserved, and a real “snap-shot” of the situation in solution can be obtained. 相似文献
19.
电喷雾质谱的非共价键蛋白质复合物研究 总被引:1,自引:0,他引:1
电喷雾质谱(ESI-MS)已经成为检测和研究生物分子弱相互作用,即非共价键作用的一个重要分析手段.ESI-MS除了具有快速、灵敏、专属的特点以外,还有能够直接得出复合物的分子量和化学计量比的优点.本文通过蛋白质与蛋白质、配体、金属离子的非共价复合物的例子阐述了ESI-MS技术的主要特性,综述了ESI-MS在非共价键蛋白质复合物方面的早期和近期应用研究成果.引用文献34篇. 相似文献
20.
Gas-phase reactions between multiply charged positive and negative protein ions are carried out in a quadrupole ion trap mass spectrometer. The ions react with one another by proton transfer and complex formation. Proton transfer products and complexes are formed via competitive processes in single ion/ion encounters. The relative contributions of proton transfer versus complex formation are dependent upon the charges of the ions as well as other characteristics of the ions yet to be clearly delineated. No fragmentation of covalent bonds of the protein reactants is observed. A model that considers the trajectories associated with ion/ion interactions appears to hold the most promise in accounting for the results. The formation of bound ion/ion orbits appears to play an important role in determining overall reaction kinetics as well as the distribution of ion/ion reaction products. Tandem mass spectrometry is used to compare protein complexes formed in the gas-phase with those formed initially in solution and subsequently liberated by electrospray; it is shown that both forms of complex dissociate similarly, but the complexes formed in the gas phase can retain a "memory" of their method of formation. 相似文献