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Article ; Online: A New Approach to Assess mAb Aggregation.

Turko, Illarion V

Methods in molecular biology (Clifton, N.J.)

2020 Volume 2131, Page(s) 245–254

Abstract: A proof of concept for new methodology to detect and potentially quantify mAb aggregation is presented. Assay development included using an aggregated mAb as bait for screening of a phage display peptide library and identifying those peptides with random ...

Abstract	A proof of concept for new methodology to detect and potentially quantify mAb aggregation is presented. Assay development included using an aggregated mAb as bait for screening of a phage display peptide library and identifying those peptides with random sequence which can recognize mAb aggregates. The selected peptides can be used for developing homogeneous quantitative methods to assess mAb aggregation. Results indicate that a peptide-binding method coupled with fluorescence polarization detection can detect mAb aggregation and potentially monitor the propensity of therapeutic protein candidates to aggregate.
MeSH term(s)	Antibodies, Monoclonal/chemistry ; Antibodies, Monoclonal/genetics ; Fluorescein-5-isothiocyanate/chemistry ; Fluorescence Polarization ; High-Throughput Nucleotide Sequencing ; Peptide Library ; Peptides/analysis ; Peptides/chemistry ; Peptides/genetics ; Proof of Concept Study ; Protein Aggregates
Chemical Substances	Antibodies, Monoclonal ; Peptide Library ; Peptides ; Protein Aggregates ; Fluorescein-5-isothiocyanate (I223NX31W9)
Language	English
Publishing date	2020-03-08
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-0389-5_12
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Article ; Online: Isolation protocols and mitochondrial content for plasma extracellular vesicles.

Nguyen, Ai / Turko, Illarion V

Analytical and bioanalytical chemistry

2022 Volume 415, Issue 7, Page(s) 1299–1304

Abstract: Mitochondrial content has been reported outside of cells either within extracellular vesicles (EVs) or as free mitochondria. Mitochondrial EVs can potentially play multiple physiological and pathophysiological roles. To understand their functions, ... ...

Abstract	Mitochondrial content has been reported outside of cells either within extracellular vesicles (EVs) or as free mitochondria. Mitochondrial EVs can potentially play multiple physiological and pathophysiological roles. To understand their functions, isolation protocols to separate mitochondrial EVs from other mitochondrial content need to be established. In the present work, we use a multiple reaction monitoring assay with isotope labeled internal standards to quantify 11 mitochondrial, 6 plasma membrane-specific, 4 endosomal membrane-specific, and 2 soluble proteins to evaluate the efficiency of chromatographic isolation of mitochondrial EVs. The isolation protocol includes ultracentrifugation, size exclusion chromatography, and chromatography on immobilized heparin. All protein concentrations were normalized to the concentration of ATP synthase alpha subunit to generate a ratio that allows comparison of different samples obtained during the isolation. We have shown that initial samples after ultracentrifugation are contaminated with non-EV mitochondrial content that cannot be separated from EVs using size exclusion chromatography, but can be efficiently separated from EVs on the column with immobilized heparin.
MeSH term(s)	Extracellular Vesicles/chemistry ; Chromatography, Gel ; Mitochondria ; Heparin/analysis ; Ultracentrifugation
Chemical Substances	Heparin (9005-49-6)
Language	English
Publishing date	2022-12-02
Publishing country	Germany
Document type	Journal Article
ZDB-ID	201093-8
ISSN	1618-2650 ; 0016-1152 ; 0372-7920
ISSN (online)	1618-2650
ISSN	0016-1152 ; 0372-7920
DOI	10.1007/s00216-022-04465-x
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Article ; Online: Isolation protocols and mitochondrial content for plasma extracellular vesicles

Nguyễn, Ái / Turko, Illarion V.

Anal Bioanal Chem. 2023 Mar., v. 415, no. 7 p.1299-1304

2023

Abstract	Mitochondrial content has been reported outside of cells either within extracellular vesicles (EVs) or as free mitochondria. Mitochondrial EVs can potentially play multiple physiological and pathophysiological roles. To understand their functions, isolation protocols to separate mitochondrial EVs from other mitochondrial content need to be established. In the present work, we use a multiple reaction monitoring assay with isotope labeled internal standards to quantify 11 mitochondrial, 6 plasma membrane-specific, 4 endosomal membrane-specific, and 2 soluble proteins to evaluate the efficiency of chromatographic isolation of mitochondrial EVs. The isolation protocol includes ultracentrifugation, size exclusion chromatography, and chromatography on immobilized heparin. All protein concentrations were normalized to the concentration of ATP synthase alpha subunit to generate a ratio that allows comparison of different samples obtained during the isolation. We have shown that initial samples after ultracentrifugation are contaminated with non-EV mitochondrial content that cannot be separated from EVs using size exclusion chromatography, but can be efficiently separated from EVs on the column with immobilized heparin.
Keywords	H-transporting ATP synthase ; gel chromatography ; heparin ; isotopes ; mitochondria ; ultracentrifugation
Language	English
Dates of publication	2023-03
Size	p. 1299-1304.
Publishing place	Springer Berlin Heidelberg
Document type	Article ; Online
ISSN	1618-2642
DOI	10.1007/s00216-022-04465-x
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Article ; Online: Quantitative proteomic analysis for evaluating affinity isolation of extracellular vesicles.

