Increasing proteome coverage for gel‐based human tear proteome maps: towards a more comprehensive profiling

In situations where the molecular mechanism of many ocular disorders is unknown, owing to the difficulties associated with sampling from ocular tissues, human tear film can be a promising medium in ophthalmic research. The present study demonstrates an in‐depth gel‐based proteome optimization survey to approach more appropriate and efficient systems in various situations such as normal and dry‐eye syndromes. Therefore, systematic and statistical evaluations were performed on different preparation methods, including acetone, acetone–methanol, chloroform–methanol–water, trichloroacetic acid (TCA)–acetone, tri‐n‐butylphosphate–acetone–methanol precipitations and ammonium sulfate fractionation at three different percentages of saturations (50, 70 and 90%). Methods were compared quantitatively on both one‐ and two‐dimensional patterns. Some important parameters such as total protein recovery yield, densitometric analysis of some protein contaminants, banding patterns and total spot numbers along with statistical models for proper clustering were considered. Findings revealed noticeable impacts of preparation methods on all aspects of gel‐based separations as well as recovery yield (ranging from 5.29 ± 0.96 to 22.56 ± 1.77 µg/mm) and banding and pattern resolution. In addition to all these, the most important point is that the total protein spot number on the final two‐dimensional patterns (varied from 528.00 ± 19.00 to 657.00 ± 21.52 for different methods) were also noticeably increased in comparison with previously published reports (maximum of 250 spots), which is essential for a more comprehensive analysis. Increasing the proteome coverage in the present study is supposed to originate from improved solubility and effective rehydration during the sample application and isoelectric focusing (IEF) procedure along with proper sample preparation. Copyright © 2014 John Wiley & Sons, Ltd.     

Recent advances in methods for the analysis of protein o‐glycosylation at proteome level

O‐Glycosylation, which refers to the glycosylation of the hydroxyl group of side chains of Serine/Threonine/Tyrosine residues, is one of the most common post‐translational modifications. Compared with N‐linked glycosylation, O‐glycosylation is less explored because of its complex structure and relatively low abundance. Recently, O‐glycosylation has drawn more and more attention for its various functions in many sophisticated biological processes. To obtain a deep understanding of O‐glycosylation, many efforts have been devoted to develop effective strategies to analyze the two most abundant types of O‐glycosylation, i.e. O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation. In this review, we summarize the proteomics workflows to analyze these two types of O‐glycosylation. For the large‐scale analysis of mucin‐type glycosylation, the glycan simplification strategies including the ‘‘SimpleCell’’ technology were introduced. A variety of enrichment methods including lectin affinity chromatography, hydrophilic interaction chromatography, hydrazide chemistry, and chemoenzymatic method were introduced for the proteomics analysis of O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation.     

Normal human mitral valve proteome: A preliminary investigation by gel‐based and gel‐free proteomic approaches

The mitral valve is a highly complex structure which regulates blood flow from the left atrium to the left ventricle (LV) avoiding a significant forward gradient during diastole or regurgitation during systole. The integrity of the mitral valve is also essential for the maintenance of normal LV size, geometry, and function. Significant advances in the comprehension of the biological, functional, and mechanical behavior of the mitral valve have recently been made. However, current knowledge of protein components in the normal human mitral valve is still limited and complicated by the low cellularity of this tissue and the presence of high abundant proteins from the extracellular matrix. We employed here an integrated proteomic approach to analyse the protein composition of the normal human mitral valve and reported confident identification of 422 proteins, some of which have not been previously described in this tissue. In particular, we described the ability of pre‐MS separation technique based on liquid‐phase IEF and SDS‐PAGE to identify the largest number of proteins. We also demonstrated that some of these proteins, e.g. αB‐Crystallin, septin‐11, four‐and‐a‐half LIM domains protein 1, and dermatopontin, are synthesised by interstitial cells isolated from human mitral valves. These initial results provide a valuable basis for future studies aimed at analysing in depth the mitral valve protein composition and at investigating potential pathogenetic molecular mechanisms. Data are available via ProteomeXchange with identifier PXD004397.     

