Quantitative Determination Of Proteins Research Articles

Biomarker discovery in breast cancer serum using 2‐D differential gel electrophoresis/ MALDI‐TOF/TOF and data validation by routine clinical assays

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.

Quantitative determination of proteins at nanogram levels by the resonance light-scattering technique with composite nanoparticles of CdS/PAA

This paper describes the development of composite nanoparticles. A novel composite nanoparticle has been prepared by an in situ polymerization method. The nano-CdS has been prepared, then the polymerization of acrylic acid (AA) was carried out by initiator potassium persulfate (KPS) under ultrasonic irradiation. The surface of the composite nanoparticles was covered with abundant carboxylic groups (–COOH). The nanoparticles are water-soluble, stable and biocompatible. Reaction of the composite nanoparticles with proteins results in an enhanced resonance light scattering (RLS) at 380 nm. Based on this, a new resonance light-scattering (RLS) method was developed for the determination of proteins including BSA, HSA and human γ-IgG. Under the optimum conditions, the enhanced RLS intensity is linearly proportional to the concentration of proteins. The liner range is 0.1–15 μg mL −1 for HSA, 0.2–20 μg mL −1 for BSA and 0.1–50.0 μg mL −1 for human γ-IgG, respectively. The method has been applied to the determination of the total protein in human serum samples collected from the hospital and the results are in good agreement with those reported by the hospital. This method proved to be very sensitive, rapid, simple and tolerant of most interfering substances.

Solid-Supported Synthesis of Polymerizable Lanthanide-Ion Chelating Lipids for Protein Detection

Usually, lipids are synthesized employing solution-phase organic synthesis techniques. Though successful, the purifications can be difficult to accomplish due to the amphiphilic nature of the molecules. Herein, we demonstrate the advantages of a solid-phase approach for preparing a variety of metal-chelating lipids. A number of saturated and polymerizable metal-chelating lipids were prepared using this methodology. This approach requires one chromatographic purification after cleaving the lipids from the solid support. We also demonstrate that the resulting polymerized liposomes (containing Eu3+) possess the appropriate luminescence properties for the qualitative and quantitative determination of proteins.

Evaluation of a rapid bedside test for the quantitative determination of C-reactive protein

Regardless of its origin, any condition associated with inflammation is accompanied by an increase in serum C-reactive protein levels. This study compared the results of a rapid test for the bedside assay of C-reactive protein (QuikRead CRP, Orion Corporation, Orion Diagnostica, Espoo, Finland) with those of a standard laboratory assay in samples taken from 231 children aged less than 14 years (126 males; median age 4.7 years) attending the Emergency Department of Milan University's Institute of Pediatrics because of acute respiratory infection. The two methods showed similar median C-reactive protein levels (standard laboratory assay: 34.7 mg/L, range 4-199 mg/L; QuikRead CRP: 33.3 mg/L, range <8-196 mg/L; p = 0.779) and a similar distribution of children with C-reactive protein levels of <20 mg/L, 20-70 mg/L and >70 mg/L. This study shows for the first time that the rapid QuikRead CRP test can be performed at the bedside or in an outpatient clinic and, in less than 5 min, gives the same quantitative results as those obtained using a more complex routine laboratory method.

Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples

Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.

Identification and determination of milk and soybean protein preparations using enzymatic hydrolysis followed by chromatography and chemometrical data analysis

This paper presents the problem of the qualitative and quantitative determination of bovine proteins in soybean protein isolate mixtures.Soybean protein isolate, bovine milk protein preparations and their mixtures with different content of milk protein preparations as described in our previous paper [J. Dziuba et al., 2004, Milchwissenschaft] were examined. The previously presented strategy of detecting and determining milk proteins in soybean protein preparation involved the statistical analysis of the chromatographic peaks with the greatest area variability which were present in the chromatograms of soybean and milk protein preparations and their mixtures. This enabled the determination of the milk protein preparation type added to soybean preparation and its content above 5%. In order to lower the detection limit for milk protein content, the present experiment was extended over five different levels of milk protein addition i.e. from 1 to 5%. However, the analysis was carried out in relation to the relative trypsin hydrolyzates obtained after specific hydrolysis.The trypsin hydrolyzates of the examined preparations of soybean and milk proteins and their mixtures had different compositions of freed peptides. At least two different peptide peaks-“markers” could be identified in each hydrolyzate. The chemometric analysis of chromatographically separated preparations of soybean and milk proteins and their trypsin hydrolyzates enabled the determination of the type of milk protein preparations added to a soybean protein preparation. Based on the results obtained, a special calculation sheet was created for the purpose of the quantitative detection of the milk protein preparation content in soybean protein isolate.

