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Rapid pre-gel visualization of proteins with mass spectrometry compatibility

ABSTRACT: BSA_nodry

INSTRUMENT(S): Agilent MSD trap XCT ultra

ORGANISM(S): Bos Taurus (bovine) Escherichia Coli Unidentified Chimeric Sequence

TISSUE(S): Blood

SUBMITTER: Robert Winkler

LAB HEAD: Robert Winkler

PROVIDER: PRD000211 | Pride | 2012-01-16

REPOSITORIES: Pride

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			Action	DRS
	PRIDE_Exp_Complete_Ac_11792.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_11792.pride.mztab.gz	Mztab
	PRIDE_Exp_Complete_Ac_11792.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_11793.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_11793.pride.mztab.gz	Mztab

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Publications

Accelerated identification of proteins by mass spectrometry by employing covalent pre-gel staining with Uniblue A.

Mata-Gómez Marco A MA Yasui Matthew T MT Guerrero-Rangel Armando A Valdés-Rodríguez Silvia S Winkler Robert R

PloS one 20120217 2

<h4>Background</h4>The identification of proteins by mass spectrometry is a standard method in biopharmaceutical quality control and biochemical research. Prior to identification by mass spectrometry, proteins are usually pre-separated by electrophoresis. However, current protein staining and de-staining protocols are tedious and time consuming, and therefore prolong the sample preparation time for mass spectrometry.<h4>Methodology and principal findings</h4>We developed a 1-minute covalent pre- ...[more]

PMID: 22363648

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Accelerated identification of proteins by mass spectrometry by employing covalent pre-gel staining with Uniblue A.

Project description:BackgroundThe identification of proteins by mass spectrometry is a standard method in biopharmaceutical quality control and biochemical research. Prior to identification by mass spectrometry, proteins are usually pre-separated by electrophoresis. However, current protein staining and de-staining protocols are tedious and time consuming, and therefore prolong the sample preparation time for mass spectrometry.Methodology and principal findingsWe developed a 1-minute covalent pre-gel staining protocol for proteins, which does not require de-staining before the mass spectrometry analysis. We investigated the electrophoretic properties of derivatized proteins and peptides and studied their behavior in mass spectrometry. Further, we elucidated the preferred reaction of proteins with Uniblue A and demonstrate the integration of the peptide derivatization into typical informatics tools.Conclusions and significanceThe Uniblue A staining method drastically speeds up the sample preparation for the mass spectrometry based identification of proteins. The application of this chemo-proteomic strategy will be advantageous for routine quality control of proteins and for time-critical tasks in protein analysis.

| S-EPMC3281962 | biostudies-literature

Gel-assisted mass spectrometry imaging.

Project description:Compatible with label-free detection and quantification, mass spectrometry imaging (MSI) is a powerful tool for spatial investigation of biomolecules in intact specimens. Yet, the spatial resolution of MSI is limited by the method's physical and instrumental constraints, which often preclude it from single-cell and subcellular applications. By taking advantage of the reversible interaction of analytes with superabsorbent hydrogels, we developed a sample preparation and imaging workflow named Gel-Assisted Mass Spectrometry Imaging (GAMSI) to overcome these limits. With GAMSI, the spatial resolution of lipid and protein MALDI-MSI can be enhanced severalfold without changing the existing mass spectrometry hardware and analysis pipeline. This approach will further enhance the accessibility to (sub)cellular-scale MALDI-MSI-based spatial omics.

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Rapid characterization of secreted recombinant proteins by native mass spectrometry.

Project description:Characterization of overexpressed proteins is essential for assessing their quality, and providing input for iterative redesign and optimization. This process is typically carried out following purification procedures that require pronounced cost of time and labor. Therefore, quality assessment of recombinant proteins with no prior purification offers a major advantage. Here, we report a native mass spectrometry method that enables characterization of overproduced proteins directly from culture media. Properties such as solubility, molecular weight, folding, assembly state, overall structure, post-translational modifications and binding to relevant biomolecules are immediately revealed. We show the applicability of the method for in-depth characterization of secreted recombinant proteins from eukaryotic systems such as yeast, insect, and human cells. This method, which can be readily extended to high-throughput analysis, considerably shortens the time gap between protein production and characterization, and is particularly suitable for characterizing engineered and mutated proteins, and optimizing yield and quality of overexpressed proteins.

