High-throughput Protein Analysis Research Articles

High throughput analysis of proteins associating with a proinvasive MT1‐MMP in human malignant melanoma A375 cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a powerful modulator of the pericellular environment, promotes migration, invasion, and proliferation of cells. To perform its potent proteolytic activity in a controlled manner, MT1-MMP has to be regulated precisely. However, our knowledge about substrates and regulatory proteins is still very limited. In this study we identify a catalog of proteins that directly or indirectly interact with MT1-MMP. We expressed a FLAG-tagged MT1-MMP stably in human malignant melanoma A375 cells. We prepared cell lysate using Brij98 and MT1-MMP was affinity purified together with associating proteins using an anti-FLAG antibody. A distinct set of membrane proteins was found to copurify with MT1-MMP when biotin-labeled proteins were monitored. The proteins were analyzed with an integrated system composed of nano-flow liquid chromatography and tandem mass spectrometry. We identified 158 proteins including several previously reported to bind MT1-MMP, although most had not previously been identified. Six of these membrane proteins, including one previously shown to interact with MT1-MMP, were co-expressed with MT1-MMP in HT1080 cells. Five of the latter were found to associate with MT1-MMP in an immunoprecipitation assay. Immunostaining of cells expressing each of these test proteins revealed that one colocalized with MT1-MMP at the ruffling membrane and the other at the perinuclear vesicles. In contrast, another protein which did not coprecipitate with MT1-MMP showed no colocalization. Recombinant MT1-MMP cleaved two of the tested proteins at least in vitro. Thus, we provide a valuable resource to identify substrates and regulators of MT1-MMP in tumor cells.

Protein microarrays: high-throughput tools for proteomics

Protein microarrays are versatile tools for parallel, miniaturized screening of binding events involving large numbers of immobilized proteins in a time- and cost-effective manner. They are increasingly applied for high-throughput protein analyses in many research areas, such as protein interactions, expression profiling and target discovery. While conventionally made by the spotting of purified proteins, recent advances in technology have made it possible to produce protein microarrays through in situ cell-free synthesis directly from corresponding DNA arrays. This article reviews recent developments in the generation of protein microarrays and their applications in proteomics and diagnostics.

Fiber-optic microsphere-based antibody array for the analysis of inflammatory cytokines in saliva.

Antibody microarrays have emerged as useful tools for high-throughput protein analysis and candidate biomarker screening. We describe here the development of a multiplexed microsphere-based antibody array capable of simultaneously measuring 10 inflammatory protein mediators. Cytokine-capture microspheres were fabricated by covalently coupling monoclonal antibodies specific for cytokines of interest to fluorescently encoded 3.1 microm polymer microspheres. An optical fiber bundle containing approximately 50,000 individual 3.1 microm diameter fibers was chemically etched to create microwells in which cytokine-capture microspheres could be deposited. Microspheres were randomly distributed in the wells to produce an antibody array for performing a multiplexed sandwich immunoassay. The array responded specifically to recombinant cytokine solutions in a concentration-dependent fashion. The array was also used to examine endogenous mediator patterns in saliva supernatants from patients with pulmonary inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). This array technology may prove useful as a laboratory-based platform for inflammatory disease research and diagnostics, and its small footprint could also enable integration into a microfluidic cassette for use in point-of-care testing.

Structure-effect relationship in the down-regulation of glutaminase in cultured human cells by phenylarsenic compounds

