Applications In Protein Analysis Research Articles

A Novel Strategy Based on Permanent Protein Modifications Induced by Formaldehyde for Food Safety Analysis.

The illegal additions of chemicals in food products are serious incidents threatening current public safety. To date, ideal methods to determine permanent traces of prohibited chemicals in foods are still lacking. For example, formaldehyde (FA) can be added illegally as a food preservative. However, most current methods that are dependent on the direct detection of FA are not able to determine if FA has ever been added once food products are rinsed completely. Herein, we present a novel approach relying upon protein modifications induced by FA (PMIF) to examine FA in foods. We reveal the entire catalog of PMIFs in food products by combining mass spectrometry analysis with unrestrictive identification of protein modifications. Consequently, four obvious PMIFs were identified and confirmed as markers to discriminate the addition of FA in foods. Our study demonstrates that the approach based on PMIFs enables detecting the imprinted trace of FA even if the food products have been washed thoroughly. Our work presents a novel strategy for analysis of chemical additives, offering broad potential applications in protein analysis and food safety.

Bioinformatics Analysis of ABCA4 Variants in Terms of Pathogenicity Prediction

ABCA4 is a retina‐specific member of the ATP‐binding cassette transporter protein family. It is responsible for the transport and clearance of retinal derivatives produced in the visual cycle in the photoreceptor cells. More than a thousand mutations in the ABCA4 gene are associated with a series of autosomal recessive inherited retinal diseases, including Stargardt disease, retinitis pigmentosa, and cone‐rod dystrophy, and also linked with susceptibility for age‐related macular degeneration. Determining disease‐causing variants is a crucial part of clinical management, yet, a limited number of the ABCA4 variants have been studied in vitro and relatively few of them have been functionally characterized. Bioinformatics approaches can be highly effective to interpret the consequences of the mutations more rapidly. In this study, we aim to analyze the pathogenicity of ABCA4 clinical and prospective variants utilizing two methods to infer mutation effects: prediction software and computational protein models. We hypothesize that these two approaches in combination will allow us to identify the consequences of the ABCA4 variants and gain insight into the overall structure and function of the protein. We found that the pathogenicity prediction software results align well with the structural analysis results in most instances. The clinical in silico prediction tools were found to predict pathogenicity more frequently than structural analyses, albeit with high sensitivity and low specificity. On the other hand, structure prediction tools are high specificity and thus, predict more accurately. Our results also indicate a correlation between disease severity and structural changes in protein models induced by genetic variations. Our findings suggest that computational analyses are promising tools to predict pathogenicity and can aid in the understanding of disease‐associated ABCA4 genetic variants. These computational approaches have additional applications in protein analysis and provide a strong framework for future in vitro studies.

Broadband laser-based mid-IR spectroscopy for analysis of proteins and monitoring of enzyme activity

Laser-based infrared (IR) spectroscopy is an emerging key technology for the analysis of solutes and for real-time reaction monitoring in liquids. Larger applicable pathlengths compared to the traditional gold standard Fourier transform IR (FTIR) spectroscopy enable robust measurements of analytes in a strongly absorbing matrix such as water. Recent advancements in laser development also provide large accessible spectral coverage thus overcoming an inherent drawback of laser-based IR spectroscopy.In this work, we benchmark a commercial room temperature operated broadband external cavity-quantum cascade laser (EC-QCL)-IR spectrometer with a spectral coverage of 400 cm−1 against FTIR spectroscopy and showcase its application for measuring the secondary structure of proteins in water, and for monitoring the lipase-catalyzed saponification of triacetin. Regarding the obtained limit of detection (LOD), the laser-based spectrometer compared well to a research-grade FTIR spectrometer employing a liquid nitrogen cooled detector. With respect to a routine FTIR spectrometer equipped with a room temperature operated pyroelectric detector, a 15-fold increase in LOD was obtained in the spectral range of 1600–1700 cm−1. Characteristic spectral features in the amide I and amide II region of three representative proteins with different secondary structures could be measured at concentrations as low as 0.25 mg mL−1. Enzymatic hydrolysis of triacetin by lipase was monitored, demonstrating the advantage of a broad spectral coverage for following complex chemical reactions. The obtained results in combination with the portability and small footprint of the employed spectrometer opens a wide range of future applications in protein analysis and industrial process control, which cannot be readily met by FTIR spectroscopy without recurring to liquid nitrogen cooled detectors.

MAESTROweb: a web server for structure-based protein stability prediction.

The prediction of change in stability upon point mutations in proteins has many applications in protein analysis and engineering. We recently adjoined a new structure-based method called MAESTRO, which is distributed as command line program. We now provide access to the most important features of MAESTRO by an easy to use web service. MAESTROweb allows the prediction of change in stability for user-defined mutations, provides a scan functionality for the most (de)stabilizing n-point mutations for a maximum of n = 5, creates mutation sensitivity profiles and evaluates potential disulfide bonds. MAESTROweb operates on monomers, multimers and biological assemblies as defined by PDB. MAESTROweb is freely available for non-commercial use at https://biwww.che.sbg.ac.at/maestro/web peter.lackner@sbg.ac.at.

High-efficiency nano/micro-reactors for protein analysis

This article reviews the recent advances regarding the development of nanomaterial-based nanoreactors and microfluidic droplet reactors and their applications in protein analysis.

AN INTRODUCTION TO DATA CLUSTERING IN BIOINOFORMATICS

Experimental methods for genome analysis are of crucial interest and have recently made a considerable progress. However, most complete orders of genomes have at least a half the number of gens with unexplicit note. There are some new trends of research in infomatics to deal with data clustering and treating. In this article, we introduce general data clustering and its application in Protein analysis, an initial step which is highly significant for the experimental study of gemone analysis. This method helps to reduce to number of prediction experiment and to perfect Protein function. Key words: Cluster analysis, bioinformatics, Protein function prediction.

