Protein phosphorylation stoichiometry by simultaneous ICP-QMS determination of phosphorus and sulfur oxide ions: A multivariate optimization of plasma operating conditions

Molecular mass spectrometry (MS) analysis of protein phosphorylation is partially limited by the molecular specie specificity of the analytical responses that might impair both qualitative and quantitative performances. Elemental MS, such as inductively coupled plasma mass spectrometry (ICP-MS) can overcome these drawbacks; in fact, analytical performance is theoretically independent of the molecular structure of a target analyte naturally containing the elements of interest. Nevertheless, isobaric interferences derived from sample matrix and laboratory environment can hinder the quantitative determination of both phosphorus (P) and sulfur (S) as ³¹P⁺ and ³²S⁺ by inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) under standard plasma conditions. These interferences may be overcome by quantifying P and S as oxide ions ³¹P¹⁶O⁺ and ³²S¹⁶O⁺, respectively.In this study, we present a systematic investigation on the effect of plasma instrumental conditions on the oxide ion responses by a design of experiment approach for the simultaneous ICP-QMS determination of P and S (³¹P¹⁶O⁺ and ³²S¹⁶O⁺, respectively) in protein samples without the use of dynamic reaction, collision reaction cells or pre-addition of oxygen as reactant gas in the torch. The proposed method was evaluated in terms of limit of detection, limit of quantification, linearity, repeatability, and trueness. Moreover, detection and quantification capabilities of the optimized method were compared to the standard plasma mode for determination of ³¹P⁺ and ³⁴S⁺. Spectral and non-spectral interferences affecting the quantification of ³¹P⁺, ³¹P¹⁶O⁺ and ³²S¹⁶O⁺ were also studied. The suitability of inorganic elemental standards for P and S quantification in proteins was assessed. The method was applied to quantify the phosphorylation stoichiometry of commercially available caseins (bovine β-casein, native and dephosphorylated α-casein) and results were confirmed by Matrix Assisted Laser Desorption Ionization Time of Flight MS analysis.We demonstrate that ICP-QMS, by quantifying P and S as oxide ions, was able to accurately calculate the degree of phosphorylation of β-casein and α-casein and to detect specific partial enzymatic dephosphorylation. The collected results might lead to further development of ICP-QMS interfaces optimized for protein phosphorylation studies and for proteomics investigations.     

Preparation of monodisperse immobilized Ti affinity chromatography microspheres for specific enrichment of phosphopeptides

This study presented an approach to prepare monodisperse immobilized Ti⁴⁺ affinity chromatography (Ti⁴⁺-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8 μm were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13 μm were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti⁴⁺ ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti⁴⁺-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of α-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5 fmol for standard tryptic digest of β-casein. The Ti⁴⁺-IMAC microspheres were compared with commercial Fe³⁺-IMAC adsorbent and homemade Zr⁴⁺-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe³⁺-IMAC, Zr⁴⁺-IMAC and Ti⁴⁺-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti⁴⁺-IMAC had the best performance for enrichment of phosphopeptides.     

IgG anti-gliadin determination with an immunological microfluidic system applied to the automated diagnostic of the celiac disease

In the present article, a novel microfluidic immunosensor coupled with electrochemical detection for anti-gliadin IgG antibody quantification is proposed. This device represents an important tool for a fast, simple, sensitive, and automated diagnostic for celiac disease, which is carried out through detection of anti-gliadin IgG antibodies present in human serum samples. Celiac disease (CD) is an autoimmune disease generated by gluten protein fractions called prolamins. This pathology affects about one in 250 people around the world, produces intestinal inflammation, villous atrophy, and crypt hyperplasia, which causes a range of symptoms including altered bowel habits, malnutrition and weight loss. Our immunosensor consists of a Plexiglas device coupled to a gold electrode, with a central channel containing 3-aminopropyl-modified controlled pore glass (AP-CPG). The quantification of anti-gliadin IgG antibodies was carried out using a heterogeneous, non-competitive enzyme-linked immunosorbent assay (ELISA) in which IgG antibodies bound to gliadin protein, immobilized on AP-CPG, were determined by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP, and the electroactive product was quantified on a gold electrode at 0.250 V. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.52 and 2.72 UR mL⁻¹, respectively, and the within- and between-assay coefficients of variation were below 5.8%. The optimized procedure was applied to the determination of anti-gliadin IgG antibodies in human serum samples.     

