Browse
Submit Data
Databases
API
Help

Dataset Information

10 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Identifying Unknown Enzyme-Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry.

ABSTRACT: The enzyme-substrate complex is inherently transient, rendering its detection difficult. In our framework designed for bisubstrate systems-isotope-labeled, activity-based identification and tracking (IsoLAIT)-the common substrate, such as S-adenosyl-l-methionine (AdoMet) for methyltransferases, is replaced by an analogue (e.g., S-adenosyl-l-vinthionine) that, as a probe, creates a tightly bound [enzyme?substrate?probe] complex upon catalysis by thiopurine-S-methyltransferase (TPMT, EC 2.1.1.67). This persistent complex is then identified by native mass spectrometry from the cellular milieu without separation. Furthermore, the probe's isotope pattern flags even unknown substrates and enzymes. IsoLAIT is broadly applicable for other enzyme systems, particularly those catalyzing group transfer and with multiple substrates, such as glycosyltransferases and kinases.

SUBMITTER: Catcott KC

PROVIDER: S-EPMC6483605 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Identifying Unknown Enzyme-Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry.

Catcott Kalli C KC Yan Jing J Qu Wanlu W Wysocki Vicki H VH Zhou Zhaohui Sunny ZS

Chembiochem : a European journal of chemical biology 20170314 7

The enzyme-substrate complex is inherently transient, rendering its detection difficult. In our framework designed for bisubstrate systems-isotope-labeled, activity-based identification and tracking (IsoLAIT)-the common substrate, such as S-adenosyl-l-methionine (AdoMet) for methyltransferases, is replaced by an analogue (e.g., S-adenosyl-l-vinthionine) that, as a probe, creates a tightly bound [enzyme⋅substrate⋅probe] complex upon catalysis by thiopurine-S-methyltransferase (TPMT, EC 2.1.1.67). ...[more]

PMID: 28140508

Similar Datasets

Cyanobacterial identification by native mass spectrometry

Project description:Cyanobacterial identification by native mass spectrometry

| PRJEB45336 | ENA

Identifying transient protein-protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry.

Project description:Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling.

| S-EPMC3563432 | biostudies-literature

pSILAC mass spectrometry reveals ZFP91 as novel IMiD dependent substrate of the CRL4CRBN ligase

Project description:Thalidomide and its derivatives lenalidomide and pomalidomide (IMiDs) are effective treatments of hematologic malignancies. It was shown that IMiDs impart gain of function properties to the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) ubiquitin ligase that enable binding, ubiquitination and degradation of key therapeutic targets such as IKFZ1, IKZF3 and CSNK1A1. While these substrates have been implicated as efficacy targets in multiple myeloma (MM) and 5q deletion associated myelodysplastic syndrome (del(5q)-MDS), other targets likely exist. Using a pulse-chase SILAC mass spectrometry-based proteomics approach, we demonstrate that lenalidomide induces the ubiquitination and degradation of ZFP91. We establish that ZFP91 is a bona fide IMiD dependent CRL4CRBN substrate and further show that ZFP91 harbors a zinc finger (ZnF) motif, related to the IKZF1/3 ZnF, critical for IMiD dependent CRBN binding. These findings demonstrate that single time point pulse-chase SILAC mass spectrometry-based proteomics (pSILAC-MS) is a sensitive approach for target identification of small molecules inducing selective protein degradation.

2017-03-01 | GSE94728 | GEO

Electrothermal supercharging in mass spectrometry and tandem mass spectrometry of native proteins.

Project description:Electrothermal supercharging of protein ions formed by electrospray ionization from buffered aqueous solutions results in significant increases to both the maximum and average charge states compared to native mass spectrometry in which ions are formed from the same solutions but with lower spray potentials. For eight of the nine proteins investigated, the maximum charge states of protonated ions formed from native solutions with electrothermal supercharging is greater than those obtained from conventional denaturing solutions consisting of water/methanol/acid, although the average charging is slightly lower owing to contributions of small populations of more folded low charge-state structures. Under these conditions, electrothermal supercharging is slightly less effective for anions than for cations. Equivalent sequence coverage (80%) is obtained with electron transfer dissociation of the same high charge-state ion of cytochrome c formed by electrothermal supercharging from native solutions and from denaturing solutions. Electrothermal supercharging should be advantageous for combining structural studies of proteins in native environments with mass spectrometers that have limited high m/z capabilities and for significantly improving tandem mass spectrometry performance for protein ions formed from solutions in which the molecules have native structures and activities.

| S-EPMC3536820 | biostudies-literature

Native Capillary Electrophoresis-Mass Spectrometry of Near 1 MDa Non-Covalent GroEL/GroES/Substrate Protein Complexes.

