Selective fluorophore-assisted light inactivation of voltage-gated calcium channels

Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Cav 1.2 (L-type) and Cav 3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Cav 1.2 calcium channels containing EYFP-labeled α 1C subunits but does not affect the EYFP-α 1G Cav 3.1 calcium channels or Cav 1.2 channels containing EYFP-labeled β subunits. Our findings limit the applicability of acceptor photobleaching for the measurements of FRET but open an opportunity to combine the fluorescent imaging of the live cell expressing labeled calcium channels with selective functional inactivation of their specific subsets.     

Selective fluorophore-assisted light inactivation of voltage-gated calcium channels

Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Ca_v1.2 (L-type) and Ca_v3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Ca_v1.2 calcium channels containing EYFP-labeled α_1C subunits but does not affect the EYFP-α_1G Ca_v3.1 calcium channels or Ca_v1.2 channels containing EYFP-labeled β subunits. Our findings limit the applicability of acceptor photobleaching for the measurements of FRET but open an opportunity to combine the fluorescent imaging of the live cell expressing labeled calcium channels with selective functional inactivation of their specific subsets.     

Spatial specificity of chromophore assisted laser inactivation of protein function.

Chromophore assisted laser inactivation (CALI) is a new technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions. An understanding of aspects of the mechanism of CALI is required for its judicious application. A critical concern for CALI is its spatial specificity because nonspecific inactivation of neighboring unbound proteins by CALI is a possibility. We show here that CALI is very dependent on the distance between the chromophore and the protein such that there is no significant effect beyond 60 A. CALI using antibodies can inactivate other proteins through a complex but its efficacy decreases approximately fourfold for each intervening protein. These data imply that CALI is spatially specific and damage to neighboring proteins is unlikely.     

Fluorophore-assisted light inactivation of calmodulin involves singlet-oxygen mediated cross-linking and methionine oxidation

Fluorophore-assisted light inactivation (FALI) permits the targeted inactivation of tagged proteins and, when used with cell-permeable multiuse affinity probes (MAPs), offers important advantages in identifying physiological function, because targeted protein inactivation is possible with spatial and temporal control. However, reliable applications of FALI, also known as chromophore-assisted light inactivation (CALI) with fluorescein derivatives, have been limited by lack of mechanistic information regarding target protein sensitivity. To permit the rational inactivation of targeted proteins, we have identified the oxidizing species and the susceptibility of specific amino acids to modification using the calcium regulatory protein calmodulin (CaM) that, like many essential proteins, regulates signal transduction through the reversible association with a large number of target proteins. Following the covalent and rigid attachment of 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) to helix A, we have identified light-dependent oxidative modifications of endogenous methionines to their corresponding methionine sulfoxides. Initial rates of methionine oxidation correlate with surface accessibility and are insensitive to the distance between the bound fluorophore and individual methionines, which vary between approximately 7 and 40 A. In addition, we observed a loss of histidines, as well as zero-length cross-linking with binding partners corresponding to the CaM-binding sites of smooth myosin light chain kinase and ryanodine receptor. Our results provide a rationale for proteomic screens using FALI to inhibit the function of many signaling proteins, which, like CaM, commonly present methionines at binding interfaces.     

Development of peptide aptamer microarrays for detection of HPV16 oncoproteins in cell extracts

Protein microarrays represent an emerging technology that promises to facilitate high-throughput proteomics. The major goal of this technology is to employ peptides, full-length proteins, antibodies, and small molecules to simultaneously screen thousands of targets for potential protein–protein interactions or modifications of the proteome. This article describes the performance of a set of peptide aptamers specific for the human papillomavirus (HPV) type 16 oncoproteins E6 and E7 in a microarray format. E6 and E7 peptide aptamer microarrays were probed with fluorescence-labeled lysates generated from HPV-infected cervical keratinocytes expressing both E6 and E7 oncoproteins. Peptide aptamer microarrays are shown to detect low levels of E6 and E7 proteins. Peptide aptamers specific for cellular proteins included on these microarrays suggested that expression of CDK2, CDK4, and BCL-6 may be affected by HPV infection and genome integration. We conclude that peptide aptamer microarrays represent a promising tool for proteomics and may be of value in biological and clinical investigations of cervical carcinogenesis.     

High-throughput expression,purification, and characterization of recombinant Caenorhabditis elegans proteins

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.     