Highly efficient enrichment of intact phosphoproteins by a cadmium ion-based co-precipitation strategy

Selective separation and enrichment of phosphoproteins are essential for understanding their important functions in almost all cellular processes. Here, taking advantage of the feature that cadmium ion (Cd²⁺) has an overwhelming preference for phosphates, we developed a robust and simple Cd²⁺ co-precipitation strategy for the selective isolation of intact phosphoproteins. After evaluating the feasibility of Cd²⁺ in phosphoprotein precipitation, we compared the washing protocols for the removal of non-specific binding proteins and then used the best-performing protocol for the isolation of phosphoproteins from different complex samples. It was found that phosphoproteins can be specifically enriched from artificial protein mixtures containing α-casein, β-casein, and bovine serum albumin or plasma, in which bovine serum albumin or plasma were served as interferences with very high molar ratios. Applying this method to enrich phosphoproteins from complex cell lysates, a high specificity was confirmed by western blotting analysis with a phosphoprotein-specific kit. Finally, we successfully applied this method to the purification of caseins from drinking milk, highlighting its potential application in the studies where purified phosphoproteins were required. In a word, this Cd²⁺ co-precipitation method enables universal and effective capture, enrichment, and detection of intact phosphoproteins, making it a powerful tool for the comprehensive analysis of the phosphoproteome.     

Proteins at liquid interfaces: I. Kinetics of adsorption and surface denaturation

The rates of change of film pressure (π) and surface concentration (Γ) of protein during the adsorption of β-casein, bovine serum albumin (BSA), and lysozyme at the air-water interface have been monitored by the Wilhelmy plate and surface radioactivity methods, respectively. The increases in π and Γ for the relatively flexible β-casein molecule occur simultaneously with both parameters attaining their steady-state values at about the same time. In contrast, π and Γ follow different time courses for the globular lysozyme molecule; Γ can reach a steady state value while π is still increasing significantly. The kinetics indicate that initially adsorption is diffusion-controlled but at higher surface coverages there is an energy barrier to adsorption. Under these conditions, the ability of the protein molecules to create space in the existing film and penetrate and rearrange in the surface is rate-determining. Two kinetic regions exist: the relaxation time τ₁ (typically ～2 hr when Γ ～2 mg m^?2) describes the adsorption when both π and Γ are increasing whereas τ₂ (in the range 1–8 hr for all three proteins) relates to the situation when π is increasing at constant Γ because the protein molecules are changing conformation in the surface.     

Ti-SBA-15介孔材料用于磷酸化肽的高效富集

结合基质辅助激光解吸飞行时间质谱(MALDI-TOF MS)检测技术,考察了Ti-SBA-15介孔材料对β-酪蛋白酶解产物中磷酸化肽的选择性富集性能。实验结果显示,含Ti和Si物质的量比为0.08的Ti-SBA-15介孔材料可选择性地对β-酪蛋白酶解产物中的磷酸化肽进行选择性富集;对于β-酪蛋白和牛血清白蛋白物质的量比为1:100的蛋白质酶解混合液,Ti-SBA-15仍能实现对其磷酸化肽的有效富集。上述结果表明,作为一种多孔、高比表面积的磷酸化多肽的选择性吸附材料,Ti-SBA-15有望在磷酸化蛋白质组的分析中得到广泛的应用。     

Stopped-flow microarray immunoassay for detection of viable <Emphasis Type="Italic">E. coli</Emphasis> by use of chemiluminescence flow-through microarrays

