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Proteomics Oct 2018In veterinary medicine, assay performance is often affected by the lack of species-specific diagnostic tools. Reliable biomarkers might be identified by investigating...
In veterinary medicine, assay performance is often affected by the lack of species-specific diagnostic tools. Reliable biomarkers might be identified by investigating biological fluids of the species of interest, but protein sequence databases are often incomplete and human-specific devices for reducing sample complexity might fail when applied to animal plasma. Here, seven commercial methods based on different capturing agents (anti-human antibodies, affinity ligands, mixture of antibodies and ligands, and combinatorial peptide ligand libraries) are applied to cat plasma and evaluated in terms of yield, identified proteins/ peptides, and relative abundance by high-resolution shotgun proteomics and label-free quantitation. As a result, anti-human antibody-based methods are unsatisfactory. Most fail in reducing albumin and immunoglobulins, and some lead to a substantial removal of other highly abundant proteins, probably because of nonspecific interactions. A protein A/dye ligand-based method is efficient in reducing immunoglobulins, fibrinogen, and apolipoprotein A1 and A2, but not albumin, and protein identifications do not increase. Only peptide ligand libraries flatten the dynamic range, and increased protein identification (59.0%). Albumin and immunoglobulins are successfully depleted (60.7% and 35.9%, respectively). Although further studies will be required for reinforcing our observations, this work can provide a useful guide for cat plasma pretreatment in biomarker discovery studies.
Topics: Animals; Biomarkers; Blood Proteins; Cats; Chromatography, Affinity; Chromatography, Liquid; Proteome; Tandem Mass Spectrometry
PubMed: 30216667
DOI: 10.1002/pmic.201800191 -
Scandinavian Journal of Immunology Jul 2021Neutropenia as an isolated clinical finding may include aetiologies ranging from severe disease to a transient condition, and differential diagnosis may be challenging....
Neutropenia as an isolated clinical finding may include aetiologies ranging from severe disease to a transient condition, and differential diagnosis may be challenging. Previous data and clinical experience suggest that low levels of the neutrophil-derived protein human 18 kDa cathelicidin antimicrobial protein (hCAP-18) in the blood are predictive of more severe forms of neutropenia. The objective of this study was to present the results from a newly developed ELISA method that has been used in clinical routine in Sweden since 2018 for quantification of hCAP-18 in blood plasma. Using this method, we report that patients with severe disease analysed during the study period presented with low or undetectable levels of blood plasma hCAP-18, validating its use as screening tool for severe neutropenia. Furthermore, neutropenic patients as a group displayed lower levels of hCAP-18 as compared to blood donors. Within the group of neutropenic patients, those with neutrophil antibodies displayed significantly higher hCAP-18 levels compared to patients with idiopathic neutropenia. By including an analysis of hCAP-18 in the primary investigation of neutropenia, an increased accuracy in differential diagnosis is achieved, thus contributing to reduced costs of neutropenia management.
Topics: Aged; Aged, 80 and over; Antibodies; Antimicrobial Cationic Peptides; Blood Proteins; Child; Child, Preschool; Enzyme-Linked Immunosorbent Assay; Female; Humans; Infant; Male; Middle Aged; Neutropenia; Neutrophils; Plasma; Cathelicidins
PubMed: 33662157
DOI: 10.1111/sji.13037 -
PloS One 2021Baseline plasma electrophoresis profiles (EPH) are important components of overall health and may aid in the conservation and captive management of species. The aim of...
Baseline plasma electrophoresis profiles (EPH) are important components of overall health and may aid in the conservation and captive management of species. The aim of this study was to establish plasma protein fractions for free-ranging Blanding's turtles (Emydoidea blandingii) and evaluate differences due to age class (adult vs. sub-adult vs. juvenile), sex (male, female, or unknown), year (2018 vs. 2019), month (May vs. June vs. July), health status, and geographical location (managed vs. unmanaged sites). Blood samples were obtained from 156 Blanding's turtles in the summer of 2018 and 129 in 2019 at two adjacent sites in Illinois. Results of the multivariate analysis demonstrated that age class, sex, year, month, health status, and geographical location all contributed to the variation observed in free-ranging populations. Adult females had the highest concentration of many protein fractions, likely associated with reproductive activity. Juveniles had lower protein concentrations. Temperature and rainfall differences between years impacted concentrations between 2018 and 2019, while May and June of both years saw higher levels in some protein fractions likely due to peak breeding and nesting season. Individuals with evidence of trauma or disease also showed increased plasma protein fractions when compared to those that were considered healthy. The two sites showed a wide/large variation over the two years. All of these factors emphasize the importance of considering multiple demographic or environmental factors when interpreting the EPH fractions. Establishing ranges for these analytes will allow investigation into disease prevalence and other environmental factors impacting this endangered species.