Automation highlights from literature

Automation highlights from literature

Quantitative determination of proteins at nanogram levels by the resonance light-scattering technique with macromolecules nanoparticles of PS–AA

The polystyrene–acrylic acid (PS–AA) nanoparticles have been prepared by ultrasonic polymerization, characterized by FT–IR and TEM. It is the first report on the determination of proteins with macromolecules nanoparticles of PS–AA by resonance light-scattering (RLS). At pH 6.9, the RLS of macromolecules nanoparticles of PS–AA can be enhanced by proteins. Based on this, a novel quantitative assay of proteins at the nanogram levels has been proposed. At pH 6.9, the RLS signals of PS–AA were greatly enhanced by proteins in the region of 250–700 nm characterized by the peak at 342 nm. Under optimal conditions, the linear ranges of the calibration curves were 0.02–11.0 μg ml −1, 0.04–10.0 μg ml −1 and 0.03–10.0 μg ml −1 for γ-globulin (γ-IgG), bovine serum albumin (BSA) and human serum albumin (HSA), respectively. The detection limits were 16.0 ng ml −1, 19.0 ng ml −1, and 15.0 ng ml −1 for γ-IgG, BSA and HSA, respectively. The method has been applied to the analysis of total proteins in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital, which indicates that the method presented here is not only sensitive, simple, but also reliable and suitable for practical application.

Total protein determinations by particle beam/hollow cathode optical emission spectroscopy.

A novel method for quantitative total protein determinations is presented. Total protein content is determined by particle beam/hollow cathode optical emission spectroscopy (PB/HC-OES) through monitoring of carbon atomic emission. The PB/HC-OES offers such advantages as ease of operation, exclusion of labor-intensive sample pretreatment processes, rapid analysis, high sensitivity, and low detection limit. The method could also be adapted to be integrated to current microfluidics devices. Parametric optimization for sample introduction, nebulization, desolvation, and hollow cathode source conditions is performed for the analysis of aqueous bovine serum albumin solutions. Response curves of C (I) 193.0-nm emission were obtained under the optimized conditions with both 10% HCl and 100 micromL KCl added to the sample matrix as potential carriers. The detection limit for triplicate injections of bovine serum albumin standards was found to be on the single-nanogram level with 200-microL injections. The addition of KCl significantly improved the sensitivity, supporting the proposed "carrier effect" of chloride salts in the particle transport process. Results obtained here suggest a range of applications for the use of the PB/HC-OES source for total protein determinations; emphasis here is future use in assessing protein quantification in microfluidic systems.

Quantitative and molecular genetic determination of protein and fat deposition

After 30 years of selection, breeding of the pig breed sus scrofa Piétrain has resulted in reduced backfat thickness (from 3.2 to 1.9 mm) and increased loin muscle area (40 to 60 cm2) which indicates high genetic determination of these body composition traits. The use of sophisticated quantitative genetic methods that include all genetic relationships of large populations has led to a high response to selection of these traits. Selection on feed intake, lean and fat tissue growth using nonlinear functions to optimise these traits during the entire growth period in a biological model offers the opportunity to further improve total genetic potential. Protein and lipid deposition rates during the entire growth period have to be known for this biological model to be applied; thus knowledge of the genetic background of these traits is of high economic value. With the use of molecular genetic methods, such as candidate gene and genome scan approaches, the identification of genes for obesity and growth can be obtained. In sus scrofa, candidate genes associated with obesity and growth include Leptin Receptor, Melanocortin-4 Receptor, Agouti related protein, Heart fatty acid binding protein 3, and Insulin-like growth factor 2. Some of these candidate genes also explain variation in obesity levels in humans. Initial genome-wide scans have identified quantitative trait loci (QTL) on chromosomes 1, 4, 5, 7 and X for obesity and on chromosomes 1, 4, 7, 8, 13 and 18 for growth. Physiological candidate genes and predispositional QTL for obesity are not always located on the same chromosome; this is known the "polygenic paradox". Use of a nonlinear growth function is recommended in order to give more insight into the physiological regulation of obesity traits. Sus scrofa is an excellent model organism to examine the genetic regulation of obesity. The conservation of DNA sequence and chromosomal segments between sus scrofa and homo sapiens will permit easy transfer of results to human studies.

Liposome Immunoassays Using Phospholipase C or Alkaline Phosphatase

This chapter summarizes the homogeneous liposome immunoassay (LIA) methods using alkaline phosphatase as a marker or using phospholipase C as a liposome lytic agent. A significantly increasing number of samples in clinical fields and in bioprocesses have increased the demand for rapid and automatic immunoassay methods. Homogeneous enzyme immunoassays are more attractive than heterogeneous ones in satisfying the increasing needs. Although the assessment studies such as cross-reactivities, recovery test, and application for many serum samples are still required and the improvement of stability of liposomes in human serum is also required for the practical application of this LIA system, the sensitivity of homogeneous LIA is comparable or somewhat better than those obtained by conventional heterogeneous techniques, suggesting a potential as a novel homogeneous immunoassay technique. Liposome immunoassays have been applied to the quantitative determination of drugs, hormones, environmental contaminants, blood proteins, and other molecules. The chapter describes two different types of LIA methods that use complement and phospholipase C as liposome lytic agents. In the complement-mediated LIA format (1) a method preparing analyte-conjugated liposomes entrapping alkaline phosphatase and (2) an LIA method using these immunoliposomes by complement are described. In the phospholipase C–mediated LIA format (1) the analyte–phospholipase C conjugation method, (2) the preparation of phospholipase C–labile liposomes, and (3) the LIA method using analyte–phospholipase C conjugates are also described.

