Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Analysis of intact protein isoforms by mass spectrometry.

ABSTRACT: The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we discuss the basic technology for "top-down" intact protein analysis. Furthermore, examples of studies involved with the qualitative and quantitative analysis of full-length polypeptides are provided.

SUBMITTER: Tipton JD

PROVIDER: S-EPMC3138281 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Analysis of intact protein isoforms by mass spectrometry.

Tipton Jeremiah D JD Tran John C JC Catherman Adam D AD Ahlf Dorothy R DR Durbin Kenneth R KR Kelleher Neil L NL

The Journal of biological chemistry 20110601 29

The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we d ...[more]

PMID: 21632550

Similar Datasets

Mass spectrometry of intact membrane protein complexes.

Project description:Mass spectrometry (MS) of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes, where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here we describe a protocol for MS of membrane protein complexes. The protocol begins with the preparation of the membrane protein complex, enabling not only the direct assessment of stoichiometry, delipidation and quality of the target complex but also the evaluation of the purification strategy. A detailed list of compatible nonionic detergents is included, along with a protocol for screening detergents to find an optimal one for MS, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a quadrupole time-of-flight (Q-TOF) mass spectrometer after the introduction of complexes from gold-coated nanoflow capillaries.

| S-EPMC4058633 | biostudies-literature

Intact microRNA analysis using high resolution mass spectrometry.

Project description:MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that post-transcriptionally regulate gene expression, and play key roles in the regulation of a variety of cellular processes and in disease. New tools to analyze miRNAs will add understanding of the physiological origins and biological functions of this class of molecules. In this study, we investigate the utility of high resolution mass spectrometry for the analysis of miRNAs through proof-of-concept experiments. We demonstrate the ability of mass spectrometry to resolve and separate miRNAs and corresponding 3' variants in mixtures. The mass accuracy of the monoisotopic deprotonated peaks from various miRNAs is in the low ppm range. We compare fragmentation of miRNA by collision-induced dissociation (CID) and by higher-energy collisional dissociation (HCD) which yields similar sequence coverage from both methods but additional fragmentation by HCD versus CID. We measure the linear dynamic range, limit of detection, and limit of quantitation of miRNA loaded onto a C18 column. Lastly, we explore the use of data-dependent acquisition of MS/MS spectra of miRNA during online LC-MS and demonstrate that multiple charge states can be fragmented, yielding nearly full sequence coverage of miRNA on a chromatographic time scale. We conclude that high resolution mass spectrometry allows the separation and measurement of miRNAs in mixtures and a standard LC-MS setup can be adapted for online analysis of these molecules.

| S-EPMC4153431 | biostudies-literature

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

Project description:One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

| S-EPMC6719561 | biostudies-literature

High-Field Asymmetric Waveform Ion Mobility Spectrometry and Native Mass Spectrometry: Analysis of Intact Protein Assemblies and Protein Complexes.

Project description:High-field asymmetric waveform ion mobility spectrometry (FAIMS) enables the separation of ions on the basis of their differential mobility in an asymmetric oscillating electric field. We, and others, have previously demonstrated the benefits of FAIMS for the analysis of peptides and denatured proteins. To date, FAIMS has not been integrated with native mass spectrometry of folded proteins and protein complexes, largely due to concerns over the heating effects associated with the high electric fields employed. Here, we demonstrate the newly introduced cylindrical FAIMS Pro device coupled with an Orbitrap Eclipse enables analysis of intact protein assemblies up to 147 kDa. No evidence for dissociation was detected suggesting that any field heating is insufficient to disrupt the noncovalent interactions governing these assemblies. Moreover, the FAIMS device was integrated into native liquid extraction surface analysis (LESA) MS of protein assemblies directly from thin tissue sections. Intact tetrameric hemoglobin (64 kDa) and trimeric reactive intermediate deiminase A (RidA, 43 kDa) were detected. Improvements in signal-to-noise of between 1.5× and 12× were observed for these protein assemblies on integration of FAIMS.

| S-EPMC7304667 | biostudies-literature

Charge reduction stabilizes intact membrane protein complexes for mass spectrometry.