Topics: Age Factors; Animals; Blood Protein Electrophoresis; Blood Proteins; Female; Health Status; Illinois; Male; Seasons; Sex Factors; Turtles
PubMed: 34648539
DOI: 10.1371/journal.pone.0258397 -
Methods in Molecular Biology (Clifton,... 2009Protein glycosylation and phosphorylation are very common posttranslational modifications. The alteration of these modifications in cancer cells is closely related to...
Protein glycosylation and phosphorylation are very common posttranslational modifications. The alteration of these modifications in cancer cells is closely related to the onset and progression of cancer and other disease states. In this protocol, strategies for monitoring the changes in protein glycosylation and phosphorylation in serum or tissue cells on a global scale and specifically characterizing these alterations are included. The technique is based on lectin affinity enrichment for glycoproteins, all liquid-phase two-dimensional fractionation, protein microarray, and mass spectrometry technology. Proteins are separated based on pI in the first dimension using chromatofocusing (CF) or liquid isoelectric focusing (IEF) followed by the second-dimension separation using nonporous silica RP-HPLC. Five lectins with different binding specificities to glycan structures are used for screening glycosylation patterns in human serum through a biotin streptavidin system. Fluorescent phosphodyes and phosphospecific antibodies are employed to detect specific phosphorylated proteins in cell lines or human tissues. The purified proteins of interest are identified by peptide sequencing. Their modifications including glycosylation and phosphorylation could be further characterized by mass-spectrometry-based approaches. These strategies can be used in biological samples for large-scale glycoproteome/phosphoproteome screening as well as for individual protein modification analysis.
Topics: Amino Acid Sequence; Analytic Sample Preparation Methods; Animals; Biomarkers, Tumor; Blood Proteins; Cattle; Cell Line, Tumor; Chemical Fractionation; Chromatography, Affinity; Chromatography, High Pressure Liquid; Glycosylation; Humans; Hydrophobic and Hydrophilic Interactions; Immunoglobulins; Lectins; Mass Spectrometry; Molecular Sequence Data; Phosphoproteins; Phosphorylation; Protein Array Analysis; Proteomics; Sequence Analysis, Protein
PubMed: 19241043
DOI: 10.1007/978-1-59745-493-3_20 -
Journal of Clinical Pathology.... 1975
Review
Topics: Animals; Blood Protein Disorders; Blood Proteins; Carbon Radioisotopes; Carbonates; Colitis, Ulcerative; Crohn Disease; Digestive System; Gastrointestinal Diseases; Humans; Kidney Diseases; Metabolic Clearance Rate; Reference Values; Serum Albumin, Radio-Iodinated; Skin Diseases
PubMed: 802869
DOI: 10.1136/jcp.s1-6.1.13 -
British Medical Journal Jul 1956
Topics: Blood Proteins; C-Reactive Protein
PubMed: 13329406
DOI: No ID Found -
Journal of Separation Science Mar 2009The binding of drugs with proteins in blood, serum, or plasma is an important process in determining the activity, distribution, rate of excretion, and toxicity of drugs... (Review)
Review
The binding of drugs with proteins in blood, serum, or plasma is an important process in determining the activity, distribution, rate of excretion, and toxicity of drugs in the body. High-performance affinity chromatography (HPAC) has received a great deal of interest as a means for studying these interactions. This review examines the various techniques that have been used in HPAC to examine drug-protein binding and discusses the types of information that can be obtained through this approach. A comparison of these techniques with traditional methods for binding studies (e.g., equilibrium dialysis and ultrafiltration) will also be presented. The use of HPAC with specific serum proteins and binding agents will then be discussed, including HSA and alpha(1)-acid glycoprotein (AGP). Several examples from the literature are provided to illustrate the applications of such research. Recent developments in this field are also described, such as the use of improved immobilization techniques, new data analysis methods, techniques for working directly with complex biological samples, and work with immobilized lipoproteins. The relative advantages and limitations of the methods that are described will be considered and the possible use of these techniques in the high-throughput screening or characterization of drug-protein binding will be discussed.