A gradient‐directed Monte Carlo approach for protein design

We develop a new global optimization strategy, gradient‐directed Monte Carlo (GDMC) sampling, to optimize protein sequence for a target structure using RosettaDesign. GDMC significantly improves the sampling of sequence space, compared to the classical Monte Carlo search protocol, for a fixed backbone conformation as well as for the simultaneous optimization of sequence and structure. As such, GDMC sampling enhances the efficiency of protein design. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

MetREx: A protein design approach for the exploration of sequence‐reactivity relationships in metalloenzymes

Metalloenzymes represent a particular challenge for any rational (re)design approach because the modeling of reaction events at their metallic cofactors requires time‐consuming quantum mechanical calculations, which cannot easily be reconciled with the fast, knowledge‐based approaches commonly applied in protein design studies. Here, an approach for the exploration of sequence‐reactivity relationships in metalloenzymes is presented (MetREx) that consists of force field‐based screening of mutants that lie energetically between a wild‐type sequence and the global minimum energy conformation and which should, therefore, be compatible with a given protein fold. Mutant candidates are subsequently evaluated with a fast and approximate quantum mechanical/molecular mechanical‐like procedure that models the influence of the protein environment on the active site by taking partial charges and van der Waals repulsions into account. The feasibility of the procedure is demonstrated for the active site of [FeFe] hydrogenase from Desulfovibrio desulfuricans. The method described allows for the identification of mutants with altered properties, such as inhibitor‐coordination energies, and the understanding of the robustness of enzymatic reaction steps with respect to variations in sequence space. © 2015 Wiley Periodicals, Inc.     

A sequential separation strategy for facile isolation and comprehensive analysis of human urine N-glycoproteome

Analytical and Bioanalytical Chemistry - Urine is an attractive and non-invasive alternative source to tissue, blood or other biofluids for biomarker screening in clinical research. In normal human...     

Ultra‐rapid non‐equilibrium solid‐phase microextraction at elevated temperatures and direct coupling to mass spectrometry for the analysis of lidocaine in urine

Solid‐phase microextraction (SPME) has been directly coupled to an ion‐trap mass spectrometer (MS) for the determination of the model compound lidocaine in urine, hereby applying MS/MS [fragmentation of [M + H]⁺ (m/z 235) to a fragment with m/z 86]. The throughput of samples has been increased using non‐equilibrium SPME with polydimethylsiloxane (PDMS) fibers. The effect of temperature on the sorption and the desorption was studied. Elevated temperatures during sorption (65°C) and desorption (55°C) had a considerable influence on the speed of the extraction. The desorption was carried out with a home‐made desorption chamber allowing thermostating. Only 1 min sorption and 1 min desorption were performed, after which MS detection took place, resulting in a total analysis time of 3 min. Detection limits below 1 ng/mL could be obtained despite yields of only 2.1 and 1.5% for a 100‐ and a 30‐μm PDMS‐coated fiber, respectively. Furthermore, the determination of lidocaine in urine had acceptable reproducibilities, i.e., relative standard deviations (RSDs) below 10%. A limit of quantitation (RSD < 15%) of about 1 ng/mL was obtained. No extra wash step of the extraction fiber was required after desorption if a 30‐μm coating was used, whereas not all the analyte was desorbed from the 100‐μm coating in a single desorption. Therefore, the SPME‐MS/MS system with a 30‐μm PDMS‐coated fiber for rapid non‐equilibrium SPME at elevated temperatures has interesting potential for high‐throughput analysis of biological samples.     