Analysis of the experimental detection of central nervous system-related genes in human brain and cerebrospinal fluid datasets

Large-scale and high-throughput proteomics experiments of specific samples provide substantial amounts of identified proteins and peptides, which increasingly find their way into centralized, public data repositories. These data typically have potential beyond the analyses performed by the original authors, and can therefore provide considerable added value by being reused for specific, unexplored enquiries. We here reanalyze two CNS-related proteomics datasets, one from the HUPO's Brain Proteome Project, and one from a comprehensive analysis of cerebrospinal fluid in light of the expression of specific splice isoforms from CNS-related genes. We also evaluate the empirically observed peptides of interest against predictions of their proteotypic character.     

A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli

Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.     

Isolation of rodent airway epithelial cell proteins facilitates in vivo proteomics studies of lung toxicity

Recent developments in genomics, proteomics, and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now considered standard procedure, but numerous publications reporting a lack of correlation between mRNA and protein expression emphasize the importance of conducting parallel proteomics studies. The cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell phenotypes are required. To address this issue, we have developed a novel method for isolation of rodent airway epithelial cell proteins that facilitates in vivo proteomics studies of two target-cell pheno-types of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly solubilized, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The method yields epithelial cell-specific proteins in fivefold higher concentrations and reduces the yield of nonepithelial cell proteins 13-fold compared with homogenates from microdissected airways. In addition, 36% more protein spots were detectable by two-dimensional gel electrophoresis.     

A simple affinity spin tube filter method for removing high-abundant common proteins or enriching low-abundant biomarkers for serum proteomic analysis

Although it is possible to identify new proteins from crude cell extracts using proteomics technology, it is often difficult to elucidate low-abundant biomarkers in the presence of a large amount of high-abundant proteins in serum. We have developed a simple and rapid method using an affinity spin tube filter to remove high-abundant common proteins and enrich the low-abundant biomarkers. The affinity spin tube filter contains protein G, coupled with antibodies against either high-abundant proteins or specific proteins of interest. After incubating with serum, the flow-through or the elute was collected and analyzed by two-dimensional gel electrophoresis. By using this affinity spin tube filter, the possibilities of identifying new biomarkers are shown. This technique could be used for large-scale sample preparation for high-throughput proteomic analysis.     

Applications of cell-based phage display panning to proteomic analysis

In the post-genomic era, proteomics together with genomic tools have led to powerful new strategies in basic and clinical research. These combined “omics” technologies are being integrated into the drug target discovery process. Unlike the genome, the proteome is a highly dynamic entity that requires techniques capable of analyzing on selected populations of proteins in specific biological conditions that reflect the proteins’ functional characteristics. Antibodies have become one of the most important reagents for the analysis of selected populations of proteins, and the application of phage-display antibody libraries to high-throughput antibody generation against large numbers of various antigens provides a tool for proteome-wide protein expression analysis. In this review, we will discuss the utility of phage-display antibodies in proteomics applications, specifically for the discovery of novel disease markers and therapeutic targets.     

p53-Dependent subcellular proteome localization following DNA damage

The nucleolus is involved in regulating several aspects of stress responses and cell cycle arrest through the tumor suppressor p53. Under normal conditions, p53 is a short-lived protein that is present in cells at a barely detectable level. Upon exposure of cells to various forms of exogenous stress, such as DNA damage, there is a stabilization of p53 which is then responsible for an ensuing cascade of events. To further investigate the effect of p53 activation, we used a MS-based proteomics method to provide an unbiased, quantitative and high-throughput approach for measuring the subcellular distribution of the proteome that is dependent on p53. The spatial proteomics method analyses a whole cell extract created by recombining differentially labeled subcellular fractions derived from cells in which proteins have been mass labeled with heavy isotopes [Boisvert, F.-M., Lam, Y. W., Lamont, D., Lamond, A. I., Mol. Cell. Proteomics 2010, 9, 457-470]. This was used here to measure the relative distribution between cytoplasm, nucleus and nucleolus of around 2000 proteins in HCT116 cells that are either expressing wild-type p53 or null for p53. Spatial proteomics also facilitates a proteome-wide comparison of changes in protein localization in response to a wide range of physiological and experimental perturbations. We used this method to study differences in protein localization in HCT116 cells either with or without p53, and studied the differences in cellular response to DNA damage following treatment of HCT116 cells with etoposide in both p53 wild-type and null genetic backgrounds.     

Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry

An important component of proteomic research is the high-throughput discovery of novel proteins and protein-protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.     

