The diagnostic value of combined detection of genetic markers and serum protein markers on breast cancer

Objective: The research is to explore the diagnostic value of several detection methods including separated and combined detection of the related genes and related proteins of breast cancer and combined detection of all genetic markers and serum protein markers on breast cancer. Method: The mRNA level expression of the related genes of breast cancer was detected by FQ-PCR technique and the ratio of BRCA-1, Myc, C-erbB2 and β2 micro-globulin was used to express levels of BRCA-1, Myc and C-erbB2; the related proteins of breast cancer were detected through ELISA. Then the research data was analyzed by SPSS19.0 software with t-test as comparison method, and ROC curve was used to calculate the sensitivity, specificity and accuracy of the diagnostic models. Result: No difference can be found among the six indexes in the control group and benign breast tumor group while compared with the benign breast tumor group and the control group, the breast cancer group was significantly different from them; combined detection of genes and that of proteins were both superior to their separated detection; all-marker combined detection was superior to separated detection, which is consistent with combined detection of genes and proteins. Conclusion: More detection indexes will not necessarily outcome better detection effect. Hence, appropriate detection indexes and number are needed to achieve better diagnosis effect. In order to conduct more specific method, more test samples are needed for further researches.     

Telomerase catalyzed fluorescent probes for sensitive protein profiling based on one-dimensional microfluidic beads array

A nucleic acid-based signal amplified method for multiple proteins detection based on one-dimensional beads array using telomerase catalyzed fluorescent probes has been developed in this paper. The biotin labeled fluorescent probes were synthesized by telomerase in homogeneous solution. Approximately 360-480 fluorescein molecules were inserted in each probe. The limit of detection for p53 protein is 1.1 pM (S/N=3) and a 3 orders of linear dynamic range is obtained. The sensitivity is nearly two orders higher than the two-site "sandwich" immunoassay using the same platform. Using this method, cellular p53 protein contents of as few as 85 CNE2 cells per mul sample can be determined specifically. The expression changes of p53, c-myc and beta-actin in CNE2 cells were further examined as a function of anti-cancer drug treatment, and the results are consistent with our previous reports. Compared with immuno-polymerase chain reaction and immuno-rolling circle amplification, this method is simple, fast, cheap and suitable for multi-protein analysis. The multiplexed proteins profiling of cellular samples should facilitate the new opportunities to the fundamental research of tumor development and progression, especially to the low abundant tumor-associated proteins analysis.     

State-of-the-art in phosphoproteomics

Presently, phosphorylation of proteins is the most studied and best understood PTM. However, the analysis of phosphoproteins and phosphopeptides is still one of the most challenging tasks in contemporary proteome research. Since not every phosphoprotein is accessible by a certain method and identification of the phosphorylated amino acid residue is required in the majority of cases, various strategies for the detection and localization of phosphorylations have been developed. Identification and localization of protein phosphorylations is mostly done by MS nowadays but phosphoproteins and -peptides are often suppressed in comparison to the unphosphorylated species if measured in complex mixtures. Thus, the isolation of pure phosphopeptide samples is a main task. This review gives an overview over the most frequently used methods in isolation and detection of phosphoproteins and -peptides such as specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques.     

Albumin depletion of human plasma also removes low abundance proteins including the cytokines

The use of proteomics for efficient, accurate, and complete analysis of clinical samples poses a variety of technical challenges. The presence of higher abundance proteins in the plasma, such as albumin, may mask the detection of lower abundance proteins such as the cytokines. Methods have been proposed to deplete the sample of these higher abundance proteins to facilitate detection of those with lower abundance. In this study, a commercially available albumin depletion kit was used to determine if removal of albumin would measurably reduce detection of lower abundance cytokine proteins in human plasma. The Montage Albumin Deplete Kit (Millipore) was used to deplete albumin from LPS-stimulated whole blood from 15 normal human donors. Albumin depletion was measured using the BCG reagent and SDS-PAGE, and cytokine recovery was determined by a microassay immunoassay that measures both pro- and anti-inflammatory cytokines. Average albumin depletion from the samples was 72%. However, several cytokines were also significantly reduced when the albumin was removed from the plasma. Additionally, there was a variable reduction in cytokine recovery from a known mixture of cytokines in a minimal amount of plasma that were loaded onto the columns. These data demonstrate that there may be a non-specific loss of cytokines following albumin depletion, which may confound subsequent proteomic analysis.     