Rapid multiplexed analysis of microorganisms is important in water analysis to control bacterial contamination for health and safety reasons. Direct quantification of bacteria by means of flow-through microarray immunoassays requires new analysis strategies for optimising sensitivity and the analysis time. For bacteria and for particles, hydrodynamic forces and sedimentation are the dominating effects for binding on surfaces in a flow-through system, whereas diffusion is insignificant. Therefore, we have implemented a stop and flow technique for quantification of viable E. coli cells. The method, with alternation of resting volume elements and pumping the elements forward, was more effective than continuous-flow approaches for analysis of bacteria. For quantification of viable E. coli cells, a chemiluminescence sandwich immunoassay test format was performed by means of antibody microarrays and flow-injection-based microarray analysis. Antibodies, which served as selective capture molecules, were immobilised on polymer-modified glass surfaces serving as microarray substrate. For the bacteria recognition step, a second detection antibody was used, forming a sandwich immunoassay at each spot of the microarray. Detection was carried out with a horseradish peroxidase catalysed chemiluminescence reaction. All assay steps were conducted with an automated flow-through chemiluminescence microarray readout system. Living E. coli cells could be detected in 67 min with a detection limit of 4 × 10⁵ cells mL⁻¹. By introduction of the stopped-flow technique and optimisation of interaction time and interaction steps the achieved detection of E. coli was faster and two orders of magnitude more sensitive than with a conventional ELISA technique in microplates.     

A highly sensitive capillary electrophoresis immunoassay strategy based on dual‐labeled gold nanoparticles enhancing chemiluminescence for the detection of prostate‐specific antigen

An enzyme and antibody dual labeled gold nanoparticles enhancing chemiluminescence strategy was developed for highly sensitive CE immunoassay (IA) of prostate‐specific antigen (PSA). In this work, gold nanoparticles were labeled with horseradish peroxidase and antiprostate specific antigen‐antibody, and used as the marker (Ab^*). After PSA (antigen, Ag) was added into the system, a noncompetitive immune reaction was happen between Ab^* and Ag to form an immune complex (Ag–Ab^*). Subsequently, the obtained Ag–Ab^* and unreacted Ab^* were separated by CE, and the chemiluminescence intensity of Ag‐Ab^* was used to estimate PSA concentration. The calibration curve showed a good linearity in the range of 0.25–10 ng/mL. Based on a S/N of 3, the detection limit for PAS was estimated to be 0.092 ng/mL. Proposed CE method was applied for PSA quantification in human serum samples from healthy volunteers and patients with prostate cancer. The obtained results demonstrated that the proposed CE method may serve as an alternative tool for clinical analysis of PSA.     

A Novel Label Free Electrochemical Magnetoimmunosensor for Human Interleukin‐6 Quantification in Serum

A novel magnetoimmunosensor, designed for sensitive and selective quantification of interleukin 6, is herein reported. The experimental design involves the covalent immobilization of anti‐interleukin 6 antibody through an amidic bond formed with the carboxyl functionalities provided at the surface of protein G‐functionalized magnetic microparticles, assuring a sandwich‐type immunoassay with electrochemical label free detection. All the experimental parameters involved in the elaboration and testing protocol were optimized. A linear calibration plot between the charge transfer resistance and the logarithmic concentration of interleukin‐6 was achieved in the 1 pg mL^?1 to 1 μg mL^?1 range. A limit of quantification of 1 pg mL^?1 and a detection limit of 0.3 pg mL^?1 were obtained. The optimized magnetoimmunosensor showed an excellent selectivity against some potentially interfering proteins and has been successfully applied for the determination of target protein in human serum, proving its clinical relevance.     