Project description:Protein complexes are essential for proteins' folding and biological function. Currently, native analysis of large multimeric protein complexes remains challenging. Structural biology techniques are time-consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis-mass spectrometry (CE-MS) method is reported to characterize, under near-physiological conditions, the conformational rearrangements of ∽1 MDa GroEL upon complexation with binding partners involved in a protein folding cycle. The developed CE-MS method is fast (30 min per run), highly sensitive (low-amol level), and requires ∽10 000-fold fewer samples compared to biochemical/biophysical techniques. The method successfully separates GroEL14 (∽800 kDa), GroEL7 (∽400 kDa), GroES7 (∽73 kDa), and NanA4 (∽130 kDa) oligomers. The non-covalent binding of natural substrate proteins with GroEL14 can be detected and quantified. The technique allows monitoring of GroEL14 conformational changes upon complexation with (ATPγS)4-14 and GroES7 (∽876 kDa). Native CE-pseudo-MS3 analyses of wild-type (WT) GroEL and two GroEL mutants result in up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE-MS performance for multimeric complexes' characterization versus direct infusion ESI-MS. This study shows the CE-MS potential to provide information on binding stoichiometry and kinetics for various protein complexes.

| S-EPMC10953559 | biostudies-literature

Quantitation of the Noncovalent Cellular Retinol-Binding Protein, Type 1 Complex Through Native Mass Spectrometry.

Project description:Native mass spectrometry (MS) has become a valuable tool in probing noncovalent protein-ligand interactions in a sample-efficient way, yet the quantitative application potential of native MS has not been fully explored. Cellular retinol binding protein, type I (CrbpI) chaperones retinol and retinal in the cell, protecting them from nonspecific oxidation and delivering them to biosynthesis enzymes where the bound (holo-) and unbound (apo-) forms of CrbpI exert distinct biological functions. Using nanoelectrospray, we developed a native MS assay for probing apo- and holo-CrbpI abundance to facilitate exploring their biological functions in retinoid metabolism and signaling. The methods were developed on two platforms, an Orbitrap-based Thermo Exactive and a Q-IMS-TOF-based Waters Synapt G2S, where similar ion behaviors under optimized conditions were observed. Overall, our results suggested that within the working range (~1-10 μM), gas-phase ions in the native state linearly correspond to solution concentration and relative ion intensities of the apo- and holo-protein ions can linearly respond to the solution ratios, suggesting native MS is a viable tool for relative quantitation in this system. Graphical Abstract ᅟ.

| S-EPMC5728378 | biostudies-literature

Recent technological developments for native mass spectrometry.

Project description:Native mass spectrometry (MS), the analysis of proteins and protein complexes from solutions that stabilize native solution structures, is a rapidly expanding area. There is strong evidence supporting the retention of proteins' native folds in the absence of solvent under the experimental timescales of MS experiments. Therefore, instrumentation has been developed to use gas-phase native-like protein ions to exploit the speed, sensitivity, and selectivity of mass spectrometry approaches to solve emerging problems in structural biology. This article reviews some of the recent advances and applications in gas-phase instrumentation for structural proteomics.

| S-EPMC9219587 | biostudies-literature

Parsimonious Charge Deconvolution for Native Mass Spectrometry.

Project description:Charge deconvolution infers the mass from mass over charge (m/z) measurements in electrospray ionization mass spectra. When applied over a wide input m/z or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new "parsimonious" charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously and, when focused on the m/z range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using m/z peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies.

| S-EPMC5838638 | biostudies-literature

Charging of Proteins in Native Mass Spectrometry.

Project description:Factors that influence the charging of protein ions formed by electrospray ionization from aqueous solutions in which proteins have native structures and function were investigated. Protein ions ranging in molecular weight from 12.3 to 79.7 kDa and pI values from 5.4 to 9.6 were formed from different solutions and reacted with volatile bases of gas-phase basicities higher than that of ammonia in the cell of a Fourier-transform ion cyclotron resonance mass spectrometer. The charge-state distribution of cytochrome c ions formed from aqueous ammonium or potassium acetate is the same. Moreover, ions formed from these two solutions do not undergo proton transfer to 2-fluoropyridine, which is 8 kcal/mol more basic than ammonia. These results provide compelling evidence that proton transfer between ammonia and protein ions does not limit protein ion charge in native electrospray ionization. Both circular dichroism and ion mobility measurements indicate that there are differences in conformations of proteins in pure water and aqueous ammonium acetate, and these differences can account for the difference in the extent of charging and proton-transfer reactivities of protein ions formed from these solutions. The extent of proton transfer of the protein ions with higher gas-phase basicity bases trends with how closely the protein ions are charged to the value predicted by the Rayleigh limit for spherical water droplets approximately the same size as the proteins. These results indicate that droplet charge limits protein ion charge in native mass spectrometry and are consistent with these ions being formed by the charged residue mechanism. Graphical Abstract ᅟ.

| S-EPMC5283922 | biostudies-literature

Native Mass Spectrometry in Fragment-Based Drug Discovery.

Project description:The advent of native mass spectrometry (MS) in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein-ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD). Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

| S-EPMC6274484 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data