Diphenylarsinic acid [DPAA(V)] was detected in ground water used as drinking water after a poisonous incident in Kamisu, Japan. An approach to define the target molecules of DPAA(V) with a high throughput analysis of proteins from cultured human cells demonstrated down-regulation of glutaminase C (GAC). GAC is a splicing variant of the kidney-type glutaminase (KGA) gene and has the enzyme activity of phosphate-activated glutaminase (PAG). To gain some insights into the mechanism of arsenic intoxication in Kamisu, the effects of various arsenic compounds, including arsenicals that were detected in ground water ([DPAA(V)], phenylarsonic acid [PAA(V)] and bis(diphenylarsine)oxide [BDPAO(III)]) and rice (phenylmethylarsinic acid [PMAA(V)]), were investigated for the expression of GAC and PAG activity. When cultured human HepG2 cells were incubated with arsenicals for 24 h, the pentavalent phenylarsenic form of PAA(V) and PMAA(V) as well as DPAA(V) suppressed the expression of GAC protein and PAG activity in a concentration-dependent manner. On the other hand, the trivalent phenylarsenic form of BDPAO(III) had no suppressive effect on GAC and PAG. In addition, trivalent phenylarsenic compounds, such as the glutathione (GSH) conjugate of DPAA(V) [DPA-GS (III)] and triphenylarsine [TPA(III)], and the inorganic arsenics, iAs(V) and iAs(III), and methylated metabolites of inorganic arsenics, dimethylarsinic acid [DMA(V)] and dimethylarsinous acid [DMA(III)], had no suppressive effect on glutaminase. Likewise, methyl substituents of the hydroxyl groups of DPAA(V), PAA(V) and PMAA(V), diphenylmethylarsine oxide [DPMAO(V)] and phenyldimethylarsine oxide [PDMAO(V)], did not have any suppressive effects. These results suggest that pentavalent arsenic compounds with both phenyl groups and hydroxyl groups are effective in the suppression of glutaminase. In addition, the fact that it was only the arsenicals detected in Kamisu that were effective in suppressing glutaminase provides insights into the cause of the arsenic intoxication at Kamisu.

Taiwan Proteomics Society: An Organization Affiliated with HUPO

Taiwan Proteomics Society: An Organization Affiliated with HUPO

On-Chip, Cell-Based Microarray Immunofluorescence Assay for High-Throughput Analysis of Target Proteins

We have developed an immunofluorescence-based assay for high-throughput analysis of target proteins on a three-dimensional cellular microarray platform. This process integrates the use of three-dimensional cellular microarrays, which should better mimic the cellular microenvironment, with sensitive immunofluorescence detection and provides quantitative information on cell function. To demonstrate this assay platform, we examined the accumulation of the alpha subunit of the hypoxia-inducible factor (HIF-1alpha) after chemical stimulation of human pancreatic tumor cells encapsulated in 3D alginate spots in volumes as low as 60 nL. We also tested the effect of the known dysregulator of HIF-1alpha, 2-methoxyestradiol (2ME2), on the levels of HIF-1alpha using a dual microarray stamping technique. This chip-based in situ Western immunoassay protocol was able to provide quantitative information on cell function, namely, the cellular response to hypoxia mimicking conditions and the reduction of HIF-1alpha levels after cell treatment with 2ME2. This system is the first to enable high-content screening of cellular protein levels on a 3D human cell microarray platform.

Parallel post‐source decay for increasing protein identification confidence levels from 2‐D gels

Peptide mass fingerprinting (PMF) has over the years become one of the most commonly used tools for high-throughput analysis and identification of proteins. This method is applicable when relatively simple samples have to be analysed and it is commonly used for analysing proteins previously separated by 2-DE. The most common type of instrument used for this approach is the MALDI-TOF that has proved to be particularly suitable for the PMF analysis because of its characteristics of speed, robustness, sensitivity and automation. We have used a MALDI-TOF equipped with a novel parallel PSD capability (MALDI micro MX), to perform the analysis of two sets of different biological samples isolated by 2-DE. By using a method that integrates the data obtained by PMF analysis with the PSD data obtained in the same experiment, we show that the new multiplexed PSD solution increases the protein identification rate compared to the normal PMF approach. We also investigated the use of a charge-directed fragmentation modification reagent to improve the identification rate and confidence levels.

Contact spotting of protein microarrays coupled with spike‐in of normalizer protein permits time‐resolved analysis of ERBB receptor signaling

Protein microarrays allow highly accurate comparison and quantification of numerous biological samples in parallel while requiring only little material. This qualifies protein arrays for systems biology and clinical research where only limited sample material is available, but a precise readout is required. With the introduction of signal normalization steps to monitor the drop size of manually contact-spotted RP protein arrays, the usefulness of normalizer proteins to ensure a high-throughput but inexpensive protein analysis was demonstrated. This approach was applied for the analysis of signaling through ERBB receptor activated kinases in the breast cancer cell line MCF-7. Activation of ERK1/2 and AKT by ERBB1 (EGFR), ERRB2 (HER2/neu), and ERBB3-4 was monitored in a time-resolved manner. Analysis of pathway activation by stimulation with epidermal growth factor and heregulin, or inhibition by blocking with gefitinib or herceptin allowed a characterization of the distinct signaling properties of the different ERBB receptor subtypes.