A CE‐LIF method based on long wavelength fluorescence labeling for the analysis of thiols in human urine

A rapid and robust CE method using a long wavelength fluorescent reagent 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(2‐maleimide)difluoroboradiaza‐s‐indacene as the labeling reagent has been developed for the simultaneous determination of thiols, including glutathione, cysteine, homocysteine, N‐acetylcysteine, cysteinylglycine, and penicillamine. The derivatization reaction was carried out in 14 mmol/L pH 8.5 borate buffer at 30°C for 6 min and the labeled thiols derivatives were separated with the running buffer containing 30 mmol/L pH 7.4 phosphate, 30% v/v acetonitrile and 8 mmol/L SDS within 12 min. Detection limits ranged from 0.4 to 2.4 nmol/L. To demonstrate the capability of this method, it was applied to the analysis of thiols in human urine with recoveries of 92.4–105.6%. The derivatization reaction was much faster at milder conditions, and the analysis was rapider. Moreover, with excitation wavelength at long wavelength region, background interference from samples was reduced effectively. The present method seems to be a potential choice for quantifying thiols in human urine.     

Surface‐activated chemical ionization time‐of‐flight mass spectrometry and labeling‐free approach: two powerful tools for the analysis of complex plant functional proteome profiles

Surface‐activated chemical ionization (SACI) has been widely used in recent years to analyze a range of different compounds (e.g., peptides, street drugs, amino acids). The main benefits of this technology are its high sensitivity and its effectiveness under different chromatographic conditions. Here, we used SACI in conjunction with a highly selective quadrupole time‐of‐flight mass analyzer to characterize a complex proteome pattern after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). The data obtained were compared with those obtained using the micro‐electrospray ionization (ESI) approach, which showed that using SACI strongly increased the number of detectable proteins. The higher sensitivity is mainly due to the ability of SACI to selectively produce singly charged species of high intensity under full‐scan conditions and doubly charged species for tandem mass spectrometric (MS/MS) peptide characterization by simply changing the ionization conditions during data acquisition. Copyright © 2010 John Wiley & Sons, Ltd.     

Investigation on Molecular Non-covalent Interaction in the Sodium Dodecyl Benzene SuIfonate-polychrome Blue B-protein Replacement Reaction

The molecular noncovalent interaction often originates from the electrostatic attraction and accords with the Langmuir isothermal adsorption. The sodium dodecyl benzene sulfonate (SDBS)‐polychrome blue B (PCB>protein bovine serum albumin (BSA), ovalbumin (OVA) and myoglobin (MB)] ternary reaction has been investigated at pH 3.88. Protein to replace PCB from the PCB‐SDBS binding product was used to characterize the assembly of an invisible‐spectral compound, SDBS, on proteins by measuring the variation of FCB light‐absorption by tbe micro‐surface adsorption‐spectral correction (MSASC) technique. The effect of ionic strength and temperature on the aggregation was studied. Results showed that the aggregates SDBS₉₂·BSA, SDBS₅₈·VA and SDBS₁₅·MB at 30 C and SDBS₈₃·BSA. SDBS₃₉·OVA and SDBS₁₀·MB at 50 °C are formed.     

Ion‐pair cloud‐point extraction: A new method for the determination of water‐soluble vitamins in plasma and urine

A novel, simple, and effective ion‐pair cloud‐point extraction coupled with a gradient high‐performance liquid chromatography method was developed for determination of thiamine (vitamin B₁), niacinamide (vitamin B₃), pyridoxine (vitamin B₆), and riboflavin (vitamin B₂) in plasma and urine samples. The extraction and separation of vitamins were achieved based on an ion‐pair formation approach between these ionizable analytes and 1‐heptanesulfonic acid sodium salt as an ion‐pairing agent. Influential variables on the ion‐pair cloud‐point extraction efficiency, such as the ion‐pairing agent concentration, ionic strength, pH, volume of Triton X‐100, extraction temperature, and incubation time have been fully evaluated and optimized. Water‐soluble vitamins were successfully extracted by 1‐heptanesulfonic acid sodium salt (0.2% w/v) as ion‐pairing agent with Triton X‐100 (4% w/v) as surfactant phase at 50°C for 10 min. The calibration curves showed good linearity (r² > 0.9916) and precision in the concentration ranges of 1‐50 μg/mL for thiamine and niacinamide, 5–100 μg/mL for pyridoxine, and 0.5–20 μg/mL for riboflavin. The recoveries were in the range of 78.0–88.0% with relative standard deviations ranging from 6.2 to 8.2%.     

