用于串联质谱鉴定多肽的计量方法

目前已有多种对串联质谱与数据库中多肽的理论质谱的一致性进行评估的高通量计量算法用于鸟枪法蛋白质组学 (shotgunproteomics)研究。然而这些方法操作时存在大量错误的多肽鉴定。这里提出一种新的串联质谱识别多肽序列的计量算法。该算法综合考虑了串联质谱中不同离子出现的概率、多肽的酶切位点数、理论离子与实验离子的匹配程度和匹配模式。对大容量的串联质谱数据集的测试表明 ,根据算法开发的软件PepSearch比目前最常用的软件SEQUEST有更好的鉴定准确性。PepSearch可从http : compbio.sibsnet.org projects pepsearch下载。     

Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.     

Accurate Protein Complex Retrieval by Affinity Enrichment Mass Spectrometry (AE-MS) Rather than Affinity Purification Mass Spectrometry (AP-MS)

Protein–protein interactions are fundamental to the understanding of biological processes. Affinity purification coupled to mass spectrometry (AP-MS) is one of the most promising methods for their investigation. Previously, complexes were purified as much as possible, frequently followed by identification of individual gel bands. However, todays mass spectrometers are highly sensitive, and powerful quantitative proteomics strategies are available to distinguish true interactors from background binders. Here we describe a high performance affinity enrichment-mass spectrometry method for investigating protein–protein interactions, in which no attempt at purifying complexes to homogeneity is made. Instead, we developed analysis methods that take advantage of specific enrichment of interactors in the context of a large amount of unspecific background binders. We perform single-step affinity enrichment of endogenously expressed GFP-tagged proteins and their interactors in budding yeast, followed by single-run, intensity-based label-free quantitative LC-MS/MS analysis. Each pull-down contains around 2000 background binders, which are reinterpreted from troubling contaminants to crucial elements in a novel data analysis strategy. First the background serves for accurate normalization. Second, interacting proteins are not identified by comparison to a single untagged control strain, but instead to the other tagged strains. Third, potential interactors are further validated by their intensity profiles across all samples. We demonstrate the power of our AE-MS method using several well-known and challenging yeast complexes of various abundances. AE-MS is not only highly efficient and robust, but also cost effective, broadly applicable, and can be performed in any laboratory with access to high-resolution mass spectrometers.Protein–protein interactions are key to protein-mediated biological processes and influence all aspects of life. Therefore, considerable efforts have been dedicated to the mapping of protein–protein interactions. A classical experimental approach consists of co-immunoprecipitation of protein complexes combined with SDS-PAGE followed by Western blotting to identify complex members. More recently, high-throughput techniques have been introduced; among these affinity purification-mass spectrometry (AP-MS)¹ (1–3) and the yeast two-hybrid (Y2H) approach (4–6) are the most prominent. AP-MS, in particular, has great potential for detecting functional interactions under near-physiological conditions, and has already been employed for interactome mapping in several organisms (7–15). Various AP-MS approaches have evolved over time, that differ in expression, tagging, and affinity purification of the bait protein; fractionation, LC-MS measurement, and quantification of the sample; and in data analysis. Recent progress in the AP-MS field has been driven by two factors: A new generation of mass spectrometers (16) providing higher sequencing speed, sensitivity, and mass accuracy, and the development of quantitative MS strategies.In the early days of AP-MS, tagged bait proteins were mostly overexpressed, enhancing their recovery in the pull-down. However, overexpression comes at the cost of obscuring the true situation in the cell, potentially leading to the detection of false interactions (17). Today, increased MS instrument power helps in the detection of bait proteins and interactors expressed at endogenous levels, augmenting the chances to detect functional interactions. In some simple organisms like yeast, genes of interest can directly be tagged in their genetic loci and expressed under their native promoter. In higher organisms, tagging proteins in their endogenous locus is more challenging, but also for mammalian cells, methods for close to endogenous expression are available. For instance, in controlled inducible expression systems, the concentration of the tagged bait protein can be titrated to close to endogenous levels (18). A very powerful approach is BAC transgenomics (19), as used in our QUBIC protocol (20), where a bacterial artificial chromosome (BAC) containing a tagged version of the gene of interest including all regulatory sequences and the natural promoter is stably transfected into a host cell line.The affinity purification step has also been subject to substantial changes over time. Previously, AP has been combined with nonquantitative MS as the readout, meaning all proteins identified by MS were considered potential interactors. Therefore, to reduce co-purifying “contaminants,” stringent two-step AP protocols using dual affinity tags like the TAP-tag (21) had to be employed. However, such stringent and multistep protocols can result in the loss of weak or transient interactors (3), whereas laborious and partially subjective filtering still has to be applied to clean up the list of identified proteins. The introduction of quantitative mass spectrometry (22–25) to the interactomics field about ten years ago was a paradigm shift, as it offered a proper way of dealing with unspecific binding and true interactors could be directly distinguished from background binders (26, 27). Importantly, quantification enables the detection of true interactors even under low-stringent conditions (28). In turn, this allowed the return to single-step AP protocols, which are milder and faster, and hence more suitable for detecting weak and transient interactors.Despite these advances, nonquantitative methods—often in combination with the TAP-tagging approach—are still popular and widely used, presumably because of reagent expenses and labeling protocols used in label-based approaches. However, there are ways to determine relative protein abundances in a label-free format. A simple, semiquantitative label-free way to estimate protein abundance is spectral counting (29). Another relative label-free quantification strategy is based on peptide intensities (30). In recent years high resolution MS has become much more widely accessible and there has been great progress in intensity-based label-free quantification (LFQ) approaches. Together with development of sophisticated LFQ algorithms, this has boosted obtainable accuracy. Intensity-based LFQ now offers a viable and cost-effective alternative to label-based methods in most applications (31). The potential of intensity-based LFQ approaches as tools for investigating protein–protein interactions has already been demonstrated by us (20, 32, 33) and others (34, 35). We have further refined intensity-based LFQ in the context of the MaxQuant framework (36) using sophisticated normalization algorithms, achieving excellent accuracy and robustness of the measured “MaxLFQ” intensities (37).Another important advance in AP-MS, again enabled by increased MS instrument power, was the development of single-shot LC-MS methods with comprehensive coverage. Instead of extensive fractionation, which was previously needed to reduce sample complexity, nowadays even entire model proteomes can be measured in single LC-MS runs (38). The protein mixture resulting from pull-downs is naturally of lower complexity compared with the entire proteome. Therefore, modern MS obviates the need for gel-based (or other) fractionation and samples can be analyzed in single runs. Apart from avoiding selection of gel bands by visual examination, this has many advantages, including decreased sample preparation and measurement time, increased sensitivity, and higher quantitative accuracy in a label-free format.In this work, we build on many of the recent advances in the field to establish a state of the art LFQ AE-MS method. Based on our previous QUBIC pipeline (20), we developed an approach for investigating protein–protein interactions, which we exemplify in Saccharomyces cerevisiae. We extended the data analysis pipeline to extract the wealth of information contained in the LFQ data, by establishing a novel concept that specifically makes use of the signature of background binders instead of eliminating them from the data set. The large amount of unspecific binders detected in our experiments rendered the use of a classic untagged control strain unnecessary and enabled comparing to a control group consisting of many unrelated pull-downs instead. Our protocol is generic, practical, and fast, uses low input amounts, and identifies interactors with high confidence. We propose that single-step pull-down experiments, especially when coupled to high-sensitivity MS, should now be regarded as affinity enrichment rather than affinity purification methods.     