350 – Judgment as a concept in psychopathology

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) being soft ionization technique, has become a method of choice for high-throughput analysis of proteins and peptides. In this study, we have explored the potential of atypical anti-psychotic drug olanzapine (OLZ) as a matrix for MALDI-MS analysis of peptides aided with the theoretical studies. Seven small peptides were employed as target analytes to check performance of olanzapine and compared with conventional MALDI matrix α-cyano-4-hydroxycinnamic acid (HCCA). All peptides were successfully detected when olanzapine was used as a matrix. Moreover, peptides angiotensin Ι and angiotensin ΙΙ were detected with better S/N ratio and resolution with this method as compared to their analysis by HCCA. Computational studies were performed to determine the thermochemical properties of olanzapine in order to further evaluate its similarity to MALDI matrices which were found in good agreement with the data of existing MALDI matrices.

Fully Automated Two-Dimensional Electrophoresis System for High-Throughput Protein Analysis

We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. The resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities.

Bacterial outer membrane protein analysis by electrophoresis and microchip technology

Outer membrane proteins are indispensable components of bacterial cells and participate in several relevant functions of the microorganisms. Changes in the outer membrane protein composition might alter antibiotic sensitivity and pathogenicity. Furthermore, the effects of various factors on outer membrane protein expression, such as antibiotic treatment, mutation, changes in the environment, lipopolysaccharide modification and biofilm formation, have been analyzed. Traditionally, the outer membrane protein profile determination was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Converting this technique to capillary electrophoresis format resulted in faster separation, lower sample consumption and automation. Coupling capillary electrophoresis with mass spectrometry enabled the fast identification of bacterial proteins, while immediate quantitative analysis permitted the determination of up- and downregulation of certain outer membrane proteins. Adapting capillary electrophoresis to microchip format ensured a further ten- to 100-fold decrease in separation time. Application of different separation techniques combined with various sensitive detector systems has ensured further opportunities in the field of high-throughput bacterial protein analysis. This review provides an overview using selected examples of outer membrane proteins and the development and application of the electrophoretic and microchip technologies for the analysis of these proteins.

Recent advances in cattle functional genomics and their application to beef quality

The advent of high-throughput DNA sequencing techniques, array technology and protein analysis has increased the efficiency of research in bovine muscle physiology, with the ultimate objective of improving beef quality either by breeding or rearing factors. For genetic purposes, polymorphisms in some key genes have been reported for their association with beef quality traits. The sequencing of the bovine genome has dramatically increased the number of available gene polymorphisms. The association of these new polymorphisms with the variability in beef quality (e.g. tenderness, marbling) for different breeds in different rearing systems will be a very important issue. For rearing purposes, global gene expression profiling at the mRNA or protein level has already shown that previously unsuspected genes may be associated either with muscle development or growth, and may lead to the development of new molecular indicators of tenderness or marbling. Some of these genes are specifically regulated by genetic and nutritional factors or differ between different beef cuts. In recognition of the potential economic benefits of genomics, public institutions in association with the beef industry are developing livestock genomics projects around the world. From the scientific, technical and economical points of view, genomics is thus reshaping research on beef quality.

Rapid and easy protein staining for SDS-PAGE using an intramolecular charge transfer-based fluorescent reagent

High-performance staining for 1-D and 2-D SDS-PAGE was carried out using a novel protein-binding fluorophore (Dye 1), which noncovalently interacts with proteins and provides a fluorescence emission response to proteins by intramolecular charge transfer. In order to achieve the high-throughput analysis of proteins for SDS-PAGE, the general protocols for in-gel protein staining (SDS-PAGE, fixation, staining, washing, and detection) were simplified to produce an easy and rapid protocol (SDS-PAGE together with staining, washing, and detection). This method was performed by preparation of an electrophoresis buffer containing Dye 1 under optimum conditions, and by the binding of Dye 1 to proteins in the gel during the SDS-PAGE. As a result, this study required only 15 min for protein staining as a minimum time. On the other hand, it takes several hours for the general protein staining method, such as SYPRO Ruby staining (18 h) and CBB staining (105 min). Moreover, the protein-to-protein variation was low, and the detection limit was 7.0 ng/band of BSA (S/N = 3.0) in this method, which was as sensitive as the short-protocol silver staining methods. On the basis of these results, this rapid and easy protocol for SDS-PAGE using Dye 1 may be widely applicable and convenient for users in the various scientific and medical fields.