Project description:The study of intact soluble protein assemblies by means of mass spectrometry is providing invaluable contributions to structural biology and biochemistry. A recent breakthrough has enabled similar study of membrane protein complexes, following their release from detergent micelles in the gas phase. Careful optimization of mass spectrometry conditions, particularly with respect to energy regimes, is essential for maintaining compact folded states as detergent is removed. However, many of the saccharide detergents widely employed in structural biology can cause unfolding of membrane proteins in the gas phase. Here, we investigate the potential of charge reduction by introducing three membrane protein complexes from saccharide detergents and show how reducing their overall charge enables generation of compact states, as evidenced by ion mobility mass spectrometry. We find that charge reduction stabilizes the oligomeric state and enhances the stability of lipid-bound complexes. This finding is significant since maintaining native-like membrane proteins enables ligand binding to be assessed from a range of detergents that retain solubility while protecting the overall fold.

| S-EPMC4594752 | biostudies-literature

Top-Down and Intact Protein Mass Spectrometry Data Visualization for Proteoform Analysis Using VisioProt-MS.

Project description:The rise of intact protein analysis by mass spectrometry (MS) was accompanied by an increasing need for flexible tools allowing data visualization and analysis. These include inspection of the deconvoluted molecular weights of the proteoforms eluted alongside liquid chromatography (LC) through their representation in three-dimensional (3D) liquid chromatography coupled to mass spectrometry (LC-MS) maps (plots of deconvoluted molecular weights, retention times, and intensity of the MS signal). With this aim, we developed a free and open-source web application named VisioProt-MS (https://masstools.ipbs.fr/mstools/visioprot-ms/). VisioProt-MS is highly compatible with many algorithms and software developed by the community to integrate and deconvolute top-down and intact protein MS data. Its dynamic and user-friendly features greatly facilitate analysis through several graphical representations dedicated to MS and tandem mass spectrometry (MS/MS) analysis of proteoforms in complex samples. Here, we will illustrate the importance of LC-MS map visualization to optimize top-down acquisition/search parameters and analyze intact protein MS data. We will go through the main features of VisioProt-MS using the human proteasomal 20S core particle as a user-case.

| S-EPMC6698994 | biostudies-literature

Direct Mass Spectrometry Analysis of Protein Complexes and Intact Proteins up to >70 kDa from Tissue.

Project description:Native liquid extraction surface analysis (LESA) mass spectrometry allows direct analysis of folded proteins and protein complexes from biological substrates, such as dried blood spots and thin tissue sections, by use of native-like extraction/ionization solvents. Previously, we have demonstrated native LESA mass spectrometry of folded proteins up to 16 kDa as well as the 64 kDa hemoglobin tetramer, from mouse tissues. With denaturing LESA solvents, the highest mass protein detected in tissue to date is ∼37 kDa. Here, we demonstrate native LESA mass spectrometry by use of a Q Exactive UHMR Hybrid Quadrupole-Orbitrap (QE-UHMR) mass spectrometer, pushing the upper mass limit of proteins detected in tissue to >70 kDa. Moreover, a protein trimer of 42 kDa was detected and its stoichiometry confirmed by higher energy collision dissociation (HCD). The benefits of inclusion of detergents in the LESA sampling solvent are also demonstrated.

| S-EPMC7006965 | biostudies-literature

Intact mass spectrometry screening to optimize hydroxyl radical dose for protein footprinting.

Project description:Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and "safe" dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.

| S-EPMC10510565 | biostudies-literature

PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.

Project description:Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

| S-EPMC8141340 | biostudies-literature

Identification of cardiac myofilament protein isoforms using multiple mass spectrometry based approaches.

Project description:PurposeThe identification of protein isoforms in complex biological samples is challenging. We, therefore, used an MS approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform.Experimental designThree different workflows were used to isolate and fractionate rat cardiac myofilament subproteomes. All fractions were analyzed on an LTQ-Orbitrap MS, proteins were identified using various search engines (MASCOT, X!Tandem, X!Tandem Kscore, and OMSSA) with results combined via PepArML Meta-Search engine, and a postsearch analysis was performed by MASPECTRAS. All MS data have been deposited in the ProteomeXchange with identifier PXD000874 (http://proteomecentral.proteomexchange.org/dataset/PXD000874).ResultsThe combination of multiple workflows and search engines resulted in a larger number of nonredundant proteins identified than with individual methods. A total of 102 myofilament annotated proteins were observed overlapping in two or three of the workflows. Literature search for myofilament presence with manual validation of the MS spectra was carried out for unambiguous identification: ten cardiac myofilament and 17 cardiac myofilament-associated proteins were identified with 39 isoforms and subisoforms.Conclusion and clinical relevanceWe have identified multiple isoforms of myofilament proteins that are present in cardiac tissue using unique tryptic peptides. Changes in distribution of these protein isoforms under pathological conditions could ultimately allow for clinical diagnostics or as therapeutic targets.

| S-EPMC4222538 | 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