Nguyen, Ai / Wang, Tingting / Turko, Illarion V

Journal of proteomics

2021 Volume 249, Page(s) 104359

Abstract: Absolute quantification with mass spectrometry and isotope labeled internal standards has found broad applications in biomedical research. In the present research, it was used for developing and evaluating a new affinity-based approach to isolate ... ...

Abstract	Absolute quantification with mass spectrometry and isotope labeled internal standards has found broad applications in biomedical research. In the present research, it was used for developing and evaluating a new affinity-based approach to isolate extracellular vesicles (EVs) from human plasma. First, a phage display peptide library was screened against EVs as a bait and absolute quantification of multiple proteins helped to select the best bait available. Then, absolute quantification was used to evaluate the efficiency of affinity chromatography on peptide-Sepharose. In summary, we have demonstrated that peptides with affinity to EVs selected from phage library screening can be valuable ligands for EVs isolation. SIGNIFICANCE: Extracellular vesicles (EVs) have an important role in intercellular communication for all cell types. This makes EVs a promising new type of therapeutics capable to deliver drugs to specific sites with no off-target side effects. However, their isolation, and correct assignment of their biological function and properties remains an obscure field of research. In this study, we proposed to use MRM quantitation of a pattern of EVs and non-EVs proteins to develop a purification protocol based on affinity peptides selected from phage library screening. MRM quantification of EVs proteins can also help in identifying those that are subpopulation specific markers for further target-specific isolation.
MeSH term(s)	Chromatography, Affinity ; Extracellular Vesicles ; Humans ; Mass Spectrometry ; Proteins ; Proteomics
Chemical Substances	Proteins
Language	English
Publishing date	2021-08-25
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2400835-7
ISSN	1876-7737 ; 1874-3919
ISSN (online)	1876-7737
ISSN	1874-3919
DOI	10.1016/j.jprot.2021.104359
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Article ; Online: Proteomic Toolbox To Standardize the Separation of Extracellular Vesicles and Lipoprotein Particles.

Wang, Tingting / Turko, Illarion V

Journal of proteome research

2018 Volume 17, Issue 9, Page(s) 3104–3113

Abstract: Circulating in blood, extracellular vesicles (EVs) and lipoprotein particles (LPs) have diagnostic and prognostic value. To unambiguously define their functions, separation protocols need to be developed. However, because of their similar size and ... ...

Abstract	Circulating in blood, extracellular vesicles (EVs) and lipoprotein particles (LPs) have diagnostic and prognostic value. To unambiguously define their functions, separation protocols need to be developed. However, because of their similar size and density, traditional approaches to separate EVs and LPs often fail to provide the required resolution. Further development and standardization of affinity-based protocols is necessary, and a quantitative method is needed to assess the efficiency of LP depletion from EV samples. In the present study, we propose the simultaneous quantification of three groups of proteins by mass spectrometry as a toolbox to evaluate prospective separation protocols. We generated
MeSH term(s)	Apolipoproteins/blood ; Apolipoproteins/chemistry ; Apolipoproteins/isolation & purification ; Blood Proteins/chemistry ; Blood Proteins/isolation & purification ; Chromatography, Affinity/methods ; Chromatography, Gel/methods ; Chromatography, Liquid ; Extracellular Vesicles/chemistry ; Extracellular Vesicles/metabolism ; Humans ; Proteomics/instrumentation ; Proteomics/methods ; Reference Standards ; Sepharose/analogs & derivatives ; Sepharose/chemistry ; Staining and Labeling/methods ; Tandem Mass Spectrometry
Chemical Substances	Apolipoproteins ; Blood Proteins ; concanavalin A-sepharose ; heparin-sepharose ; Sepharose (9012-36-6)
Language	English
Publishing date	2018-08-16
Publishing country	United States
Document type	Journal Article
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021/acs.jproteome.8b00225
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Article ; Online: Quantitative proteomic analysis for evaluating affinity isolation of extracellular vesicles

Nguyễn, Ái / Wang, Tingting / Turko, Illarion V.