Fluorescence-mediated analysis of mitochondrial preprotein import in vitro

Mitochondrial biogenesis is a crucial element of the functional maintenance of a eukaryotic cell. The organelle must import the majority of its proteins from the cytosol where they are synthesized as precursors. In vitro import assays have been developed in which isolated mitochondria are incubated with precursor proteins, that are generated either by in vitro translation systems or by expression and purification as recombinant proteins. The detection of imported proteins is performed by autoradiography or by Western blot. We have now established a novel detection system for imported precursor proteins that is based on fluorescent labeling. We constructed a mitochondrial preprotein containing a C-terminal SNAP-tag that can label itself with a single fluorescein molecule in an enzymatic reaction. The fluorescent preproteins were efficiently imported into isolated mitochondria and showed kinetic behavior similar to that of standard preproteins. The fluorescence detection was sensitive and significantly faster than other comparable procedures. We also showed that precursor proteins containing a SNAP-tag domain could be successfully labeled in a postimport reaction in intact mitochondria. In summary, the use of a reporter domain modified with a fluorescent dye provides a novel, sensitive, and fast detection method to analyze the properties of the mitochondrial import reaction in vitro.     

Efficient isolation and elution of cellular proteins using aptamer-mediated protein precipitation assay

Protein precipitation is one of the most widely used methods for antigen detection and purification in biological research. We developed a reproducible aptamer-mediated magnetic protein precipitation method that is able to efficiently capture, purify and isolate the target proteins. We discovered DNA aptamers having individually high affinity and specificity against human epidermal growth factor receptor (EGFR) and human insulin receptor (INSR). Using aptamers and magnetic beads, we showed it is highly efficient technique to enrich endogenous proteins complex and is applicable to identify physiologically relevant protein–protein interactions with minimized nonspecific binding of proteins. The results presented here indicate that aptamers would be applicable as a useful and cost-effective tool to identify the presence of the particular target protein with their specific protein partners.     

蛋白免疫印迹法同时检测大、小分子蛋白的实验条件改进

目的:蛋白免疫印迹法是现代生物医学研究中广泛应用于蛋白定性和半定量分析的实验技术。然而,常规采用传统单一浓度凝胶的蛋白免疫印迹法在应用过程中仍有不足之处,如不能同时检测分子量很大和很小的蛋白,因而有必要探索一种增大凝胶有效分离范围的检测方法。本文提出采用组合凝胶来实现更大范围分子量蛋白的同时检测。方法:比较双浓度的组合凝胶与单一浓度凝胶的分离范围以及分析采用组合凝胶,蛋白免疫印迹法对大、小分子蛋白的检测效果。结果:12%/7.5%组合凝胶和15%/7.5%组合凝胶的分离范围显著大于相应的单一浓度凝胶。通过12%/7.5%组合凝胶,蛋白免疫印迹法同时检测到15-300 k Da范围内的大、小分子蛋白。结论:组合凝胶有助于蛋白免疫印迹法对分子量相差很大的蛋白进行同时检测分析,具有很强的实用性。     

蛋白质芯片技术进展

人类基因组测序工作的完成 ,引起人们对蛋白质组研究的热忱。蛋白质作为生命活动的执行者 ,种类繁多 ,结构复杂 ,并且其活性与空间结构密切相关 ,需要更为先进的技术去研究和探索。近来出现的蛋白质芯片以并行、高通量检测、分析和处理蛋白质样品 ,发展迅速 ,应用前景广泛。介绍蛋白质芯片的种类、蛋白质固定的表面化学以及不同的检测方法 ,简述蛋白质芯片在不同领域的应用 ,并讨论蛋白质芯片目前存在的问题。     

Sedimentation velocity analysis of amyloid oligomers and fibrils using fluorescence detection