Topics: Binding Sites; Blood Proteins; Chromatography, High Pressure Liquid; Pharmaceutical Preparations
PubMed: 19278006
DOI: 10.1002/jssc.200800640 -
Parasites & Vectors Sep 2019Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diverse diseases of public health significance. Blood protein digestion by midgut proteases...
BACKGROUND
Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diverse diseases of public health significance. Blood protein digestion by midgut proteases provides anautogenous mosquitoes with the nutrients essential for oocyte maturation and egg production. Midgut-specific miR-1174 affects the functions of the midgut through its target gene serine hydroxymethyltransferase (SHMT). However, less is known about SHMT-regulated processes in blood digestion by mosquitoes.
METHODS
RNAi of SHMT was realized by injection of the double-stranded RNA at 16 h post-eclosion. The expression of SHMT at mRNA level and protein level was assayed by real-time PCR and Western blotting, respectively. Statistical analyses were performed with GraphPad7 using Student's t-test.
RESULTS
Here, we confirmed that digestion of blood was inhibited in SHMT RNAi-silenced female A. aegypti mosquitoes. Evidence is also presented that all SHMT-depleted female mosquitoes lost their flight ability and died within 48 h of a blood meal. Furthermore, most examined digestive enzymes responded differently in their transcriptional expression to RNAi depletion of SHMT, with some downregulated, some upregulated and some remaining stable. Phylogenetic analysis showed that transcriptional expression responses to SHMT silence were largely unrelated to the sequence similarity between these enzymes.
CONCLUSIONS
Overall, this research shows that SHMT was expressed at a low level in the midgut of Aedes aegypti mosquitoes, but blood-meal digestion was inhibited when SHMT was silenced. Transcriptional expressions of different digestive enzymes were affected in response to SHMT depletion, suggesting that SHMT is required for the blood-meal digestion in the midgut and targeting SHMT could provide an effective strategy for vector mosquito population control.
Topics: Aedes; Animals; Blood Proteins; Digestion; Gastrointestinal Tract; Gene Expression Profiling; Gene Silencing; Glycine Hydroxymethyltransferase
PubMed: 31551071
DOI: 10.1186/s13071-019-3714-2 -
Journal of Biomedical Materials... Apr 2011The aim of this study was to create polymeric materials with known properties to study the preconditions for complement activation. Initially, 22 polymers were screened...
The aim of this study was to create polymeric materials with known properties to study the preconditions for complement activation. Initially, 22 polymers were screened for complement activating capacity. Based on these results, six polymers (P1-P6) were characterized regarding physico-chemical parameters, for example, composition, surface area, pore size, and protein adsorption from human EDTA-plasma. P2, P4, and reference particles of polystyrene and polyvinyl chloride, were hydrophobic, bound low levels of protein and were poor complement activators. Their accessible surface was limited to protein adsorption in that they had pore diameters smaller than most plasma proteins. P1 and P3 were negatively charged and adsorbed IgG and C1q. A 10-fold difference in complement activation was attributed to the fact that P3 but not P1 bound high amounts of C1-inhibitor. The hydrophobic P5 and P6 were low complement activators. They selectively bound apolipoproteins AI and AIV (and vitronectin), which probably limited the binding of complement activators to the surface. We demonstrate the usefulness of the modus operandi to use a high-throughput procedure to synthesize a great number of novel substances, assay their physico-chemical properties with the aim to study the relationship between the initial protein coat on a surface and subsequent biological events. Data obtained from the six polymers characterized here, suggest that a complement-resistant surface should be hydrophobic, uncharged, and have a small available surface, accomplished by nanostructured topography. Additional attenuation of complement can be achieved by selective enrichment of inert proteins and inhibitors.
Topics: Adsorption; Blood Proteins; Blotting, Western; Complement Activation; Complement System Proteins; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Endotoxins; Flow Cytometry; Hirudins; Humans; Hydrogen-Ion Concentration; Materials Testing; Microscopy, Electron, Scanning; Polymers; Porosity
PubMed: 21319295
DOI: 10.1002/jbm.a.33030 -
Journal of Clinical Pathology.... 1973
Topics: Adolescent; Adult; Age Factors; Aged; Blood Protein Electrophoresis; Blood Proteins; Cholesterol; Female; Heparin; Humans; Lipoproteins; Lipoproteins, HDL; Lipoproteins, LDL; Lipoproteins, VLDL; Male; Methods; Middle Aged; Sex Factors; Triglycerides; Ultracentrifugation
PubMed: 4354846
DOI: 10.1136/jcp.s1-5.1.32