Surface plasmon resonance aided electrochemical immunosensor for CK-MB determination in undiluted serum samples

This article presents a simple chronoamperometric immunosensor for the quantitative assessment of creatine kinase MB (CK-MB) in 50 μL undiluted serum samples. The immunosensor consists of gold working and counter electrodes patterned onto a glass chip by thin-film photolithography and an external Ag|AgCl reference electrode. The detection limit (DL) of the chronoamperometric method is 13 ng mL⁻¹ (DL = 2×RMSD/S, where RMSD is the residual mean standard deviation of the measured points around a calibration curve with a slope of S). In spiked serum samples, the response was linear up to 300 ng mL⁻¹ of CK-MB. A surface plasmon resonance (SPR) system with simultaneous electrochemical detection (EC-SPR) aided the development of the sandwich immunoassay. Real-time monitoring of the SPR signal was used to optimize the capture antibody immobilization, CK-MB and detection antibody binding, as well as to minimize the nonspecific adsorption of serum proteins to the sensor surface. The detection antibody has been labeled with alkaline phosphatase (ALP) enzyme for sensitive electrochemical detection. ALP catalyzes the hydrolysis of ascorbic acid phosphate and generates ascorbic acid, which is measured chronoamperometrically. The electrochemical immunoassay for CK-MB was less sensitive to nonspecific adsorption related interferences, had a better detection limit, and required a lower volume of sample than the SPR method.     

Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity

The β-lactam antibiotics, including penicillins, are the most important antimicrobial substances used for mastitis treatment. Consequently, this is also the most frequently occurring type of antibiotic residues in milk. Today, in addition to the traditional microbial inhibitor tests, rapid and sensitive receptor and immunoassays are used in residue control. Due to the limitations in throughput capacity of these tests, recent applications of automated biosensor technology in food analysis are of great interest.A surface plasmon resonance (SPR)-based biosensor (Biacore) was used to design an inhibition assay to detect β-lactam antibiotics in milk. A microbial receptor protein with carboxypeptidase activity was used as detection molecule. One advantage of using this receptor protein over antibodies that are more commonly used is that only the active, intact β-lactam structure is recognized, whereas most antibodies detect both active and inactive forms. In the presence of β-lactam antibiotics the formation of a stable complex between receptor protein and antibiotic inhibits the enzymatic activity of the protein. The decrease in enzymatic activity was measured using an antibody against the degraded substrate and penicillin G in milk samples was quantitatively determined. The limit of detection of the assay for penicillin G was determined to 2.6 μg kg⁻¹ for antibiotic-free producer milk, which is below the European maximum residue limit (MRL) of 4 μg kg⁻¹. The coefficient of variation at 4 μg kg⁻¹ penicillin G, ranged between 7.3 and 16% on three different days.     

Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis

Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.     

Polymer nanoparticles as fluorescent labels in a fluoroimmunoassay for human chorionic gonadotropin

A sensitive fluoroimmunoassay (FIA) was developed for the determination of human chorionic gonadotropin (β-HCG). It is based on fluorescent polymer nanoparticles (PFNPs) coated with anti-β-HCG monoclonal antibodies in a sandwich type of fluoroimmunoassay. The PFNPs were synthesized by precipitation polymerization using methacrylic acid (MAA) as the monomer, trimethylolpropane trimethacrylate as the cross-linker, azobisisobutyronitrile as the radical initiator, and fluorescein as the fluorophore. Anti-β-HCG monoclonal antibody was labeled with the PFNPs and then used in a FIA of β-HCG in human serum samples using low-fluorescent transparent 96-well microtiter plates. The calibration graph for β-HCG is linear over the range from 1.25 to 300 mIU mL-¹ with a detection limit of 0.3 mIU mL-¹ (3σ). The relative standard deviation for seven parallel measurements of 10 mIU mL-¹ of β-HCG is 3.8%. The method has the specificity of an immunoassay and the sensitivity of fluorescent nanoparticle label technology.     