Targeting Ferroptosis as a Novel Approach to Alleviate Aortic Dissection

A variety of programmed cell death types have been shown to participate in the loss of smooth muscle cells (SMCs) during the development of aortic dissection (AD), but it is still largely unclear whether ferroptosis is involved in the development of AD. In the present study, we found that the expression of key ferroptosis regulatory proteins, solute carrier family 7 member 11 (SLC7A11), ferroptosis suppressor protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) were downregulated in aortas of Stanford type A AD (TAAD) patients, and liproxstatin-1, a specific inhibitor of ferroptosis, obviously abolished the β-aminopropionitrile (BAPN)-induced development and rupture of AD in mice. Furthermore, the expression of methyltransferase-like 3 (METTL3), a major methyltransferase of RNA m⁶A, was remarkably upregulated in the aortas of TAAD patients, and the protein levels of METTL3 were negatively correlated with SLC7A11 and FSP1 levels in human aortas. Overexpression of METTL3 in human aortic SMCs (HASMCs) inhibited, while METTL3 knockdown promoted SLC7A11 and FSP1 expression. More importantly, overexpression of METTL3 facilitated imidazole ketone erastin- and cystine deprivation-induced ferroptosis, while knockdown of METTL3 repressed ferroptosis of HASMCs. Overexpression of either SLC7A11 or FSP1 largely abrogated the effect of METTL3 on HASMC ferroptosis. Therefore, we have revealed that ferroptosis is a critical cause of AD in both humans and mice and that METTL3 promotes ferroptosis of HASMCs by inhibiting the expression of SLC7A11 and FSP1. Thus, targeting ferroptosis or m⁶A RNA methylation is a potential novel strategy for the treatment of AD.     

Novel Cartilage Oligomeric Matrix Protein (COMP) Neoepitopes Identified in Synovial Fluids from Patients with Joint Diseases Using Affinity Chromatography and Mass Spectrometry

To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDS-PAGE followed by in-gel digestion and mass spectrometric identification and characterization. Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser⁷⁷, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser⁷⁷ were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.     

Nucleic Acids in Protein Samples Interfere with Phosphopeptide Identification by Immobilized-Metal-Ion Affinity Chromatography and Mass Spectrometry

Immobilized-metal-ion affinity chromatography (IMAC) is used extensively for phosphopeptide enrichment in phosphoproteomics. However, the effect of nucleic acids in protein samples on phosphopeptide enrichment by IMAC has not yet been well clarified. In this study, we demonstrate that IMAC beads possess a strong adsorption of nucleic acids, especially single-stranded or single-stranded-region-containing nucleic acids, leading to approximately 50% loss of phosphopeptides during the process of IMAC enrichment. Therefore, nucleic acids must be removed from protein samples prior to IMAC. Acetonitrile (ACN) precipitation, a simple and efficient procedure, was established to remove nucleic acids from the protein samples. We showed that ACN precipitation approximately doubled the phosphopeptide number identified by IMAC and mass spectrometry, indicating that nucleic acid removal significantly improves the identification of phosphopeptides. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems^{1, 2}. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria^1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes^{1, 2, 7}. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast^{8, 9}, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system¹⁰. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.     

An Integrated Approach of Differential Mass Spectrometry and Gene Ontology Analysis Identified Novel Proteins Regulating Neuronal Differentiation and Survival

