Protein folding/refolding analysis by mass spectrometry. Scrambling of disulphide bridges in insulin.
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ABSTRACT:
SUBMITTER: Morris HR
PROVIDER: S-EPMC1131513 | biostudies-other | 1990 Jun
REPOSITORIES: biostudies-other
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Project description:Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) has become a powerful method to characterize protein conformational dynamics. Workflows typically utilize pepsin digestion prior to MS analysis to yield peptide level structural resolution. Tandem mass spectrometry (MS/MS) can potentially facilitate determination of site-specific deuteration to single-residue resolution. However, to be effective, MS/MS activation must minimize the occurrence of gas-phase intramolecular randomization of solution-generated deuterium labels. While significant work has focused on understanding this process in positive-ion mode, little is known about hydrogen/deuterium (H/D) scrambling processes in negative-ion mode. Here, we utilize selectively deuterated model peptides to investigate the extent of intramolecular H/D scrambling upon several negative-ion mode MS/MS techniques, including negative-ion collision-induced dissociation (nCID), electron detachment dissociation (EDD), negative-ion free radical-initiated peptide sequencing (nFRIPS), and negative-ion electron capture dissociation (niECD). H/D scrambling was extensive in deprotonated peptides upon nCID and nFRIPS. In fact, the energetics required to induce dissociation in nCID are sufficient to allow histidine C-2 and Cβ hydrogen atoms to participate in the scrambling process. EDD and niECD demonstrated moderate H/D scrambling with niECD being superior in terms of minimizing hydrogen migration, achieving ~ 30% scrambling levels for small c-type fragment ions. We believe the observed scrambling is likely due to activation during ionization and ion transport rather than during the niECD event itself.
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Project description:Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra performance liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS. In this review, we focus on studies that have used a combination of approaches to reveal (re)folding of proteins as well as on long-distance allosteric changes upon interaction.
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Project description:The precise mechanism of protein folding remains elusive and there is a deficiency of biophysical techniques that are capable of monitoring the individual behavior of copopulated protein conformers during the folding process. Herein, an ion mobility spectrometry (IMS) device integrated with electrospray ionization mass spectrometry (ESI-MS) has been used to successfully separate and analyze protein conformers differing in cross section and/or charge state. In an initial test, an ensemble of folded and partially folded conformers of the protein cytochrome c was separated. A detailed study undertaken on the amyloidogenic protein beta(2)-microglobulin (beta(2)m), which forms fibrils by protein unfolding followed by self-aggregation and is responsible for the disease dialysis-related amyloidosis, has generated important insights into its folding landscape. Initially, a systematic titration of beta(2)m over the pH range 2 to 7 using ESI-IMS-MS allowed individual conformers to be monitored and quantified throughout the acid denaturation process. Furthermore, a comparison of wild-type beta(2)m with single and double amino acid variants with a range of folding stabilities and propensities for amyloid fibril formation has provided illuminating evidence of the role of different conformers in protein stability and amyloidogenic aggregation. The ESI-IMS-MS data presented here not only demonstrate an important and informative further dimension to ESI-MS, but also illustrate the potential of the ESI-IMS-MS technique for unravelling protein folding enigmas in general and studying protein misfolding diseases in particular.
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Project description:Described here is a mass spectrometry-based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the chemical denaturant dependence of the slow H/D exchange reaction of the imidazole C(2) proton in histidine side chains. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin (Hb), and the hemoglobin-haptoglobin (Hb-Hp) protein complex. Folding free energies consistent with those previously determined by other more conventional techniques were obtained for the two-state folding proteins, Rnase A and myoglobin. The protocol successfully detected a previously observed partially unfolded intermediate stabilized in the BCA II folding/unfolding reaction, and it could be used to generate a K(d) value of 0.24 nM for the Hb-Hp complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods was also demonstrated in an experiment in which the protocol was used to detect the binding of zinc to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein's three-dimensional structure for successful application.
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Project description:Cellular membranes are composed of proteins and glyco- and phospholipids and play an indispensible role in maintaining cellular integrity and homeostasis, by physically restricting biochemical processes within cells and providing protection. Membrane proteins perform many essential functions, which include operating as transporters, adhesion-anchors, receptors, and enzymes. Recent advancements in proteomic mass spectrometry have resulted in substantial progress towards the determination of the plasma membrane (PM) proteome, resolution of membrane protein topology, establishment of numerous receptor protein complexes, identification of ligand-receptor pairs, and the elucidation of signaling networks originating at the PM. Here, we discuss the recent accelerated success of discovery-based proteomic pipelines for the establishment of a complete membrane proteome.