Quantitative determination of proteins by the Rayleigh light-scattering technique after optimization of the derivation reaction with Arsenazo-DBS

This paper researched the determination of proteins with 2-(2-arsonophehenylazo)-7-[(2,6-dibromo-4-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid (Arsenazo-DBS) by Rayleigh light-scattering (RLS). The reaction parameters, such as acidity, volume of buffer solution and concentration of Arsenazo-DBS, were examined by orthogonal array design (OAD). Under optimal conditions, the weak RLS of Arsenazo-DBS and BSA can be enhanced greatly and two enhanced RLS signals were produced at 340–350 and 400–420 nm. Based on this reaction, a new quantitative determination method for proteins has been developed. This method is proved to be very sensitive (the determination limits are 0.077 μg ml −1 for bovine serum albumen (BSA) and 0.074 μg ml −1 for human serum albumen (HSA)), rapid (<2 min), simple (one step) and tolerance of most interfering substances. The effects of different surfactants were also examined. The amount of proteins in human serum samples was determined and the maximum relative error was no more than 2% and the recovery was between 95 and 105%.

Quantitative determination of protein of bacterial origin on the basis ofd-aspartic acid andd-glutamic acid content

In recent decades several methods have been developed for determination of the proportion of nitrogen-containing substances passed from the rumen into the abomasum, or small intestine, which are of microbial origin. Recently, when examining thed-amino acid content of foodstuffs, particularly milk and milk products, it was observed that, in addition tod-alanine (d-Ala,d-glutamic acid (d-Glu) andd-aspartic acid (d-Asp) can also be detected in similar quantities, primarily in products which have links with bacterial activity. This gave rise to the idea of examining the diaminopimelic acid (DAPA),d-Glu, andd-Asp content of bacteria extracted from the rumen of cattle, and that of chyme from the same cattle, to establish whetherd-Asp andd-Glu can be used to estimate protein of bacterial origin.

Hydroxyurea-induced partial mushroom body ablation in the honeybee Apis mellifera: volumetric analysis and quantitative protein determination.

Hydroxyurea (HU) treatment of first instar honeybee larvae was previously shown to cause mushroom body (MB) ablations. Predominantly, either one or both median MB subunits were ablated. This prompted us to analyze the effects of asymmetrical or symmetrical HU-induced MB ablation on both the morphology of the brain and on the level of three proteins (synapsin, PKA RII, and PKC), which are considered to play a role in synaptic plasticity, learning, and memory. In brains with one median MB subunit missing the volume of the overall MB calyx neuropil in the lesioned side was diminished by 35%. This strong reduction occurred although the remaining lateral MB calyx of the lesioned brain side was found to be significantly larger than that of the intact side. Accordingly, in brains with both median MB subunits missing the size of the remaining lateral calyces increased. The various types of MB ablation differentially affected the amounts of synapsin, PKA RII, and PKC expressed in the central brain. In animals with bilateral and thus symmetrical MB ablation (both median calyces ablated) the protein amount was found to be similar to that in control animals. However, unilateral MB ablation causes an increase in the amounts of the tested proteins in the intact brain side, while the levels in the ablated side were the same as in control animals. These findings not only show that HU-induced ablation of MB subunits is accompanied by volume changes and by changes in protein expression, but also suggest that these processes are highly regulated between the brain sides. The latter is of general importance in understanding the potential contribution of the MB subunits to learning and memory and their interaction between the brain sides.

Microdetermination of proteins with the arsenazo-DBN-Al(III) complex by Rayleigh light-scattering technique and application of the method.

The determination of proteins with arsenazo-DBN and Al3+ by Rayleigh light-scattering (RLS) is described. The weak RLS of arsenazo-DBN and BSA can be enhanced greatly by addition of Al3+ in the pH range 5.3-7.0; this resulted in two enhanced RLS signals at 420-440 nm and 460-480 nm. The reaction between arsenazo-DBN, Al3+, and proteins was studied and a new method was developed for quantitative determination of proteins. This method is very sensitive (0.34-41.71 microg mL(-1) for bovine serum albumin, BSA, and 0.29-53.41 microg mL(-1) for human serum albumin, HSA), rapid (< 2 min), simple (one step), and tolerant of most interfering substances. The effects of different surfactants were also examined. When these proteins were determined in four human serum samples the maximum relative error was not more than 2% and the recovery was between 97 and 103%.