MS-based quantitative proteomics is widely used for large scale identification of proteins. However, an integrated approach that offers comprehensive proteome coverage, a tool for the quick categorization of the identified proteins, and a standardized biological study method is needed for helping the researcher focus on investigating the proteins with biologically important functions. In this study, we utilized isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative differential LC/MS/MS, functional annotation with a proprietary gene ontology tool (Molecular Annotation by Gene Ontology (MANGO)), and standard biochemical methods to identify proteins related to neuronal differentiation in nerve growth factor-treated rat pheochromocytoma (PC12) cells, which serve as a representative model system for studying neuronal biological processes. We performed MS analysis by using both nano-LC-MALDI-MS/MS and nano-LC-ESI-MS/MS for maximal proteome coverage. Of 1,482 non-redundant proteins semiquantitatively identified, 72 were differentially expressed with 39 up- and 33 down-regulated, including 64 novel nerve growth factor-responsive PC12 proteins. Gene ontology analysis of the differentially expressed proteins by MANGO indicated with statistical significance that the up-regulated proteins were mostly related to the biological processes of cell morphogenesis, apoptosis/survival, and cell differentiation. Some of the up-regulated proteins of unknown function, such as PAIRBP1, translationally controlled tumor protein, prothymosin α, and MAGED1, were further analyzed to validate their significant functions in neuronal differentiation by immunoblotting and immunocytochemistry using each antibody combined with a specific short interfering RNA technique. Knockdown of these proteins caused abnormal cell morphological changes, inhibition of neurite formation, and cell death during each course of the differentiation, confirming their important roles in neurite formation and survival of PC12 cells. These results show that our iTRAQ-MANGO-biological analysis framework, which integrates a number of standard proteomics strategies, is effective for targeting and elucidating the functions of proteins involved in the cellular biological process being studied.MS-based quantitative proteomics strategies such as iTRAQ¹ (1) and stable isotope labeling with amino acids in cell culture (2) are powerfully effective for the comprehensive characterization of biological phenomena (1–5). Although these methods have been applied for cancer biomarker (6, 7) and drug target (8) discovery, their use in the elucidation of biological and functional processes has been limited because of certain technical problems that arise when attempting to meaningfully process the immense amount of data obtained from such experiments. The following four main issues are typically the sources of such difficulties. 1) Quantitative identification by one type of MS system may fail to cover the total proteome because of ionization efficiency differences, such as those between ESI and MALDI, for certain peptides, leading to theoretical limitations in proteome coverage. 2) The public protein databases are often insufficient for searching non-human species because of the limited available genomic information. 3) The identification of the functions and biological processes of thousands of proteins is a formidable task because of the lack of simple and user-friendly software to automate gene ontology (GO) annotation. Furthermore it is difficult to convert large lists of taxonomically diverse proteins into their human orthologs to obtain the richest GO information available. 4) Lastly biological validation strategies for identified proteins have not been standardized. Therefore, we believe an analysis framework that provides (a) comprehensive proteome data; (b) a simple and quick tool for organizing, enriching, and sorting those data to reveal candidate molecules for relation to certain processes; and (c) a standardized biological validation technique would greatly benefit this field. We therefore designed a concise, three-step, sequential proteomics strategy that addresses the above concerns and utilized it successfully in studying the mechanism of neuronal differentiation in PC12 cells.PC12 cells (9) have been widely used as a model of neurons because of their unique advantages, such as stability, homogeneity, strong nerve growth factor (NGF) responsiveness, high differentiation potential, and a wealth of accessible background material, which help to facilitate their manipulation (10). This cell line has also been used for studying the mechanisms of neuronal disorders such as Alzheimer (11), Huntington (12), and Parkinson diseases (13) and neurofibromatosis type 1 (14–16). Here we used PC12 cells as a model for characterizing the mechanisms of neuronal differentiation and neurodegenerative disorders by means of MS-based quantitative proteomics.NGF is one member of a family of structurally and functionally related dimeric polypeptides, neurotrophins, that are essential for the development and maintenance of distinct neuronal populations in the central and peripheral nervous systems (17). The initial signaling cascades in the neuronal cells right after NGF stimulation have been subjected to thorough investigation and characterization by using PC12 cells. After binding of extracellular NGF to the cell membrane-localized tropomyosin-related kinase A (TrkA) receptor, TrkA receptors dimerize and subsequently autophosphorylate each other. Then the phosphorylated receptors recruit a complex of signaling molecules and induce a number of intracellular signaling cascades involving the signaling molecules, such as phosphoinositide 3-kinase, phospholipase C-γ, and Ras (18). The posttranslational modifications, such as phosphorylation cascades, triggered by NGF stimulation play important roles in PC12 cell differentiation. However, knowledge of the precise dynamic molecular events of protein expression in response to NGF signaling in PC12 cells after an interval that allows the stimulation to take full effect and produce morphological changes remains far from complete.Several reported studies have applied such methods as expressed sequence tag (19), restriction landmark cDNA scanning (20), targeted display (21), serial analysis of gene expression (22), and cDNA microarray (23) to survey the global change of differentially expressed genes in PC12 cells before and after NGF treatment (19–23). However, the genes and underlying mechanisms associated with the acquisition of a neuronal phenotype in these cells have not been clarified. Also a few proteomics approaches have been used for identifying the proteins related to NGF-inducible neurite formation in PC12 cells. For example, 2-D electrophoresis was applied in whole-cell extract separation to study the NGF modulation of protein synthesis (24); however, only two peptides were identified (25). Even currently available PC12 cell 2-D databases include merely a few proteins related to NGF stimulation (26–29). There is thus a paucity of functional proteomic information related to PC12 cell biological processes that may be attributed to technical limitations such as those listed above.In this study, we performed the first proteomics survey of proteins differentially expressed in PC12 cells during NGF treatment by using a semiquantitative differential LC shotgun method, namely isobaric tagging for relative and absolute quantitation (iTRAQ) coupled with concurrent use of two tandem MS/MS systems, namely nano-LC-MALDI-TOF-TOF and nano-LC-ESI-Quadrupole/quadrupole/time-of-flight mass spectrometers. The total list of proteins identified was converted into a new file linked to the GO database by our proprietary GO analysis tool for proteomes (MANGO) and categorized by biological process and function using specific classification methods. Thereafter we classified the subset of proteins that were up- or down-regulated during neurite formation into specific molecular categories by combining the differential data obtained by iTRAQ with the proteomic GO analysis results. We then attempted to characterize the functional mechanism of NGF-induced PC12 cell neuronal differentiation. Interestingly the specific up-regulated groups classified in this study were related to apoptosis/cell survival in addition to cell motility, differentiation, stress stimulation, and morphogenesis. To investigate the molecular functions of the up-regulated proteins in relation to both PC12 cell differentiation and apoptosis/survival during neurite formation, some of them were further analyzed with a biochemical and cellular biological strategy using a combined antibody and siRNA technique. Lastly we demonstrated the advantages that our concise, sequential proteomics strategy offers for studying the molecular mechanisms of cellular biological events such as cell differentiation and survival/apoptosis.     