A pH‐dependent computational approach to the effect of mutations on protein stability

This article describes a novel software implementation for high‐throughput scanning mutagenesis with a focus on protein stability. The approach combines molecular mechanics calculations with calculations of protein ionization and a Gaussian‐chain model of electrostatic interactions in unfolded state. Comprehensive testing demonstrates a state‐of‐the‐art accuracy for predicted free energy differences on single, double, and triple mutations with a correlation coefficient R above 0.7, which takes about 1.5 min per mutation on a single CPU. Unlike most of existing in silico methods for fast mutagenesis, the stability changes are reported as a continuous function of solution pH for wide pH intervals. We also propose a novel in silico strategy for searching stabilized protein variants that is based on combinatorial scanning mutagenesis using representative amino acid types. Our in silico predictions are in excellent agreement with the hyper‐stabilized variants of mesophilic cold shock protein found using the Proside method of direct evolution. © 2016 Wiley Periodicals, Inc.     

A study of the deformation‐induced whitening phenomenon for cavitating and non‐cavitating semicrystalline polymers

In this work, we used two techniques to study the deformation‐induced whitening phenomenon that occurs when certain semicrystalline polymers (SCPs) are subjected to tensile drawing: (1) IPLST (Incoherent Polarized Steady Light Transport) was used for characterizing the light scatterers and in particular for determining their size. (2) SRXTM (Synchrotron Radiation X‐Ray Tomographic Microscopy) was used to visualize the internal structure of the deformed SCPs. In particular, with this technique the possible presence of micrometric cavities can be detected. In the early whitening stage of a cavitating polypropylene (PP), the IPLST technique was found to show that the size of the light scatterers is larger than 1 μm. At the same time, the SRXTM measurements showed that no void larger than 1 μm was present in the material. The micrometric light scatterers responsible for the whitening phenomenon may thus not be simple cavities. In fact, this experimental study suggests that they correspond to areas where smaller objects (possibly nanovoids) are highly confined. At the scale of visible wavelengths, these regions could scatter visible light like individual entities of micrometric size. The study also showed that the size of cavities observable using SRXTM for a very deformed PP is dependent on the initial dimensions of the spherulites. Results previously obtained for a non‐cavitating high density polyethylene are also briefly presented in this article to confirm the theory that deformation‐induced‐whitening phenomenon may have various origins for such complex microstructuring. © 2013 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys., 2013     

A quantitative and comprehensive method to analyze human milk oligosaccharide structures in the urine and feces of infants

Human milk oligosaccharides (HMOs), though non-nutritive to the infant, shape the intestinal microbiota and protect against pathogens during early growth and development. Infant formulas with added galacto-oligosaccharides have been developed to mimic the beneficial effects of HMOs. Premature infants have an immature immune system and a leaky gut and are thus highly susceptible to opportunistic infections. A method employing nanoflow liquid chromatography time-of-flight mass spectrometry (MS) is presented to simultaneously identify and quantify HMOs in the feces and urine of infants, of which 75 HMOs have previously been fully structurally elucidated. Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS was employed for high-resolution and rapid compositional profiling. To demonstrate this novel method, samples from mother–infant dyads as well as samples from infants receiving infant formula fortified with dietary galacto-oligosaccharides or probiotic bifidobacteria were analyzed. Ingested oligosaccharides are demonstrated in high abundance in the infant feces and urine. While the method was developed to examine specimens from preterm infants, it is of general utility and can be used to monitor oligosaccharide consumption and utilization in term infants, children, and adults. This method may therefore provide diagnostic and therapeutic opportunities.

