Comparison of commercial probe labeling kits for microarray: towards quality assurance and consistency of reactions

Microarray technology is readily available to scientists interested in gene expression. Commensurate with this availability is the growing market in accessory products offering convenience but potentially variable performance. Here we evaluate seven commercial kits for probe labeling against a human apoptosis oligonucleotide array. All kits were found to label probes successfully using the manufacturers' instructions. The Stratagene Fairplay Microarray Labeling Kit was the most sensitive, with an overall call rate of 74% and the lowest rate of indeterminant calls for the HEK and HepG2 cell lines. The Invitrogen SuperScript Indirect cDNA Labeling System showed the most reproducible gene expression pattern and the least technical variation, both in terms of signal strength and between replicates on each array. The Promega Pronto! Plus System showed the least dye bias however, a higher level of variation between replicates was observed. Pairwise comparisons revealed that the Promega Pronto! Plus System and Invitrogen SuperScript Indirect cDNA Labeling System had the most similarity in their patterns of gene expression. Results obtained suggest variability in the performance of commercial kits between different manufacturers. This study supports the need to conduct comparative evaluations of commercial microarray probe labeling kits and the need for validation prior to use.     

Design of recombinant antibody microarrays for membrane protein profiling of cell lysates and tissue extracts

Generating global protein expression profiles, including also membrane proteins, will be crucial for our understanding of biological processes in health and disease. In this study, we have expanded our antibody microarray technology platform and designed the first human recombinant antibody microarray for membrane proteins targeting crude cell lysates and tissue extracts. We have optimized all key technological parameters and successfully developed a setup for extracting, labeling and analyzing non-fractionated membrane proteomes under non-denaturing conditions. Finally, the platform was also extended and shown to be compatible with simultaneous profiling of both membrane proteins and water-soluble proteins.     

Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (101 cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 102 spores and 75 % for 101 spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.     

Methods for mapping of interaction networks involving membrane proteins

Nearly one-third of all genes in various organisms encode membrane-associated proteins that participate in numerous protein-protein interactions important to the processes of life. However, membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is limited with respect to the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.     

Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis

The solubilizing power of various nonionic and zwitterionic detergents as membrane protein solubilizers for two-dimensional electrophoresis was investigated. Human red blood cell ghosts and Arabidopsis thaliana leaf membrane proteins were used as model systems. Efficient detergents could be found in each class, i.e. with oligooxyethylene, sugar or sulfobetaine polar heads. Among the commercially available nonionic detergents, dodecyl maltoside and decaethylene glycol mono hexadecyl ether proved most efficient. They complement the more classical sulfobetaine detergents to widen the scope of useful detergents for the solubilization of membrane proteins in proteomics.     

Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples

Aim:  To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples.
Methods and Results:  Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples.
Conclusion:  Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples.
Significance and Impact of the Study:  The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.     

Comparison of novel and familiar commercial kits for detection of C-reactive protein levels

In this study, randomized patient sera were used to simultaneously evaluate an automated C-reactive protein (CRP) assay and a commercial semi-automated microCRP assay with respect to correlation, linearity, and accuracy. Patient specimens were analyzed; two independent assay runs were performed on i-CHROMA (Boditech Med Inc., Korea) and IMMAGE 800 (Beckman Coulter Inc., USA) analyzers to estimate the between- and within-run precision. All systems were calibrated, and quality-control materials were analyzed according to the manufacturer’s instructions. The results using the control materials were within the respective manufacturers’ specified limits. The comparison studies were designed using the CLSI EP9-2A guidelines. The mean serum CRP concentrations were 123.2 ± 123.5 mg/L (95 % confidence of interval (CI) 97.9–148.3) using the CRP assay and 130.1 ± 109.3 mg/L (95 % CI 107.9–152.4) using the microCRP assay. The variance values were σ = 15,252.6 and 11,935.8 for the CRP and microCRP assays, respectively. The concordance correlation coefficient value was calculated as 0.8314 (95 % CI 0.7594–0.8833). There was a significant correlation between the CRP and microCRP assays: r = 0.8392 and 95 % CI 0.7675–0.8902 (p < 0.0001). The CRP and microCRP detection methods were well correlated. The i-CHROMA has many advantages over the IMMAGE 800 with respect to space required, analysis time, and system setup/application costs in a laboratory. It may be an attractive instrument for small and intermediate medical centers.     

Targeting of the mitochondrial membrane proteins to the cell surface for functional studies

Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K(+) and Na(+) ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.     

Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues

A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA.     

Redesign of a plugged beta-barrel membrane protein

The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust β-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded β-barrel protein from the outer membrane of Escherichia coli. FhuA has a luminal cross-section of 3.1 × 4.4 nm and is filled by a globular N-terminal cork domain. Various redesigned FhuA proteins were investigated, including single, double, and multiple deletions of the large extracellular loops and the cork domain. We identified four large extracellular loops that partially occlude the lumen when the cork domain is removed. The newly engineered protein, FhuAΔC/Δ4L, was the result of a removal of almost one-third of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. This extensive protein engineering encompassed the entire cork domain and four extracellular loops. Remarkably, FhuAΔC/Δ4L forms a functional open pore in planar lipid bilayers, with a measured unitary conductance of ～4.8 nanosiemens, which is much greater than the values recorded previously with other engineered FhuA protein channels. There are numerous advantages and prospects of using such an engineered outer membrane protein not only in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating, but also in more applicative areas of stochastic single-molecule sensing of proteins and nucleic acids.     

