scholarly journals Targeted proteomics analysis of plasma proteins using recombinant protein standards for addition only workflows

BioTechniques ◽  
2021 ◽  
Author(s):  
David Kotol ◽  
Andreas Hober ◽  
Linnéa Strandberg ◽  
Anne-Sophie Svensson ◽  
Mathias Uhlén ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Plasma Proteins ◽  
Mass Spectrometry Analysis ◽  
Targeted Proteomics ◽  
Plasma Samples ◽  
Blood Proteins ◽  
Proteomics Analysis ◽  
Protein Targets ◽  
Spectrometry Analysis ◽  
Absolute Concentrations

Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation <10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed.

scholarly journals Evidence for Direct Protein-Protein Interaction between Members of the Enterobacterial Hha/YmoA and H-NS Families of Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 629-635 ◽  
Author(s):  
J. M. Nieto ◽  
C. Madrid ◽  
E. Miquelay ◽  
J. L. Parra ◽  
S. Rodríguez ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein A ◽  
Nitrilotriacetic Acid ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Protein Protein Interaction ◽  
New Family ◽  
Missense Mutant ◽  
First Time

ABSTRACT Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin α-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni2+-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha′ proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.

Presence of orally administered rice bran oil γ-oryzanol in its intact form in mouse plasma

2016 ◽  
Vol 7 (12) ◽  
pp. 4816-4822 ◽  
Author(s):  
Eri Kobayashi ◽  
Junya Ito ◽  
Shunji Kato ◽  
Kazue Sawada ◽  
Midori Matsuki ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Oral Administration ◽  
Rice Bran ◽  
Rice Bran Oil ◽  
Mass Spectrometry Analysis ◽  
Plasma Samples ◽  
Spectrometry Analysis ◽  
Mouse Plasma ◽  
Chromatography Mass Spectrometry ◽  
Rice Oil

We prepared OZ concentrate from purified rice bran oil (Rice Oil OZ) and carried out chromatography-mass spectrometry analysis of plasma samples from mice after oral administration of the Rice Oil OZ.

Elevated Plasma Levels of Crosslinked Fibrinogen Gamma-chain Dimer Indicate Cancer-related Fibrin Deposition and Fibrinolysis

2001 ◽  
Vol 85 (03) ◽  
pp. 494-501 ◽  
Author(s):  
Werner Steinkellner ◽  
Klaus Holzmann ◽  
Andrea Gsur ◽  
Rudolf Grimm ◽  
Christian Ensinger ◽  
...  
Keyword(s):  
Solid Tumor ◽  
Acute Phase Proteins ◽  
Mass Spectrometry Analysis ◽  
Plasma Samples ◽  
Fibrin Deposition ◽  
Gamma Chain ◽  
Two Dimensional Gel Electrophoresis ◽  
Spectrometry Analysis ◽  
Chain Dimer ◽  
Potential Marker

SummaryCancer-related fibrin deposition and fibrinolysis were investigated by two-dimensional gel electrophoresis of human solid tumor and effusion specimen in addition to plasma samples. Fibrinogen gamma-chain dimer indicating fibrin deposition and plasmin-generated fibrinogen beta-chain fragments were identified in various solid tumor types by amino acid sequencing, mass spectrometry analysis and Western blotting. In tumor-associated effusions, these techniques allowed to observe plasmin-generated fragments of fibrinogen alpha, beta and gamma-chains in addition to elevated levels of acute-phase proteins. Similar observations were made in case of inflammation-associated effusions. No fibrin degradation product was observed in plasma samples, however, high amounts of fibrinogen gamma-chain dimer crosslinked by transglutaminase were detected in plasma from tumor patients, but not in plasma from controls and patients suffering acute infections and/or inflammations. This finding demonstrated that high transglutaminase activity may be associated with cancer. The presented data indicate that the amount of crosslinked fibrinogen gamma-chain dimer in plasma may correlate with tumor-associated fibrin deposition. The tumor-biological relevance of this potential marker protein is discussed.

scholarly journals Conversion of spent CHO cell culture media waste to new fermentation feed efficiently supports production of recombinant protein by Escherichia coli

2021 ◽  
Author(s):  
Ciara Lynch ◽  
David J O'Connell
Keyword(s):  
Cell Culture ◽  
Recombinant Protein ◽  
Culture Media ◽  
Mass Spectrometry Analysis ◽  
Microbial Fermentation ◽  
Differential Protein Expression ◽  
Cell Culture Media ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Rich Media

Deriving new value from waste streams is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be effectively recycled and used as a feed in microbial fermentation to produce new recombinant protein products. Our results show that 1) CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich media (LB) used as a baseline reference. 2) The amount of recombinant protein produced following induction in an expression screen was approximately two-fold higher in the CDSM fed cultures than that of baseline. 3) Mass spectrometry analysis of the proteome of E. coli cultures fed in CDSM revealed a greater or lesser differential protein expression pattern depending on supplementation conditions. Further, in a 16 hr fermentation the optimised CDSM-fed culture delivered a protein yield of more than double that achieved by the baseline media. We conclude that spent cell culture media, which represents millions of litres of waste generated by the bioprocessing industry annually, has the potential to be a valuable feed resource for the production of recombinant proteins in secondary microbial fermentation.