Journal of Proteomics. 2021 Oct., v. 249 p.104359-

2021

Abstract	Absolute quantification with mass spectrometry and isotope labeled internal standards has found broad applications in biomedical research. In the present research, it was used for developing and evaluating a new affinity-based approach to isolate extracellular vesicles (EVs) from human plasma. First, a phage display peptide library was screened against EVs as a bait and absolute quantification of multiple proteins helped to select the best bait available. Then, absolute quantification was used to evaluate the efficiency of affinity chromatography on peptide-Sepharose. In summary, we have demonstrated that peptides with affinity to EVs selected from phage library screening can be valuable ligands for EVs isolation. Extracellular vesicles (EVs) have an important role in intercellular communication for all cell types. This makes EVs a promising new type of therapeutics capable to deliver drugs to specific sites with no off-target side effects. However, their isolation, and correct assignment of their biological function and properties remains an obscure field of research. In this study, we proposed to use MRM quantitation of a pattern of EVs and non-EVs proteins to develop a purification protocol based on affinity peptides selected from phage library screening. MRM quantification of EVs proteins can also help in identifying those that are subpopulation specific markers for further target-specific isolation.
Keywords	DNA libraries ; affinity chromatography ; biomedical research ; cell communication ; humans ; isotopes ; ligands ; mass spectrometry ; peptide libraries ; proteomics ; therapeutics ; Extracellular vesicles ; Targeted proteomics ; QconCATs ; Multiple reaction monitoring ; α-2-MG ; EV ; MRM ; SEC
Language	English
Dates of publication	2021-10
Publishing place	Elsevier B.V.
Document type	Article ; Online
ZDB-ID	2400835-7
ISSN	1876-7737 ; 1874-3919
ISSN (online)	1876-7737
ISSN	1874-3919
DOI	10.1016/j.jprot.2021.104359
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Article: Quantitative Proteomic Analysis of Biogenesis-Based Classification for Extracellular Vesicles.

Zhang, Linwen / Parot, Jeremie / Hackley, Vincent A / Turko, Illarion V

Proteomes

2020 Volume 8, Issue 4

Abstract: Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it ...

Abstract	Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it is increasingly clear that the actual composition of a particular EV preparation cannot be adequately described with these two simple terms and is much more complex. Since the cell membrane origin of EVs predetermines their biological functions, the understanding of EV biogenesis is important for accurate interpretation of observed results. In the present study, we propose to take advantage of selective expression of some proteins in plasma or endosomal membranes and to use these proteins as plasma membrane-specific or endosomal membrane-specific markers. We have demonstrated that a quantitative mass spectrometry analysis allows simultaneous measurement of plasma membrane-specific and endosomal membrane-specific proteins in microvesicles and exosomes obtained after differential ultracentrifugation. Before mass spectrometry analysis, we also used sonicated platelets as a model of mixed EVs and multidetector asymmetrical-flow field-flow fractionation as an analytical method to verify a possible cross contamination of obtained microvesicles and exosomes. Based on the quantitative appearance of membrane-specific protein markers in EV preparations from human plasma and from human ARPE-19 cell medium, we concluded that there is no actual size limitation and both microvesicles and exosomes can be represented by large and small vesicles.
Language	English
Publishing date	2020-11-06
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2720995-7
ISSN	2227-7382
ISSN	2227-7382
DOI	10.3390/proteomes8040033
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Article ; Online: Mass spectrometry enumeration of filamentous M13 bacteriophage.

Wang, Tingting / Nguyen, Ai / Zhang, Linwen / Turko, Illarion V

Analytical biochemistry

2019 Volume 582, Page(s) 113354

Abstract: In the last decade, filamentous M13 bacteriophage has emerged into numerous biotechnological applications as a promising nontoxic and self-assembling biomaterial with specific binding properties. This raises a question about its upscale production that ... ...

Abstract	In the last decade, filamentous M13 bacteriophage has emerged into numerous biotechnological applications as a promising nontoxic and self-assembling biomaterial with specific binding properties. This raises a question about its upscale production that consequently requires an accurate phage enumeration during the various protocol developments. However, traditional methods of measuring phage concentration are mainly biological in nature and therefore time and labor intensive. These traditional methods also demonstrate poor reproducibility and are semi-quantitative at best. In the present work, we capitalized on mass spectrometry based absolute protein quantitation. We have optimized the quantitation conditions for a major coat protein, pVIII. Enumeration of M13 bacteriophage can be further performed using the determined molar concentration of pVIII, Avogadro's number, and known copy number of pVIII per phage. Since many different phages have well-defined copy number of capsid proteins, the proposed approach can be simply applied to any phage with known copy number of a specific capsid protein.
MeSH term(s)	Bacteriophage M13/isolation & purification ; Capsid Proteins/analysis ; Mass Spectrometry/methods
Chemical Substances	Capsid Proteins
Language	English
Publishing date	2019-07-02
Publishing country	United States
Document type	Journal Article
ZDB-ID	1110-1
ISSN	1096-0309 ; 0003-2697
ISSN (online)	1096-0309
ISSN	0003-2697
DOI	10.1016/j.ab.2019.113354
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Article: Histone post-translational modifications in frontal cortex from human donors with Alzheimer's disease.