Isolation of phosphopeptides using zirconium-chlorophosphonazo chelate-modified silica nanoparticles

Due to the low abundance of phosphoproteins and substoichiometry of phosphorylation, the elucidation of protein phosphorylation requires highly specific materials for isolation of phosphopeptides from biological samples prior to mass spectrometric analysis. In this study, chlorophosphonazo type derivatives of chromotropic acid including p-hydroxychlorophosphonazo (HCPA) and chlorophosphonazo I (CPA I), traditionally used in the photometric determination of transition metal ions, have been employed as chelating ligands in the preparation of novel affinity materials for phosphopeptide enrichment. The chromogenic reagents of HCPA and CPA I were chemically modified on the surface of silica nanoparticles, and the functionalized materials were charged with zirconium ions through the strong complexation between chelating ligands and Zr(4+). The obtained zirconium-chlorophosphonazo chelate-modified silica nanoparticles (Zr-HCPA-SNPs and Zr-CPA I-SNPs) were applied to the selective enrichment of phosphopeptides, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The purification procedures were optimized using α-casein digest at first, and then the performance of these two affinity materials for efficient and specific enrichment of phosphopeptides was evaluated with the tryptic digests of standard proteins (α-casein, β-casein, ovalbumin and bovine serum albumin). It is found that Zr-HCPA-SNPs are superior to Zr-CPA I-SNPs in phosphopeptide enrichment. Using Zr-HCPA-SNPs to trap phosphopeptides in α-casein digest, the detection limit was close to 50fmol based on MALDI-TOF MS analysis. Finally, Zr-HCPA-SNPs were used to directly isolate phosphopeptides from diluted human serum of healthy, diabetes and hypertension persons, respectively. Our results show that the constitution and level of phosphopeptides are remarkably different among the three groups, which indicate the powerful potentials of Zr-HCPA-SNPs in disease diagnosis and biomarker screening.     

Quantification of unadsorbed protein and surfactant emulsifiers in oil-in-water emulsions

Unadsorbed emulsifiers affect the physical and chemical behaviour of oil-in-water (O/W) emulsions. A simple methodology to quantify unadsorbed emulsifiers in the aqueous phase of O/W emulsions has been developed. Emulsions were centrifuged and filtered to separate the aqueous phase from the oil droplets and the concentration of unadsorbed emulsifiers in the aqueous phase determined. The quantification of unadsorbed surfactants based on the direct transesterification of their fatty acids was validated for Tween 20, Tween 80, citric acid ester (Citrem), Span 20 and monolauroyl glycerol. To determine unadsorbed proteins, results obtained with Folin-Ciocalteu reagent or UV-spectrophotometry were compared on emulsions stabilized by β-lactoglobulin (BLG), β-casein (BCN) or bovine serum albumin (BSA). The first method gave more accurate results especially during aging of emulsions in oxidative conditions. The whole methodology was applied to emulsions stabilized with single or mixed emulsifiers. This approach enables optimization of emulsion formulations and could be useful to follow changes in the levels of unadsorbed emulsifiers during physical or chemical aging processes.     

Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca²⁺ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC-CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the μIMER were investigated for the substrate β-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of β-casein could be achieved in a few minutes (86 s residence time in the μIMER followed by a wash step).     

Determination of proline in human serum by a robust LC‐MS/MS method: application to identification of human metabolites as candidate biomarkers for esophageal cancer early detection and risk stratification

Altered serum proline levels are related to cancer metabolism. This study developed and validated a LC‐MS/MS method to analyze proline in human serum. Surrogate blank serum, coupled with stable isotope l ‐proline‐¹³C₅,¹⁵ N as internal standard, was used for generating standard curves ranging from 2.5 to 100 μg/mL. Proline was extracted from serum samples using methanol. A Phenomenex Lux 5u Cellulose‐1 column (250 × 4.6 mm) was used for chromatographic separation with 40% methanol in 0.05% formic acid aqueous solution as a mobile phase. Mass detection was performed under positive ionization electrospray. Intra‐ and inter‐day accuracy and precision were <10%. The extraction recovery and matrix factor were 99.17 and 1.47%, respectively. Our study showed that the chiral column had high specificity and selectivity for separating proline from serum components. The assay was successfully applied for the quantification of human serum proline levels from 30 esophageal cancer patients and 30 healthy volunteers. Statistical analyses showed significantly lower levels of serum proline in the patients as compared with the healthy volunteers (p‐value = 0.011). We report here a simple, specific and reproducible LC‐MS/MS method for the quantification of proline in human serum as a potential screening biomarker for esophageal cancer. Copyright © 2014 John Wiley & Sons, Ltd.     