Preparation of highly hydrophilic strong cation exchangers and their applications in protein analysis

Based on the needs of new packing materials for rapid and efficient separation, purification and analysis of biomacromolecules, a novel sulfonic acid-type strong cation exchange resin (SP-G-PGMA SCX resin) was prepared. The porous poly(glycidyl methacrylate) microspheres (PGMA) were selected as the matrix and glucose was used as the hydrophilic modifier to block the hydrophobic domains of PGMA beads. Glucose modification on PGMA beads improved the biocompatibility and reduced the non-specific adsorption so as to increase the recoveries of protein. The PGMA beads possess the porous structure and the relatively high specific surface area, which make the PGMA-based resins good permeability and high loading capacity. The application of such SP-G-PGMA SCX resin for the chromatographic separation of biomacromolecules was explored. Four basic proteins were baseline separated within 6 min with the column size of 100 mm x 4.6 mm. The adsorption capacity of lysozyme on SP-G-PGMA SCX resin was determined as 39.5 g/L. The results make the material promising for the separation and purification of biomacromolecules.

A novel procedure for protein extraction from formalin‐fixed paraffin‐embedded tissues

Most of the archived pathological specimens in hospitals are kept as formalin-fixed paraffin-embedded tissues (FFPE) for long-term preservation. Up to now, these samples are only used for immunohistochemistry in a clinical routine as it is difficult to recover intact protein from these FFPE tissues. Here, we report a novel, short time-consuming and cost-effective method to extract full-length, non-degraded proteins from FFPE tissues. This procedure is combined with an effective and non-toxic deparaffinisation process and an extraction method based on antigen-retrieval, high concentration of SDS and high temperature. We have obtained enough intact protein to be detected by Western blotting analysis. This technique will allow utilising these stored FFPE tissues in several applications for protein analysis helping to advance the translational studies in cancer and other diseases.

Proteomics: Technologies for Protein Analysis

Proteomics technologies have produced an abundance of drug targets, which is creating a bottleneck in drug development process. There is an increasing need for better target validation for new drug development and proteomic technologies are contributing to it. Identifying a potential protein drug target within a cell is a major challenge in modern drug discovery; techniques for screening the proteome are, therefore, an important tool. Major difficulties for target identification include the separation of proteins and their detection. These technologies are compared to enable the selection of the one by matching the needs of a particular project. There are prospects for further improvement, and proteomics technologies will form an important addition to the existing genomic and chemical technologies for new target validation. Proteomics is applicable for protein analysis and bioinformatics based analysis gives the comprehensive molecular description of the actual protein component. Bioinformatics is being increasingly used to support target validation by providing functionally predictive information mined from databases and experimental datasets using a variety of computational tools. This review is focused on key technologies for proteomics strategy and their application in protein analysis.

Fiber‐packed channel bioreactor for microfluidic protein digestion

The fabrication and performance of a fiber-packed channel bioreactor in microchip along with its application in protein analysis were reported. The feasibility and performance of the unique microfluidic bioreactor were demonstrated by the tryptic digestion of myoglobin (MYO) and BSA. The on-chip digestion was carried out at a flow rate of 2.0 microL/min and the digestion time was significantly reduced to less than 5 s. The digests were identified by MALDI-TOF MS with sequence coverages of 66% (MYO) and 40% (BSA) that were comparable to those obtained by the conventional in-solution tryptic digestion. The present fiber-based microchip bioreactor provides a promising platform for the high-throughput protein identification.

Flow-through sampling for electrophoresis-based microchips and their applications for protein analysis.

This work presents a model behind the operation of a flow-through sampling chip and its application for immunoseparation, as well as its integration with a wash/elution bed for protein purification, concentration, and detection. This device used hydrodynamic pressure to drive the sample flow, and a gating voltage was applied to the electrophoretic channel on the microchip to control the sample loading for the separation and to inhibit sample leakage. The deduced model indicates that the critical gating voltage (VC) that is defined as the minimum gating voltage applied to the microchip for sampling is a function of the pump flow rate, the configuration of the microchannel on the chip, and the electroosmosis of the buffer solution. It was found that the theoretical V(C) values calculated from the measured electroosmotic mobilities and flow split ratios were comparable to those experimentally obtained from two microchips with different sampling channel sizes. This had an error percentage ranging from 1 to 20%. Because the hydrodynamic flow is insensitive to electrophoretic mobility, this electrophoresis-based microchip device was free of injection bias due to different ionic strength and electrophoretic mobility in the sample. Additionally, the usefulness of this device was demonstrated for the study of affinity interactions. Mixtures of Cy5-labeled bovine serum albumin (Cy5-BSA) and anti-BSA in various proportions were introduced into the microchip via a syringe pump, and the immunocomplex was electrophoretically separated from the free Cy5-BSA on the microchip. Based on the relative intensity of the free and complex BSA, the binding constant of BSA and anti-BSA was estimated as 3.3 x 10(7) M(-1). Furthermore, a C18 microcartridge (20 microL) was connected to the hydrodynamic inlet of the microchip. Using this device, the wash/elution step can be integrated on-line with the electrophoretic separation and detection on the microchip. Results show that the calibration curve of Cy5-BSA obtained from this integrated device has an R2 value greater than 0.99 and a minimum of quantitation at approximately 10 ng. This direct sampling method is another means of subfractionation, resulting in a relatively greater concentration factor than the average concentration of the whole fraction. Moreover, the electrical field-free bed ensures that the protein interaction will not be affected by the electric field during the wash/elution step.