scholarly journals The associations between p,p’-DDE levels and plasma levels of lipoproteins and their subclasses in an elderly population determined by analysis of lipoprotein content

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Juliann Jugan ◽  
P. Monica Lind ◽  
Samira Salihovic ◽  
Jordan Stubleski ◽  
Anna Kärrman ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Solid Phase ◽  
Density Lipoprotein ◽  
Apolipoprotein A1 ◽  
Mass Spectrometry Analysis ◽  
Lipid Lowering ◽  
Resonance Spectroscopy ◽  
Plasma Samples ◽  
Spectrometry Analysis ◽  
Lipoprotein Content

Abstract Background Lipoproteins at aberrant levels are known to play a role in cardiovascular disease. The metabolite of the insecticide dichlorodiphenyltrichloroethane (DDT), p,p’-dichlorodiphenyldichloroethylene (p,p’-DDE), physically associates with lipids and accumulates in adipose tissue. Little is known about which lipoproteins associate with p,p’-DDE. An association between p,p’-DDE exposure and altered levels of circulating lipids was assessed in a large human cohort using a detailed analysis of lipoprotein content. Methods Plasma samples were collected from the subset of 75-year old Swedes in the Prospective Investigation of the Vasculature of Uppsala Seniors (PIVUS) cohort who were not prescribed lipid lowering medication (n = 571). p,p’-DDE concentrations in plasma were measured using high-throughput solid phase extraction and gas chromatography-high resolution mass spectrometry. Analysis of plasma lipoprotein content was performed with nuclear magnetic resonance spectroscopy. Results Detectable levels of p,p’-DDE were found in the plasma samples of all subjects. Elevated p,p’-DDE levels were associated with increased concentrations of lipoproteins of all diameters, with the exception of high density lipoprotein (HDL) of diameters between 14.3 nm–10.9 nm. Of the lipoprotein constituents, triglycerides were most uniformly associated with elevated p,p’-DDE across lipoproteins. p,p’-DDE was furthermore associated with apolipoprotein B, but not apolipoprotein A1. Conclusions The positive associations observed between each lipoprotein class and elevated p,p’-DDE support previous data suggesting that p,p’-DDE interacts with lipoproteins within plasma. It is speculated that both physio-chemical and biological mechanisms may explain why p,p’-DDE does not uniformly associate with lipids across lipoproteins.

scholarly journals Autoimmune septin-5 cerebellar ataxia

2018 ◽  
Vol 5 (5) ◽  
pp. e474 ◽  
Author(s):  
Josephe A. Honorat ◽  
A. Sebastian Lopez-Chiriboga ◽  
Thomas J. Kryzer ◽  
James P. Fryer ◽  
Michelle Devine ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Recombinant Protein ◽  
Western Blot ◽  
Cerebellar Ataxia ◽  
Molecular Layer ◽  
Immunofluorescence Assay ◽  
Mass Spectrometry Analysis ◽  
Antigen Specificity ◽  
Spectrometry Analysis ◽  
Protein Assays

ObjectiveTo report a form of autoimmune cerebellar ataxia in which antibodies target septin-5, a guanosine triphosphate (GTP)-binding neural protein involved in neurotransmitter exocytosis.MethodsArchived sera and CSF specimens with unclassified synaptic antibodies were re-evaluated by tissue-based indirect immunofluorescence assay. Autoantigens were identified by Western blot and mass spectrometry. Recombinant protein assays (Western blot, cell based, and protein screening array) confirmed antigen specificity.ResultsSerum and CSF from 6 patients produced identical synaptic immunoglobulin G (IgG) staining patterns of synaptic regions (neuropil) of the mouse cerebrum and cerebellum. The molecular layer of the cerebellum and the thalamus demonstrated stronger immunoreactivity than the midbrain, hippocampus, cortex, and basal ganglia. The antigen revealed by mass spectrometry analysis of immunoprecipitated cerebellar proteins and confirmed by recombinant protein assays was septin-5. All 4 patients with records available had subacute onset of cerebellar ataxia with prominent eye movement symptoms (oscillopsia or vertigo). None had cancer detected. Improvements occurred after immunotherapies (2) or spontaneously (1). One patient died.ConclusionSeptin-5 IgG represents a biomarker for a potentially fatal but treatable autoimmune ataxia.

scholarly journals Detecting Drug-Target Binding in Cells using Fluorescence Activated Cell Sorting Coupled with Mass Spectrometry Analysis

2017 ◽  
Author(s):  
Kris Wilson ◽  
Scott P Webster ◽  
John P Iredale ◽  
Xiaozhong Zheng ◽  
Natalie Z Homer ◽  
...  
Keyword(s):  
Drug Target ◽  
Drug Targets ◽  
Mass Spectrometry Analysis ◽  
Protein Targets ◽  
Target Proteins ◽  
Spectrometry Analysis ◽  
Cellular Permeability ◽  
Target Binding ◽  
Hit Validation

AbstractThe assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity - the Toxicity-Affinity-Permeability-Selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

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