The assembly of proteins into large fibrillar aggregates, known as amyloid fibrils, is associated with a number of common and debilitating diseases. In some cases, proteins deposit extracellularly, while in others the aggregation is intracellular. A common feature of these diseases is the presence of aggregates of different sizes, including mature fibrils, small oligomeric precursors, and other less well understood structural forms such as amorphous aggregates. These various species possess distinct biochemical, biophysical, and pathological properties. Here, we detail a number of techniques that can be employed to examine amyloid fibrils and oligomers using a fluorescence-detection system (FDS) coupled with the analytical ultracentrifuge. Sedimentation velocity analysis using fluorescence detection is a particularly useful method for resolving the complex heterogeneity present in amyloid systems and can be used to characterize aggregation in exceptional detail. Furthermore, the fluorescence detection module provides a number of particularly attractive features for the analysis of aggregating proteins. It expands the practical range of concentrations of aggregating proteins under study, which is useful for greater insight into the aggregation process. It also enables the assessment of aggregation behavior in complex biological solutions, such as cell lysates, and the assessment of processes that regulate in-cell or extracellular aggregation kinetics. Four methods of fluorescent detection that are compatible with the current generation of FDS instrumentation are described: (1) Detection of soluble amyloid fibrils using a covalently bound fluorophore. (2) Detection of amyloid fibrils using an extrinsic dye that emits fluorescence when bound to fibrils. (3) Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates. (4) Detection of green fluorescence protein (GFP) constructs and their interactions within mammalian cell lysates.     

Proteomic identification of Listeria monocytogenes surface‐associated proteins

This study aimed to identify proteins exposed on the surface of Listeria monocytogenes cells for diagnostic reagent development. Brief trypsin treatment of L. monocytogenes cells followed by peptide separation and identification by nano‐LC and online‐MS/MS was performed. In parallel, as a negative control, proteins secreted into the digest buffer as well as proteins from cell lysis were identified. One hundred and seventy‐four proteins were identified in at least two of three trials in either the negative control or during cell digest. Nineteen surface, 21 extracellularly secreted, 132 cytoplasmic, and two phage proteins were identified. Immunofluorescence microscopy of L. monocytogenes cells revealed the surface localization of two potential candidates for L. monocytogenes isolation and detection: lipoprotein LMOf2365_0546 and PBPD1 (LMOf2365_2742). In this report, we present the first data set of surface‐exposed L. monocytogenes proteins currently available. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000035.     

The movement of proteins across the insect and tick digestive system

The movement of intact proteins across the digestive system was shown in a number of different blood-feeding and non-blood-feeding insects in the orders Blattaria, Coleoptera, Diptera, Hemiptera, Lepidoptera, Orthoptera, Neuroptera and Siphonaptera, as well as in two tick families Ixodidae and Argasidae. Protein movement was observed for both normal dietary and xenobiotic proteins, which suggest that the mechanism for transfer is not substrate specific. The number of studies on the mechanism of movement is limited. The research so far suggests that movement can occur by either a transcellular or an intercellular pathway in the ventriculus with most of the research describing the former. Transfer is by continuous diffusion with no evidence of pinocytosis or vesicular transport common in mammalian systems. Proteins can move across the digestive system without modification of their primary or multimeric structure and with retention of their functional characteristics. Accumulation in the hemolymph is the result of the protein degradation rate in the gut and hemolymph and transfer rate across the digestive system and can be highly variable depending on species. Research on the development of delivery systems to enhance protein movement across the insect digestive system is in its infancy. The approaches so far considered with some success include the use of lipophilic-polyethylene glycol (PEG) polymers, the development of fusion proteins with lectins, reduced gut protease activity and the development of amphiphilic peptidic analogs. Additional research on understanding the basic mechanisms of protein delivery across the insect digestive system, the importance of structure activity in this transfer and the development of technology to improve movement across the gut could be highly significant to the future of protein and nucleic acid-based insecticide development as well as traditional chemical insecticidal technologies.     

Origins of globular structure in proteins

Since natural proteins are the products of a long evolutionary process, the structural properties of present-day proteins should depend not only on physico-chemical constraints, but also on evolutionary constraints. Here we propose a model for protein evolution, in which membranes play a key role as a scaffold for supporting the gradual evolution from flexible polypeptides to well-folded proteins. We suggest that the folding process of present-day globular proteins is a relic of this putative evolutionary process. To test the hypothesis that membranes once acted as a cradle for the folding of globular proteins, extensive research on membrane proteins and the interactions of globular proteins with membranes will be required.     