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Chromatin proteins linked to dosage compensation in Drosophila captured by ChIP-mass spectrometry

Project description:We report the usage of ChIP-mass spectrometry in identifying proteins and histone modifications involved in Drosophila dosage compensation. We identified a chromatin targeting factor, CG4747, that is involved in recognition of H3K36me3 and robust recruitment of the Drosophila MSL complex to its correct targets on the male X chromosome. ChIP-seq with PAP antibody of Drosophila larvae expressing C-terminally TAP-tagged CG4747.

2012-11-06 | E-GEOD-42025 | biostudies-arrayexpress

Improved mass spectrometry compatibility is afforded by ammoniacal silver staining.

Project description:Sequence coverage in MS analysis of protein digestion-derived peptides is a key issue for detailed characterization of proteins or identification at low quantities. In gel-based proteomics studies, the sequence coverage greatly depends on the protein detection method. It is shown here that ammoniacal silver detection methods offer improved sequence coverage over standard silver nitrate methods, while keeping the high sensitivity of silver staining. With the development of 2D-PAGE-based proteomics, another burden is placed on the detection methods used for protein detection on 2-D-gels. Besides the classical requirements of linearity, sensitivity, and homogeneity from one protein to another, detection methods must now take into account another aspect, namely their compatibility with MS. This compatibility is evidenced by two different and complementary aspects, which are (i) the absence of adducts and artefactual modifications on the peptides obtained after protease digestion of a protein detected and digested in - gel, and (ii) the quantitative yield of peptides recovered after digestion and analyzed by the mass spectrometer. While this quantitative yield is not very important per se, it is however a crucial parameter as it strongly influences the S/N of the mass spectrum and thus the number of peptides that can be detected from a given protein input, especially at low protein amounts. This influences in turn the sequence coverage and thus the detail of the analysis provided by the mass spectrometer.

| S-EPMC2797540 | biostudies-literature

Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry.

Project description:SummaryHydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, analysis of HDX-MS data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. We present Deuteros which is a standalone software designed to be coupled with Waters DynamX HDX data analysis software, allowing the rapid analysis and visualization of data from differential HDX-MS.Availability and implementationDeuteros is open-source and can be downloaded from https://github.com/andymlau/Deuteros, under the Apache 2.0 license. Written in MATLAB and supported on both Windows and MacOS. Requires the MATLAB runtime library. According to the Wellcome Trust and UK research councils' Common Principles on Data Policy on data, software and materials management and sharing, all data supporting this study will be openly available from the software repository.

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Matrix Selection for the Visualization of Small Molecules and Lipids in Brain Tumors Using Untargeted MALDI-TOF Mass Spectrometry Imaging

Project description:Matrix-assisted laser desorption/ionization mass spectrometry imaging allows for the study of metabolic activity in the tumor microenvironment of brain cancers. The detectable metabolites within these tumors are contingent upon the choice of matrix, deposition technique, and polarity setting. In this study, we compared the performance of three different matrices, two deposition techniques, and the use of positive and negative polarity in two different brain cancer types and across two species. Optimal combinations were confirmed by a comparative analysis of lipid and small-molecule abundance by using liquid chromatography-mass spectrometry and RNA sequencing to assess differential metabolites and enzymes between normal and tumor regions. Our findings indicate that in the tumor-bearing brain, the recrystallized α-cyano-4-hydroxycinnamic acid matrix with positive polarity offered superior performance for both detected metabolites and consistency with other techniques. Beyond these implications for brain cancer, our work establishes a workflow to identify optimal matrices for spatial metabolomics studies.

2024-10-09 | GSE279139 | GEO

Chromatin proteins linked to dosage compensation in Drosophila captured by ChIP-mass spectrometry

2012-11-06 | GSE42025 | GEO

Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry.

Project description:High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats. In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market.

| S-EPMC4966600 | biostudies-literature

Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry.

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