Comparison of proteomic profiles of serum, plasma, and modified media supplements used for cell culture and expansion

BackgroundThe culture and expansion of human cells for clinical use requires the presence of human serum or plasma in culture media. Although these supplements have been extensively characterized in their chemical composition, only recently it has been possible to provide by high throughput protein analysis, a comprehensive profile of the soluble factors contributing to cell survival. This study analyzed and compared the presence of 100 proteins including chemokines, cytokines and soluble factors in six different types of media supplements: serum, plasma, recalcified plasma, heat inactivated serum, heat inactivated plasma and heat inactivated recalcified plasma.MethodsSerum, plasma, recalcified plasma, and heat inactivated supplements were prepared from ten healthy subjects. The levels of 100 soluble factors were measured in each sample using a multiplexed ELISA assay and compared by Eisen hierarchical clustering analysis.ResultsA comparison of serum and plasma levels of soluble factors found that 2 were greater in plasma but 18 factors were greater in serum including 11 chemokines. The levels of only four factors differed between recalcified plasma and plasma. Heat inactivation had the greatest effect on soluble factors. Supervised Eisen hierarchical clustering indicated that the differences between heat inactivated supplements and those that were not were greater than the differences within these two groups. The levels of 36 factors differed between heat inactivated plasma and plasma. Thirty one of these factors had a lower concentration in heat inactivated plasma including 12 chemokines, 4 growth factors, 4 matrix metalloproteases, and 3 adhesion molecules. Heat inactivated decalcified plasma is often used in place of heat inactivated serum and the levels of 19 soluble factors differed between these two supplements.ConclusionOur report provides a comprehensive protein profile of serum, plasma recalcified plasma, and heat inactivated supplements. This profile represents a qualitative and quantitative database that can aid in the selection of the appropriate blood derived supplement for human cell cultures with special requirements.

Assessment of the integral membrane protein topology in living cells

The bimolecular fluorescence complementation (BiFC) phenomenon has been successfully applied for in vivo protein-protein interaction studies and protein tagging analysis. Here we report a novel BiFC-based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non-fluorescent fragment of the yellow fluorescent protein (YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C-terminus or inserted into the internal loop(s). We employed this technique for topological studies of beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C-termini localized to the cytosol. We conclude that the BiFC-based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high-throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.

Application of proteomic strategies to the identification of urinary biomarkers for prostate cancer: A review

In the post-genomic era, genes and proteins are now studied on a more comprehensive scale. Studying disease processes at only the genetic or transcriptomic level will give an incomplete amount of information. A proteomic approach potentially allows for a more global overview of how disease processes affect the proteins present in cells, tissues and organisms. The challenge arises in determining which proteins are affected in specific diseases and establishing which of these changes are unique to a particular disease. Existing and emerging proteomic technologies allow for high throughput analysis of proteins in a variety of sample types. Prostate cancer is a significant male health problem in the Western world. It is widely accepted that more specific prognostic and diagnostic markers of prostate cancer are urgently required. The present paper suggests that urine may be an attractive biofluid in which to pursue the identification of novel biomarkers of prostate cancer. This review introduces some proteomic techniques including mass spectrometry and the newer, quantitative proteomic strategies. It focuses on the potential application of these platforms to novel urinary biomarker identification in prostate malignancy. It also includes a synopsis of the current literature on urinary proteomics.

A Pilot Application of SELDI-TOF Mass Spectrometry in Bone Marrow Failure Syndromes.