Immobilization of a Ce(IV)-substituted polyoxometalate on ethylenediamine-functionalized graphene oxide for selective extraction of phosphoproteins

A sorbent for selective extraction of phosphoproteins was obtained by immobilization of a Ce(IV)-substituted polyoxometalate on ethylenediamine-functionalized graphene oxide (CeEGO). The resulting composites exhibit an adsorption capacity of 981 mg g^?1 for β-casein due to the synergistic effect of metal-affinity interaction between Ce(IV) and phosphate groups and π-stacking interaction between the polyoxometalate framework and the phosphate groups. The results of LC-MS and SDS-PAGE analysis show that the CeEGO composites can be applied to the extraction of phosphoproteins from protein mixture, and as little as 50 μg mL^?1 of the phosphoprotein β-casein can be detected by SDS-PAGE. It was also applied to the extraction of β-casein from spiked biological samples such as drinking milk, whole blood and swine heart tissue extract.

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Graphical abstract An efficient sorbent is obtained by immobilization of a Ce(IV)-substituted polyoxometalate on ethylenediamine-functionalized graphene oxide (CeEGO). The resulting composites exhibit highly selective capture capacity towards phosphoproteins due to the synergistic effect of metal-affinity interaction between Ce(IV) and phosphate groups and π-stacking interaction between the polyoxometalate framework and the phosphate groups.

     

Highly selective and sensitive enrichment of phosphopeptides via NiO nanoparticles using a microwave-assisted centrifugation on-particle ionization/enrichment approach in MALDI-MS

The strategy to concentrate phosphopeptides has become a critical issue for mapping protein phosphorylation sites, which are well known as posttranslational modifications in proteomics. In this study, we propose a simple and highly sensitive method for phosphopeptide enrichment on NiO nanoparticles (NPs) from a trypsin predigested phosphoprotein complex solution in a microwave oven. Furthermore, this technique was combined with centrifugation on-particle ionization/enrichment of phosphopeptides and phosphopeptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Weak magnetism of these NPs and a positive surface charge effect at low pH accomplished rapid and selective phosphopeptide enrichment within 30s. Trypsin-digested products of phosphoproteins such as α-casein and β-casein, human blood serum, nonfat milk, and egg white were also investigated to explore their phosphopeptide enrichment from complex samples by this approach. The results demonstrate that NiO NPs exhibit good affinity to trace the phosphopeptides even in the presence of 30 times higher molar concentration of complex solution of non-phosphopeptide proteolytic predigested bovine serum albumin. The detection limits of NiO NPs for α-casein and β-casein were 2.0?×?10(-9) M, with good signal-to-noise ratio in the mass spectrum. NiO NPs were found to be effective and selective for enrichment of singly and multiply phosphorylated peptides at a trace level in complex samples in a microwave oven. The cost of preparing NiO NPs is low, the NiO NPs are thermally stable, and therefore, they hold great promise for use in phosphopeptide enrichment.     

Kinetics of competitive adsorption of β-casein and methylene blue on hydrophilic glass

The competitive adsorption of methylene blue (MB) and β-casein on hydrophilic glass from an aqueous mixed solution was directly detected at the solution pH smaller than the protein isoelectric point (pI) by means of the waveguide-based broadband time-resolved evanescent wave absorption spectroscopy. The competitive adsorption causes the MB coverage to exponentially decrease with time from its peak value and prevents MB aggregation at the interface. The kinetic equation for the competitive adsorption of binary adsorbates was theoretically deduced based on the Langmuir model, and was used for creating the best fit to the experimental data. In the case of a fixed concentration of MB in the mixed solution, the best-fit parameter τ(-1) increases with the protein concentration at a specific pH and decreases with the solution pH at a given concentration of protein. The findings suggest that the β-casein concentration in sub-μM level can be rapidly determined by the time-resolved waveguide absorptiometry based on the competitive adsorption of MB and protein.