A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria

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Highlights

• •XL-MS reveals new PPIs in yeast mitochondria under glycerol and glucose condition.
• •Significant but limited results from quantitative XL-MS experiments.
• •Ndi1 participates in a CIII₂CIV₂ respiratory supercomplex.
• •Min8 promotes assembly of Cox12 into an intermediate complex IV.

     

Mass Spectrometry Metabolomics Approach Reveals Anti-Trichomonas vaginalis Scaffolds from Marine Fungi

Marine Biotechnology - Trichomoniasis is the most common non-viral sexually transmitted infection (STI) in the world caused by Trichomonas vaginalis. Failures in the treatment with the...     

Comparative Proteomics Studies of Insect Cuticle by Tandem Mass Spectrometry: Application of a Novel Proteomics Approach to the Pea Aphid Cuticular Proteins

The cuticle is a biological composite material consisting principally of N‐acetylglucosamine polymer embedded in cuticular proteins (CPs). CPs have been studied and characterized by mass spectrometry in several cuticular structures and in many arthropods. Such analyses were carried out by protein extraction using SDS followed by electrophoresis, allowing detection and identification of numerous CPs. To build a repertoire of cuticular structures from Bombyx mori, Apis mellifera and Anopheles gambiae the use of SDS and electrophoresis was avoided. Using the combination of hexafluoroisopropanol and of a surfactant compatible with MS, a high number of CPs was identified in An. gambiae wings, legs and antennae, and in the thoracic integument cuticle of Ap. mellifera pupae. The exoskeleton analysis of B. mori larvae allowed to identify 85 CPs from a single larva. Finally, the novel proteomics approach was tested on cuticles left behind after the molt from the fourth instar of Acyrthosiphon pisum. Analysis of these cast cuticles allowed to identify 100 Ac. pisum CPs as authentic cuticle constituents. These correspond to 68% of the total putative CPs previously annotated for this pea aphid. While this paper analyzes only the recovered cuticular proteins, peptides from many other proteins were also detected.     