Myelodysplastic syndrome (MDS), aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are a heterogeneous group of disorders with significant clinical overlap. Current nosologic classifications schemes based mostly on morphology may not reflect pathophysiologic relationship between individual entities. The recent refinement of surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) technology allows for a high-throughput protein analysis of various biologic samples. Plasma samples from patients with MDS (N=134), AA (N=41), PNH (N=34), and healthy controls (N=75) were subjected to SELDI analyses using H4 chips. We limited analysis to spectra with more than 80 peaks. Reproducibility for each array run was monitored by repeating measurement of one standard sample on each chip. All spectra with peaks characterized by mass-to-charge ratios between 1.5–30 kDa were examined. Peaks with amplitude of at least > 3 times the average background noise were considered as meaningful. We had previously attempted SELDI analysis in bone marrow failure in a direct, manual, non-parametric fashion; this approach did not result in satisfactory discrimination. Here we have designed a new method utilizing a "binning" algorithm of peaks fulfilling selection criteria. A non-parametric approach was applied to select peaks within the bin defined by specific m/z amplitude. For each condition the "binned" signals were screened using Fisher's exact test to determine significant peaks; p<0.001 was used as the cutoff for selecting signals for further study. Logistic regression models with step-wise and backward variable selection was then used to determine peaks that had independent prognostic value and could therefore discriminate between the patient groups. The resulting model was validated internally using a bootstrap re-sampling procedure; random samples from the original population were drawn with replacement in such a way that the resulting "bootstrap population" had the same number of subjects as the original study population. This process was repeated 500 times and the frequency of each factor's inclusion in the resulting models was calculated. Factors present in ≥ 50% of the models were considered significant and used to build a final "bootstrap-based" model. Discriminating signals were identified and a scoring system was used to derive a score for each condition relative to controls (+1 for the presence of the signal in a bin being associated with the condition and −1 for the absence of the signal being associated with the condition). A score of 3 was used as a cutoff in MDS and a score of 2 in other diseases to differentiate from normal controls (Table 1). The observed discrimination obtained using only one chip surface and crude disease definitions encourages future studies. Currently non-overlapping sample sets are being generated for further analysis to identify proteomic patterns in specific cytogenetic defects and morphological categories; thereby validating the pilot results in more stringently defined disease phenotypes.Disease versus normal controlsGroupNumberScore + in %Score - in %psensitivity/specificityControl758317MDS1341783<0.00183%/83%Control758812AA412476<0.00176%/88%Control75928PNH342179<0.00179%/92%

Label-Free Detection of Proteins in Crude Cell Lysate with Antibody Arrays by a Surface Plasmon Resonance Imaging Technique

We established a label-free method of measuring proteins in crude cell lysate using antibody arrays and surface plasmon resonance (SPR) imaging. The refractivity of the running buffer was adjusted with that of the lysate to overcome the bulk effect. The chemistries of the fabricated arrays were investigated to reduce nonspecific adsorption on the array surface. We found that the hydrophilicity of the poly(ethylene glycol) moiety and lower electrostatic charge on the surface provided a specific measurement of antigen-antibody interaction. We validated the system by measuring the expression of eight proteins in the mouse brain and comparing the results to those by conventional Western blotting. The detection limit of the antibody array was approximately 30 ng/mL in crude cell lysate, on the same order as that of previous SPR research. The system enabled quick, label-free, and high-throughput analysis of abundant proteins with minimal sample volume ( approximately 200 muL). It is expected that our SPR antibody array will be applicable for direct protein expression profiling of cell lysate, as well as for cell phenotyping, food analysis, discovery of new biomarkers, and immunological disease diagnostics.

High-Throughput Protein and DNA Analysis Based on Microfluidic On-Chip Electrophoresis

Microfluidic technology applied to on-chip electrophoresis provides high-throughput DNA or protein analysis in an automated, unattended mode, which is currently not possible with any other technology. The 5100 Automated Lab-on-a-Chip Platform automates all the required experimental steps, including sample loading from multiple sample plates, electrophoresis, staining/destaining, and detection. The analysis of the digital data is completely automated as well and the results together with all other information, such as sample names, are directly fed into a database. The article describes in detail the design of the microfluidic system, including instrumentation, chips, DNA and protein assays, as well as the structure and the main features of the software.

Charge State Estimation for Tandem Mass Spectrometry Proteomics

High-throughput protein analysis by tandem mass spectrometry produces anywhere from thousands to millions of spectra that are being used for peptide and protein identifications. Though each spectrum corresponds only to one charged peptide (ion) state, repetitive database searches of multiple charge states are typically conducted since the resolution of many common mass spectrometers is not sufficient to determine the charge state. The resulting database searches are both error-prone and time-consuming. We describe a straightforward, accurate approach on charge state estimation (CHASTE). CHASTE relies on fragment ion peak distributions, and by using reliable logistic regression models, combines different measurements to improve its accuracy. CHASTE's performance has been validated on data sets, comprised of known peptide dissociation spectra, obtained by replicate analyses of our earlier developed protein standard mixture using ion trap mass spectrometers at different laboratories. CHASTE was able to reduce number of needed database searches by at least 60% and the number of redundant searches by at least 90% virtually without any informational loss. This greatly alleviates one of the major bottlenecks in high throughput peptide and protein identifications. Thresholds and parameter estimates can be tailored to specific analysis situations, pipelines, and instrumentations. CHASTE was implemented in Java GUI-based and command-line-based interfaces.