RAYLEIGH LIGHT SCATTERING STUDY ON INTERACTION OF PROTEIN WITH BERYLLON II AND ITS APPLICATION

A new quantitative determination of proteins using 2-(8-hydroxy-3,6-dislpho-1-naphthylazo)-1,8-dihydroxynapphthalene-3,6-disulphonic acid (Beryllon II) by Rayleigh Light Scattering (RLS) has been developed.Under weak acidic condition (pH 3.80) proteins can enhance the weak RLS of Beryllon greatly and quantitatively at 380nm. This method is characterized with very high sensitivity, wide linear range of determination (0.10—32.40μg ml-1 for bovine serum albumin), rapidity, and simplicity(one step and common fluorimeter). There is little or no interference from amino acids, most of the metal ions or other coexistent substances. The method was applied to the determination of protein in human plasma and the results are very close to those of the Coomassie Brilliant Blue method assay (CBB method).

Determination of albumin and myoglobin in dialysate and ultrafiltrate samples by high-performance size-exclusion chromatography

A high-performance size-exclusion chromatographic method was developed, validated and implemented for simultaneous and quantitative determination of albumin and myoglobin along with inulin, vancomycin and creatinine in dialysate and ultrafiltrate samples from in vitro hemodialysis experiments. The experimental parameters including mobile phase pH, ionic strength, detection wavelength, flow-rate, injection volume were first optimized for the determination of albumin, myoglobin, inulin, vancomycin and creatinine. The peak height ratio and detection limits of the proteins were then comparatively studied at 210, 254 and 280 nm by UV and diode array detection. The method was further validated by evaluating the linearity, precision and accuracy of the proteins. The assay was finally implemented to the simultaneous and quantitative determination of the proteins in dialysate and ultrafiltrate samples.

Protein standardization III: Method optimization basic principles for quantitative determination of human serum proteins on automated instruments based on turbidimetry or nephelometry.

Quantitative protein determinations in routine laboratories are today most often carried out using automated instruments. However, slight variations in the assay principle, in the programming of the instrument or in the reagents may lead to different results. This has led to the prerequisite of method optimization and standardization. The basic principles of turbidimetry and nephelometry are discussed. The different reading principles are illustrated and investigated. Various problems are identified and a suggestion is made for an integrated, fast and convenient test system for the determination of a number of different proteins on the same instrument. An optimized test system for turbidimetry and nephelometry should comprise high-quality antibodies, calibrators, controls, and buffers and a protocol with detailed parameter settings in order to program the instrument correctly. A good user program takes full advantage of the optimal reading principles for the different instruments. This implies--for all suitable instruments--sample preincubation followed by real sample blanking, which automatically corrects for initial turbidity in the sample. Likewise it is recommended to measure the reagent blank, which represents any turbidity caused by the antibody itself. By correcting all signals with these two blank values the best possible signal is obtained for the specific analyte. An optimized test system should preferably offer a wide measuring range combined with a wide security range, which for the user means few re-runs and maximum security against antigen excess. A non-linear calibration curve based on six standards is obtained using a suitable mathematical fitting model, which normally is part of the instrument software.

Quantitative determination of protein of bacterial origin

Several methods have been developed for the determination of the proportion of nitrogen-containing substances of microbial origin in the digestive tracts of cattle. These include assays that use nucleic acids and adenosine triphosphate as indicators, radioisotopes 35S, 15N, 32P and 33P incorporated into bacterial protein and phospholipids, duodenal amino acid composition with amino-ethylphosphonic acid, diaminopimelic acid and d-alanine (d-Ala) contents as indicators. On the basis of the data in the literature on d-amino acid content of milk and milk products the authors came to the conclusion that d-glutamic acid (d-Glu) and d-aspartic acid (d-Asp) can be considered as markers for proteins of bacterial origin. To demonstrate this, experiments have been carried out and some of the results are reported here.

Quantitative determination of protein of bacterial origin

Several methods have been developed for the determination of the proportion of nitrogen-containing substances of microbial origin in the digestive tracts of cattle. These include assays that use nucleic acids and adenosine triphosphate as indicators, radioisotopes 35S, 15N, 32P and 33P incorporated into bacterial protein and phospholipids, duodenal amino acid composition with amino-ethylphosphonic acid, diaminopimelic acid and d-alanine ( d-Ala) contents as indicators. On the basis of the data in the literature on d-amino acid content of milk and milk products the authors came to the conclusion that d-glutamic acid ( d-Glu) and d-aspartic acid ( d-Asp) can be considered as markers for proteins of bacterial origin. To demonstrate this, experiments have been carried out and some of the results are reported here.