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Project description:Ion mobility spectrometry (IMS) coupled with gas-phase hydrogen deuterium exchange (HDX)-mass spectrometry (MS) and molecular dynamic simulations (MDS) has been used for structural investigation of anions produced by electrospraying a sample containing a synthetic peptide having the sequence KKDDDDDIIKIIK. In these experiments the potential of the analytical method for locating charge sites on ions as well as for utilizing collision-induced dissociation (CID) to reveal the degree of deuterium uptake within specific amino acid residues has been assessed. For diffuse (i.e., more elongated) [M - 2H](2-) ions, decreased deuterium content along with MDS data suggest that the D4 and D6 residues are charge sites, whereas for the more diffuse [M - 3H](3-) ions, the data suggest that the D4, D7, and the C-terminus are deprotonated. Fragmentation of mobility-selected, diffuse [M - 2H](2-) ions to determine deuterium uptake at individual amino acid residues reveals a degree of deuterium retention at incorporation sites. Although the diffuse [M - 3H](3-) ions may show more HD scrambling, it is not possible to clearly distinguish HD scrambling from the expected deuterium uptake based on a hydrogen accessibility model. The capability of the IMS-HDX-MS/MS approach to provide relevant details about ion structure is discussed. Additionally, the ability to extend the approach for locating protonation sites on positively-charged ions is presented.
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Project description:The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on native-like interfoldon interactions orders the pathway.
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Project description:Chlorination of tyrosine is a commonly known effect/consequence of myeloperoxidase activity at sites of inflammation, and detection of 3-chlorotyrosine has been used as biomarker for inflammatory diseases. However, few studies have addressed site specific chlorination in proteins, and no methods for large scale chloroproteomics studies have yet been published. In this study, we present an optimized mass spectrometry based protocol to identify and quantify chlorinated peptides from single proteins modified by HOCl (100 and 500 μM, within estimated pathophysiological levels), at a high level of sensitivity and accuracy. Particular emphasis was placed on 1) sensitive and precise detection of modification sites, 2) the avoidance of loss or artefactual creation of modifications, 3) accurate quantification of peptide abundance and reduction of missing values problem, 4) monitoring the dynamics of modification in samples exposed to different oxidant concentrations and 5) development of guidelines for verification of chlorination sites assignment. A combination of an optimised sample preparation protocol, and improved data analysis approaches have allowed identification of 33 and 15 chlorination sites in laminin and fibronectin, respectively, reported in previous manuscripts [1,2]. The method was subsequently tested on murine basement membrane extract, which contains high levels of laminin in a complex mixture. Here, 10 of the major chlorination sites in laminin were recapitulated, highlighting the utility of the method in detecting damage in complex samples.
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Project description:[(35)S]Cystine-labelled immunoglobulin MOPC21 (IgG1) was prepared from myeloma cells in tissue culture. Carrier myeloma protein was added and the protein was digested with pepsin. The digest was fractionated on Sephadex G-50 into two fractions, further digested with trypsin and again fractionated on Sephadex. Disulphide-bridge peptides were purified by electrophoresis and chromatography and identified by radioautography. A peptide of 96 residues was isolated, which contains both the heavy-light interchain disulphide bridge and all the inter-heavy-chain disulphide bridges. Other peptides were isolated, accounting for all the intrachain disulphide bridges (which could be placed by homology with proteins of other species), except for the variable section of the light chain. Sequences describing this missing disulphide bridge were obtained from totally reduced and alkylated light chains. Peptides related to the interchain disulphide-bridge peptide were isolated from partially reduced and alkylated myeloma protein and from totally reduced heavy chain. The interchain disulphide-bridge peptide was placed at the C-terminal position of the F(ab')(2) fragment, prepared by digestion of the protein with pepsin at pH4.0. Sequences from the heavy-chain intrachain disulphide bridges of MOPC 21 immunoglobulin are compared with homologous sequences from mouse myeloma proteins of other subclasses and proteins of other species.
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Project description:Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). The S40 and S2 strains were grown in 14N and 15N media with acetate separately. A biological replicate was made by switching the media. Mass spectrometry based quantitative proteomics were done on the mixtures to investigate the differences in expression patterns between S40 and S2. Keywords: isotope labeling mass spectrometry, quantitative proteomics
2005-09-20 | GSE2744 | GEO