Experimental standards for high-throughput proteomics

Proteome analysis, utilizing high-throughput proteomics approaches, involves studying proteins that a whole organism (or specific tissue or cellular compartment) expresses under certain conditions. Intrinsic difficulties of these studies, as well as the enormous volumes of data they typically produce, make the proteome analysis and interpretation very difficult. As with any high-throughput approach, proteomics experiments should be carefully designed, analyzed, and verified. In addition to computational standards,experimental standards--simple and complex mixtures of known proteins--for high-throughput proteomics have to be developed and utilized. This article discusses such experimental standards and their implementations.     

Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry

An important component of proteomic research is the high-throughput discovery of novel proteins and protein–protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.     

Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.     

Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins within cells. Here, we demonstrate that GFP can be used as a CALI reagent to locally inactivate proteins in living cells. We show that focused laser irradiation of EGFP-alpha-actinin expressed in Swiss 3T3 fibroblasts results in the detachment of stress fibres from focal adhesions (FAs), whereas the integrity of FAs, as determined by interference reflection microscopy (IRM), is preserved. Moreover, consistent with a function for focal adhesion kinase (FAK) in FA signalling and not FA structure, laser irradiation of EGFP-FAK did not cause either visible FA damage or stress fibre detachment, although in vitro CALI of isolated EGFP-FAK decreased its kinase activity, but not its binding to paxillin. These data indicate that CALI of specific FA components may be used to precisely dissect the functional significance of individual proteins required for the maintenance of this cytoskeletal structure. In vitro CALI experiments also demonstrated a reduction of EGFP-alpha-actinin binding to the cytoplasmic domain of the beta(1) integrin subunit, but not to actin. Thus, alpha-actinin is essential for the binding of microfilaments to integrins in the FA. CALI-induced changes in alpha-actinin result in the breakage of that link and the subsequent retraction of the stress fibre.     

Cisplatin treatment leads to changes in nuclear protein and microRNA expression

Using a proteomic approach, we have previously shown that exposure to different concentrations of cisplatin during a 12-h period can lead to changes in nuclear protein expression and alternative splicing in HeLa cells. To further shed light on the DNA damage response (DDR) induced by cisplatin, we examined the nuclear proteome profiles of HeLa cells treated with 5μM cisplatin for different times (2, 12, and 24h). Two-dimensional electrophoresis (2-DE) identified 98 differentially expressed proteins in cisplatin-treated cells as compared to control cells. Among them, 54 spots (55%) were down-regulated and 44 spots (45%) were up-regulated. 51 spots were subjected to Matrix-assisted-laser-desorption-ionization Time-of-flight/time-of-flight Mass spectrometry (MALDI-TOF/TOF MS) identification, and 40 spots were identified. Among these, 22 proteins were located in nucleus. These proteins were involved in stress response, cell cycle and division, apoptosis, mRNA processing, transport, splicing and microRNA (miRNA) maturation. The changed expression of Annexin A1 and Lamin B1 were confirmed by Western blot. The role of Annexin A1 in the response to cisplatin-induced DNA damage was further analyzed, and it was shown that after Annexin A1 knockdown, cisplatin-induced DNA damage was significantly increased. In addition, the changed expression of several miRNAs was also observed by quantitative real-time PCR (qRT-PCR). Taken together, these data indicate that cisplatin-induced DDR is a complex process, and that those proteins identified by proteomics can lead to new directions for a better understanding of this process.     

Immunoisolation and subfractionation of synaptic vesicle proteins

Within recent years, the advances in proteomics techniques have resulted in considerable novel insights into the protein expression patterns of specific tissues, cells, and organelles. The information acquired from large-scale proteomics approaches indicated, however, that the proteomic analysis of whole cells or tissues is often not suited to fully unravel the proteomes of individual organellar constituents or to identify proteins that are present at low copy numbers. In addition, the identification of hydrophobic proteins is still a challenge. Therefore, the development of techniques applicable for the enrichment of low-abundance membrane proteins is essential for a comprehensive proteomic analysis. In addition to the enrichment of particular subcellular structures by subcellular fractionation, the spectrum of techniques applicable for proteomics research can be extended toward the separation of integral and peripheral membrane proteins using organic solvents, detergents, and detergent-based aqueous two-phase systems with water-soluble polymers. Here, we discuss the efficacy of a number of experimental protocols. We demonstrate that the appropriate selection of physicochemical conditions results in the isolation of synaptic vesicles of high purity whose proteome can be subfractionated into integral membrane proteins and soluble proteins by several phase separation techniques.