scholarly journals Mass-Tag Labeling Using Acyl-PEG Exchange for the Determination of Endogenous Protein S-Fatty Acylation

2017 ◽  
pp. 14.17.1-14.17.11 ◽  
Author(s):  
Avital Percher ◽  
Emmanuelle Thinon ◽  
Howard Hang
Keyword(s):  
Protein S ◽  
Fatty Acylation ◽  
Endogenous Protein ◽  
Mass Tag

scholarly journals Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation

2016 ◽  
Vol 113 (16) ◽  
pp. 4302-4307 ◽  
Author(s):  
Avital Percher ◽  
Srinivasan Ramakrishnan ◽  
Emmanuelle Thinon ◽  
Xiaoqiu Yuan ◽  
Jacob S. Yount ◽  
...  
Keyword(s):  
Protein Function ◽  
Transmembrane Protein ◽  
Protein S ◽  
Cysteine Residues ◽  
Site Specific ◽  
Immune Effector ◽  
Fatty Acylation ◽  
Acylated Proteins ◽  
Mass Tag ◽  
In Cells

Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.

Determination of Protein C Antigen Levels in Hospitalized Patients with Blood Coagulative Defects

2021 ◽  
Vol 2 (1) ◽  
pp. 1-04
Author(s):  
George Zhu
Keyword(s):  
Plasma Protein ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Promyelocytic Leukemia ◽  
Hospitalized Patients ◽  
Cancer Cell Proliferation ◽  
Healthy Individuals ◽  
Acute Leukemias

Protein C, a vitamin K-dependent anticoagulant serine protease, is involved in blood coagulation. Activated protein C inactivates Va and VIIIa in purified protein systems and stimulates fibrinolysis by indirectly increasing the level of circulating plasminogen activator. In this process, protein S serve as an important factor for activated protein C. In recent years, excess protein S drives cancer cell proliferation and cell survival through oncogenic receptor Axl (Anexelekto). We determined changes of plasma protein C antigen by using rocket immunoassay both in 50 healthy individuals and 103 distinct hospitalized patients. In healthy individuals protein C antigen(PC:Ag) ranges o.6439- 1.4752 µ/ml. The results showed that plasma protein C antigen was considerably high in 22 diabetes mellitus. In contrast, the PC:Ag was significantly decreased in 19 liver cirrhosis(p< 0.001) and in closely line with serum albumin levels(p< 0.05). In 31 acute leukemias, on the average, there was slightly lower values in PC:Ag, and accompanied with the distribution of significant decrease of PC:Ag values in 5 FAB M5 subtype and in 9 hyperleukocytic leukemias. However, the 3 acute promyelocytic leukemia (APL) with overt laboratory criteria of disseminated intravascular coagulation (DIC) had protein C concentration no lower than the remaining 2 patients with infectious DIC, which suggested the coagulopathy in APL might be due to mechanisms different from other forms of DIC.

scholarly journals Comparison of the isotope dilution method for determination of the ileal endogenous amino acid losses with labelled diet and labelled pigs

2000 ◽  
Vol 83 (2) ◽  
pp. 123-130 ◽  
Author(s):  
Vincent Hess ◽  
Philippe Ganier ◽  
Jean-Noel Thibault ◽  
Bernard Sève
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Latin Square ◽  
Isotope Dilution Method ◽  
Dilution Method ◽  
First Case ◽  
Endogenous Protein ◽  
The Individual ◽  
Protein Free Diet

The aims of the present study were first to compare the amino acid dilution method performed using labelled animals with that using labelled diets, and second to determine real digestibilities and total ileal endogenous losses of N and amino acids. Two diets containing pea cultivars (Solara and Amino) and a protein-free diet were compared in a 3 × 3 Latin-square experiment. The three pigs were each prepared with an ileo-rectal anastomosis and were continuously infused with [1-13C]leucine. For each cultivar,15N-labelled and unlabelled diets were formulated. The real digestibility and endogenous losses of leucine were higher when obtained by labelling the pig than by labelling the foodstuff. This was due either to the inadequate estimation of the endogenous protein enrichment in the first case or to the importance of dietary N recycling in the second case. However, in both cases the ileal endogenous losses of N and amino acids were higher than the basal losses determined with the protein-free diet. There were significant differences between the two pea cultivars in terms of phenylalanine and leucine when measured with labelled diets. It is suggested that, although ileal endogenous losses may be underestimated, using labelled feedstuffs is of great interest due to the direct estimation of the individual amounts of amino acids.

scholarly journals Exploring peptide capture by anti-protein antibodies for LC-MS-based biomarker determination

2018 ◽  
Author(s):  
Maren Levernæs ◽  
Bassem Farhat ◽  
Inger Oulie ◽  
Sazan S. Abdullah ◽  
Elisabeth Paus ◽  
...  
Keyword(s):  
Protein Extraction ◽  
High Sensitivity ◽  
Intact Protein ◽  
Protein Biomarkers ◽  
Promising Technique ◽  
Intact Proteins ◽  
Protein Levels ◽  
Endogenous Protein ◽  
Sensitivity And Selectivity