Optimization of IPG strip equilibration for the basic membrane protein mABC1

Many proteins with extreme physical properties, including basic and acidic proteins, membrane proteins, and very large proteins, present specific challenges to 2-DE separation. Using a pressure-blotting approach, we demonstrate that a basic integral membrane protein, mitochondrial ATP-binding cassette protein 1 (mABC1), focuses in the IPG strip, but fails to enter into the 2-D SDS-PAGE gel. Through modifying the equilibration conditions between the IPG strip and 2nd dimension, we demonstrate that only by increasing both the volume (from 3 to 6 mL for a 7-cm strip) and SDS concentration (from 2 to 10%) of the equilibration buffer is migration of mABC1 into the 2nd dimension achieved. While 2-DE remains one of the core separation technologies of proteomic analysis, proteins that are extremely basic, hydrophobic, or of large mass present significant challenges to 2-DE separation due to aggregation, oxidation, precipitation, and the physical limitations of the 1-D IPG strip. Since the advent of commercially available IPG strips, numerous groups have experimented with IEF conditions using various detergents alone or in combination, alternative denaturants, and thiol oxidation agents to improve protein focusing. Effective 2-DE separation of membrane proteins has been affected dramatically by these advances in protein solubilization, as well as improvements in isolation of membranes, delipidation, and active in-gel rehydration. Since the development of commercially available basic IPG strips, the most significant advance in the separation of basic proteins has been the introduction of hydroxyethyldisulfides, either alone or in combination with DTT. While hydrophobic proteins were once virtually absent from the 2-D gel, and basic proteins were only visible as dense streaks, now many groups are undertaking large-scale analyses of membranes and basic proteins. Using this base of experimental tools, we embarked on a proteomic analysis of cardiac mitochondrial membranes, hoping to combine the knowledge gained from ongoing targeted protein chemistry and molecular biology studies with a broader-based proteomic analysis. Of particular interest is the inner mitochondrial membrane protein mABC1 (mitochondrial ATP-binding cassette protein 1), which may play a significant role in cardioprotection as part of the mitochondrial ATP-sensitive potassium channels. Therefore, in designing our 2-DE approach, it was crucial to ensure that mABC1 is focused, observable, and quantifiable, despite being an integral membrane protein of pI 9.37.     

Structural basis of outer membrane protein biogenesis in bacteria

In Escherichia coli, a multicomponent BAM (β-barrel assembly machinery) complex is responsible for recognition and assembly of outer membrane β-barrel proteins. The functionality of BAM in protein biogenesis is mainly orchestrated through the presence of two essential components, BamA and BamD. Here, we present crystal structures of four lipoproteins (BamB-E). Monomeric BamB and BamD proteins display scaffold architectures typically implied in transient protein interactions. BamB is a β-propeller protein comprising eight WD40 repeats. BamD shows an elongated fold on the basis of five tetratricopeptide repeats, three of which form the scaffold for protein recognition. The rod-shaped BamC protein has evolved through the gene duplication of two conserved domains known to mediate protein interactions in structurally related complexes. By contrast, the dimeric BamE is formed through a domain swap and indicates fold similarity to the β-lactamase inhibitor protein family, possibly integrating cell wall stability in BAM function. Structural and biochemical data show evidence for the specific recognition of amphipathic sequences through the tetratricopeptide repeat architecture of BamD. Collectively, our data advance the understanding of the BAM complex and highlight the functional importance of BamD in amphipathic outer membrane β-barrel protein motif recognition and protein delivery.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

Proteomics-based consensus prediction of protein retention in a bacterial membrane

The availability of complete bacterial genome sequences allows proteome-wide predictions of exported proteins that are potentially retained in the cytoplasmic membranes of the corresponding organisms. In practice, however, major problems are encountered with the computer-assisted distinction between (Sec-type) signal peptides that direct exported proteins into the growth medium and lipoprotein signal peptides or amino-terminal membrane anchors that cause protein retention in the membrane. In the present studies, which were aimed at improving methods to predict protein retention in the bacterial cytoplasmic membrane, we have compared sets of membrane-attached and extracellular proteins of Bacillus subtilis that were recently identified through proteomics approaches. The results showed that three classes of membrane-attached proteins can be distinguished. Two classes include 43 lipoproteins and 48 proteins with an amino-terminal transmembrane segment, respectively. Remarkably, a third class includes 31 proteins that remain membrane-retained despite the presence of typical Sec-type signal peptides with consensus signal peptidase recognition sites. This unprecedented finding indicates that unknown mechanisms are involved in membrane retention of this class of proteins. A further novelty is a consensus sequence indicative for release of certain lipoproteins from the membrane by proteolytic shaving. Finally, using non-overlapping sets of secreted and membrane-retained proteins, the accuracy of different signal peptide prediction algorithms was assessed. Accuracy for the prediction of protein retention in the membrane was increased to 82% using a majority-vote approach. Our findings provide important leads for future identification of surface proteins from pathogenic bacteria, which are attractive candidate infection markers and potential targets for drugs or vaccines.     

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%–19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.     

Comparison of metalloproteinase protein and activity profiling

Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The current study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer cell lines and 1 normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media (in both amount and variety). TIMP-1 was produced in all cell lines. MMP activity assays with three different fluorescence resonance energy transfer (FRET) substrates were then used to determine whether protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was confirmed only in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays.     

利用胶束电动毛细管电泳比较五种提取鲫鱼总蛋白方法研究

毛细管电泳又称高效毛细管电泳(High-performance capillary electrophoresis,HPCE),是 20 世纪 80 年代问世的一种高效液相分离法.