A combinatorial chemistry approach to new materials for non‐linear optics. I. Five Schiff bases

A combinatorial chemistry approach has been used to synthesize an array of Schiff bases, five of which, namely N‐[(E,2E)‐3‐(4‐methoxy­phenyl)‐2‐propenyl­idene]‐3‐nitro­aniline, C₁₆H₁₄N₂O₃, (1a), N‐[(E,2E)‐3‐(4‐methoxy­phenyl)‐2‐propenyl­idene]‐4‐nitro­aniline, C₁₆H₁₄N₂O₃, (2a), N‐{(E,2E)‐3‐[4‐(di­methyl­amino)­phenyl]‐2‐propenyl­idene}‐3‐nitro­aniline, C₁₇H₁₇N₃O₂, (1b), N‐{(E,2E)‐3‐[4‐(di­methyl­amino)­phenyl]‐2‐propenyl­idene}‐4‐nitro­aniline, C₁₇H₁₇N₃O₂, (2b), and N‐{(E,2E)‐3‐[4‐(di­methyl­amino)­phenyl]‐2‐propenyl­idene}‐2‐methyl‐4‐nitro­aniline, C₁₈H₁₉N₃O₂, (3b), have been structurally characterized. A stack structure is observed for (1a) and (1b) in the crystal phase. Experimental and calculated molecular structures are discussed for these compounds which belong to a chemical class having potential applications as non‐linear optical materials.     

Hypercrosslinked polystyrene: A polymer in a non‐classical physical state

Deformation and relaxation properties of hypercrosslinked polystyrene networks have been studied by thermomechanical method at a uniaxial compression using individual spherical beads of the polymer. The networks examined were prepared by postcrosslinking of highly swollen beads of a styrene‐0.3% DVB copolymer with 0.3–0.75 mole of monochlorodimethyl ether, which results in the introduction of 0.6–1.5 methylene bridges between each two polystyrene phenyl rings. The polymers obtained are shown to belong neither to typical glassy materials, nor to typical elastomers. Though no characteristic plateau of rubberlike elasticity was observed on the deformation curves of the beads, the polymers exhibit two fundamentally important features of the rubberlike state: The deformations are large (up to 30–40% of the initial diameter) and reversible. Relaxation of residual deformations, however, requires prolonged heating of the sample, or a cycle of swelling and drying. The deformation can start in the temperature range from −70 to +150°C depending on pressure applied. The crosslinking degree in the range from 40–100% and higher does not affect noticeably the behavior of the hypercrosslinked polystyrene. Nature of the high mobility of the hypercrosslinked network is discussed. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 2324–2333, 1999     

HPLC‐MS/MS analysis of anthocyanins in human plasma and urine using protein precipitation and dilute‐and‐shoot sample preparation methods,respectively

A high‐performance liquid chromatography tandem–mass spectrometry (HPLC‐MS/MS) method has been developed to analyze anthocyanins in urine and plasma to further understand their absorption, distribution, metabolism and excretion. The method employed a Synergi RP‐Max column (250 × 4.6 mm, 4 μm) and an API 4000 mass spectrometer. A gradient elution system consisted of mobile phase A (water–1% formic acid) and mobile phase B (acetonitrile) with a flow rate of 0.60 mL/min. The gradient was initiated at 5% B, increased to 21% B at 20 min, and then increased to 40% B at 35 min. The analysis of anthocyanins presents a challenge because of the poor stability of anthocyanins during sample preparation, especially during solvent evaporation. In this method, the degradation of anthocyanins was minimized using protein precipitation and dilute‐and‐shoot and sample preparation methods for plasma and urine, respectively. No interferences were observed from endogenous compounds. The method has been used to analyze anthocyanin concentrations in urine and plasma samples from volunteers administered saskatoon berries. Cyanidin‐3‐galactoside, cyanidin‐3‐glucoside, cyanidin‐3‐arabinoside, cyanidin‐3‐xyloside and quercetin‐3‐galactoside, the five major flavonoid components in saskatoon berries, were identified in plasma and urine samples.