Anderson, Kyle W / Turko, Illarion V

Clinical proteomics

2015 Volume 12, Page(s) 26

Abstract: Background: Alzheimer's disease (AD) is the sixth leading cause of death and the most costly disease in the US. Despite the enormous impact of AD, there are no treatments that delay onset or stop disease progression currently on the market. This is ... ...

Abstract	Background: Alzheimer's disease (AD) is the sixth leading cause of death and the most costly disease in the US. Despite the enormous impact of AD, there are no treatments that delay onset or stop disease progression currently on the market. This is partly due to the complexity of the disease and the largely unknown pathogenesis of sporadic AD, which accounts for the vast majority of cases. Epigenetics has been implicated as a critical component to AD pathology and a potential "hot spot" for treatments. Histone post-translational modifications (PTMs) are a key element in epigenetic regulation of gene expression and are known to be associated with the pathology of numerous diseases. Investigation of histone PTMs can help elucidate AD pathology and identify targets for therapies. Results: A multiple reaction monitoring mass spectrometry assay was used to measure changes in abundance of several histone PTMs in frontal cortex from human donors affected with AD (n = 6) and age-matched, normal donors (n = 6). Of the changes observed, notable decreases in methylation of H2B residue K108 by 25 % and H4 residue R55 by 35 % were measured and are likely associated with hydrogen bonding networks important for nucleosome stability. Additionally, a 91 % increase in ubiquitination of K120 on H2B was measured as well as an apparent loss in acetylation of the region near the N-terminus of H4. Our method of quantification was also determined to be precise and robust, signifying measured changes were representative of true biological differences between donors and sample groups. Conclusion: We are the first to report changes in methylation of H2B K108, methylation of H4 R55, and ubiquitination of H2B K120 in frontal cortex from human donors with AD. These notable PTM changes may be of great importance in elucidating the epigenetic mechanism of AD as it relates to disease pathology. Beyond the structural and functional impacts of the changes we have measured, the sites of altered PTMs may be used to identify enzymes responsible for their modulation, which could be used as prospective drug targets for highly specific AD therapies.
Language	English
Publishing date	2015-10-01
Publishing country	England
Document type	Journal Article
ZDB-ID	2205154-5
ISSN	1542-6416
ISSN	1542-6416
DOI	10.1186/s12014-015-9098-1
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Article ; Online: Assessment of Extracellular Vesicles Purity Using Proteomic Standards.

Wang, Tingting / Anderson, Kyle W / Turko, Illarion V

Analytical chemistry

2017 Volume 89, Issue 20, Page(s) 11070–11075

Abstract: The increasing interest in extracellular vesicles (EVs) research is fueled by reports indicating their unique role in intercellular communication and potential connection to the development of common human diseases. The unique role assumes unique protein ...

Abstract	The increasing interest in extracellular vesicles (EVs) research is fueled by reports indicating their unique role in intercellular communication and potential connection to the development of common human diseases. The unique role assumes unique protein and nucleic acid cargo. Unfortunately, accurate analysis of EVs cargo faces a challenge of EVs isolation. Generally used isolation techniques do not separate different subtypes of EVs and even more, poorly separate EVs from non-EVs contaminants. Further development of EVs isolation protocols urgently needs a quantitative method of EVs purity assessment. We report here that multiple reaction monitoring assay using internal standards carrying peptides for quantification of EVs and non-EVs proteins is a suitable approach to assess purity of EVs preparations. As a first step in potential standardization of EVs isolation, we have evaluated polymer-based precipitation techniques and compared them to traditional ultracentrifugation protocol.
MeSH term(s)	Chromatography, High Pressure Liquid ; Dynamic Light Scattering ; Extracellular Vesicles/chemistry ; Extracellular Vesicles/metabolism ; Humans ; Nitrogen Isotopes/chemistry ; Peptides/chemistry ; Proteomics/methods ; Tandem Mass Spectrometry ; Ultracentrifugation
Chemical Substances	Nitrogen Isotopes ; Nitrogen-15 ; Peptides
Language	English
Publishing date	2017-10-05
Publishing country	United States
Document type	Journal Article
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.7b03119
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