Quantum dot-based protein micro- and nanoarrays for detection of prostate cancer biomarkers

In this article, we demonstrate the fabrication and detection of cancer protein biochips consisting of micro- and nanoarrays whereby pegylated quantum dots (QDs) conjugated to antibodies (Abs) of prostate specific antigens (PSA) were used for the detection of clinical biomarkers such as PSA. BSA which acts as an efficient blocking layer in microarrays, tends to show an interaction with QDs. In view of this fact, we investigated two series of samples which were fabricated in the presence and absence of BSA blocking layer. Variation in the incubation time required for the antigen-antibody interaction to take place, different proteins as controls and the effect of bare QDs on these microarrays, were the three main parameters which were studied in these two series. Samples fabricated in the absence of BSA blocking layer exhibited an extremely high specificity in the detection of cancer proteins and were also marked by negligible nonspecific binding effects of QDs, in stark contrast to the samples fabricated using BSA as a blocking layer. Fabrication of nanoarrays of QD-conjugated PSA Abs having a spot size of nearly 900 nm has also been demonstrated. Thus, we show the potential offered by QDs in in vitro analysis of cancer biomarker imaging.     

蛋白质二级结构指定

蛋白质二级结构是指蛋白质骨架结构中有规律重复的构象。由蛋白质原子坐标正确地指定蛋白质二级结构是分析蛋白质结构与功能的基础,二级结构的指定对于蛋白质分类、蛋白质功能模体的发现以及理解蛋白质折叠机制有着重要的作用。并且蛋白质二级结构信息广泛应用到蛋白质分子可视化、蛋白质比对以及蛋白质结构预测中。目前有超过20种蛋白质二级结构指定方法,这些方法大体可以分为两大类:基于氢键和基于几何,不同方法指定结果之间的差异较大。由于尚没有蛋白质二级结构指定方法的综述文献,因此,本文主要介绍和总结已有蛋白质二级结构指定方法。     

Advances in the production of membrane proteins in Pichia pastoris

Membrane proteins play key roles in diverse cellular functions and have become the target for a large number of pharmacological drugs. Despite representing about 20-30% of cellular proteins, their characterization is long overdue since they are difficult to handle, to purify from their natural source or to obtain as recombinant proteins. Pichia pastoris is a methylotrophic yeast species increasingly used as a host for heterologous protein expression for both research and industrial purposes. Over the past few years many efforts have allowed important advances in the development of this expression system for the expression and production of membrane proteins. The most recent achievements in improving yield and proper folding of integral membrane proteins are summarized in this review.     

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.     

Comparison of diverse affinity based high-abundance protein depletion strategies for improved bio-marker discovery in oral fluids

Oral fluids (OF) have been suggested as a source of biomarkers for oral and systemic health, but as with other bio-fluids, the presence of high-abundance proteins interferes with the detection of lower-abundance biomarkers. Here, we compared the performance of four depletion treatments: triple depletion (TD) of amylases, albumins and immunoglobulin G; multiple depletion (MD) of amylases and a panel of 20 proteins, a combination of the two (EMD) and combinatorial peptide ligand library based depletion termed CPLL. TD, MD, EMD and CPLL removed 76%, 83%, 85% and 94% of total proteins, respectively, coupled with increased low abundance protein detection and narrowed dynamic range. 2-DE revealed that all depletion pretreatments successfully clarified areas hampered by high-abundance proteins; however, EMD and CPLL exposed the highest number of proteins. Quantitative MS of EMD samples relative to none treated samples indicated that most of downregulated proteins (>90%) were EMD target proteins. In conclusion, a multiple step EMD and CPLL depletion approaches bring about the highest number of protein detection ability and the best hampered-area clearance. As CPLL requires at least 10 fold more protein starting material, we suggest EMD pretreatment as a new detection tool in instances of low protein starting material.     

Tandem mass spectrometry for the detection of plant pathogenic fungi and the effects of database composition on protein inferences

LC-MS/MS has demonstrated potential for detecting plant pathogens. Unlike PCR or ELISA, LC-MS/MS does not require pathogen-specific reagents for the detection of pathogen-specific proteins and peptides. However, the MS/MS approach we and others have explored does require a protein sequence reference database and database-search software to interpret tandem mass spectra. To evaluate the limitations of database composition on pathogen identification, we analyzed proteins from cultured Ustilago maydis, Phytophthora sojae, Fusarium graminearum, and Rhizoctonia solani by LC-MS/MS. When the search database did not contain sequences for a target pathogen, or contained sequences to related pathogens, target pathogen spectra were reliably matched to protein sequences from nontarget organisms, giving an illusion that proteins from nontarget organisms were identified. Our analysis demonstrates that when database-search software is used as part of the identification process, a paradox exists whereby additional sequences needed to detect a wide variety of possible organisms may lead to more cross-species protein matches and misidentification of pathogens.