<p>Immunocapture LC-MS/MS is a promising technique to ensure high sensitivity and selectivity of low-abundant protein biomarkers. For this purpose, the use of monoclonal antibodies (mAb) is especially attractive as they are renewable reagents that can be standardized. In this article we investigated the possibility of using mAbs developed against intact proteins (anti-protein antibodies) to capture proteotypic epitope peptides. Three mAbs were tested, and all selectively extracted proteotypic epitope peptides from a complex sample. Compared to intact protein extraction, this concept which we call peptide capture by anti-protein antibodies provided cleaner extracts, which further improved the sensitivity. Analysis of three patient samples demonstrated that p can be used for the determination of different endogenous protein levels. </p><p></p>

Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays (ELISA) for the Measurement of Vitamin K-Dependent Protein S: The Effect of Antibody Immunoreactivity on Plasma Protein S Antigen Determinations

1992 ◽  
Vol 67 (06) ◽  
pp. 631-638 ◽  
Author(s):  
Silvana Vigano D’Angelo ◽  
Sandra Tombesi ◽  
Santica Marcovina ◽  
Alberto Albertini ◽  
Patrizia Della Valle ◽  
...  
Keyword(s):  
Protein S ◽  
Plasma Samples ◽  
Potential Influence ◽  
Polyclonal Igg ◽  
Dependent Protein ◽  
Capture Antibody ◽  
Antigen Determination ◽  
Sample Dilution ◽  
Selection Of

SummaryTwo monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution cuiyes obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution -plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA. In addition, plasma PS and C4b-BP levels were significantly correlated only when it was measured by EIA or the ELISAs employing S10, 3B10.25 or polyclonal IgG. These results highlight the potential influence of high C4b-BP on plasma PS antigen determination. Accurate measurement of PS by ELISA requires selection of antibodies with identical affinity for all plasma PS forms.

scholarly journals Determination of Protein S-Acylation State by Enhanced Acyl-Switch Methods

2019 ◽  
pp. 3-11
Author(s):  
Charlotte H. Hurst ◽  
Dionne Turnbull ◽  
Piers A. Hemsley
Keyword(s):  
Protein S

An Impedimetric Approach for COVID-19 Detection

The Analyst ◽  
2021 ◽  
Author(s):  
Yudum Tepeli ◽  
Burak Ekrem Citil ◽  
U. Anik
Keyword(s):  
Protein S ◽  
Spike Protein ◽  
S Protein ◽  
Infection Mechanism ◽  
Biosensor System ◽  
Electrochemical Approach

In this study, an electrochemical approach for the determination of coronavirus disease (COVID-19) was developed. The biosensor system relied on the spike protein (S-protein) based infection mechanism of the virus...

scholarly journals Postnatal changes in dolichol-pathway enzyme activities in cerebral cortex neurons

1982 ◽  
Vol 202 (1) ◽  
pp. 87-95 ◽  
Author(s):  
V Idoyaga-Vargas ◽  
H Carminatti
Keyword(s):  
Cerebral Cortex ◽  
Postnatal Development ◽  
Enzyme Activities ◽  
Synapse Formation ◽  
Adult Stage ◽  
Protein S ◽  
Rat Cerebral Cortex ◽  
Dolichyl Phosphate ◽  
Endogenous Protein ◽  
Dolichol Pathway

Neuronal perikarya were isolated from rat cerebral cortex at different stages of postnatal development. Membranes sedimenting at 100000 g were obtained from these neurons to study several glycosyltransferases of the dolichol pathway. Enzyme activities from stages before and during synapse formation were compared (days 5 and 15 respectively). Dolichyl diphosphate (Dol-P-P) N-acetylglucosamine, dolichyl phosphate mannose and dolichyl phosphate glucose synthases and the enzymes catalysing Dol-P-P-GlcNAc2Man9Glc3 formation were higher at day 15 of postnatal development. The glycosyl transfer of the latter compound to endogenous protein(s) as well as to a dinitrophenyl-heptapeptide was also measured. The activity was higher at day 15. Furthermore, the activity of dolichyl phosphate mannose synthase was also measured during the time when the number of synapses ceased to increase (day 36) and in the adult stage. The activity of dolichyl phosphate mannose synthase was higher at day 36 than at day 15, and declined in the adult stage. From these results it may be concluded that there is an increase in the glycosylation of asparagine-type glycoproteins during synapse formation in the neurons of the cerebral cortex.

First experience with a new assay for activity determination of protein S

1994 ◽  
Vol 8 ◽  
pp. 124 ◽  
Author(s):  
C. Wagner ◽  
A. Girolami ◽  
P. Simeoni ◽  
F. Dati
Keyword(s):  
Protein S ◽  
Activity Determination
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