The cAMP Capture Compound Mass Spectrometry as a Novel Tool for Targeting cAMP-binding Proteins: FROM PROTEIN KINASE A TO POTASSIUM/SODIUM HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED CHANNELS

The profiling of subproteomes from complex mixtures on the basis of small molecule interactions shared by members of protein families or small molecule interaction domains present in a subset of proteins is an increasingly important approach in functional proteomics. Capture Compound^TM Mass Spectrometry (CCMS) is a novel technology to address this issue. CCs are trifunctional molecules that accomplish the reversible binding of target protein families to a selectivity group (small molecule), covalent capturing of the bound proteins by photoactivated cross-linking through a reactivity group, and pullout of the small molecule-protein complexes through a sorting function, e.g. biotin. Here we present the design, synthesis, and application of a new Capture Compound to target and identify cAMP-binding proteins in complex protein mixtures. Starting with modest amounts of total protein mixture (65–500 μg), we demonstrate that the cAMP-CCs can be used to isolate bona fide cAMP-binding proteins from lysates of Escherichia coli, mammalian HepG2 cells, and subcellular fractions of mammalian brain, respectively. The identified proteins captured by the cAMP-CCs range from soluble cAMP-binding proteins, such as the catabolite gene activator protein from E. coli and regulatory subunits of protein kinase A from mammalian systems, to cAMP-activated potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels from neuronal membranes and specifically synaptosomal fractions from rat brain. The latter group of proteins has never been identified before in any small molecule protein interaction and mass spectrometry-based proteomics study. Given the modest amount of protein input required, we expect that CCMS using the cAMP-CCs provides a unique tool for profiling cAMP-binding proteins from proteome samples of limited abundance, such as tissue biopsies.cAMP is an important biological second messenger molecule involved in many biological processes, such as adaptation of bacteria to low glucose growing conditions, chemotaxis in slime molds, and various signal transduction processes in metazoa downstream of the activation of hormone receptors (1). The concentration level of cAMP in biological systems is tightly controlled by the activity of adenylyl cyclases that catalyze the formation of cAMP and by the activity of phosphodiesterases, which catalyze the degradation of cAMP. Given the importance of signaling cascades downstream of hormone or neurotransmitter receptors that involve increased formation or degradation of cAMP, the identification and profiling of cAMP effector proteins can be expected to be an essential contribution to elucidate the molecular basis of physiological as well as pathophysiological signaling events.Bona fide effectors of cAMP are proteins that contain a cyclic nucleotide binding domain (CNBD).1 This motif represents a protein domain initially defined and characterized by the crystal structure of the major known cAMP-binding protein from Escherichia coli, the catabolite gene activator protein (2). This domain is present in all known mammalian cAMP-binding proteins as well. Three major classes of proteins exist that contain CNBDs. The first group contains protein kinase A subunits, namely regulatory subunits of protein kinase A isozymes (3), as well as the cGMP-dependent protein kinases (4). A group of Rap guanine nucleotide exchange factors (Epac proteins) that contain CNBDs (5) comprises the second group. Both groups contain key proteins involved in signaling cascades. A number of ion channels that can be directly regulated by cAMP contain CNBDs, such as the cyclic nucleotide-gated channels (6), make up the third group. In particular, potassium/sodium hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a crucial role in the pacemaking of heart and brain activity (7). A relatively small number of further proteins that contain CNBDs, such as phosphodiesterase isoforms and a sodium-hydrogen exchange transporter, can be retrieved from searches in databases such as Swiss-Prot.Among the methodological repertoire applied in functional proteomics, small molecule affinity-based techniques seem to be ideal for the task of profiling the cAMP binding proteome subset. Established strategies make use of cAMP affinity beads. These beads comprise cAMP derivatives covalently attached to the polymer backbone via an aminoalkyl linker. The linker may vary in length of the alkyl chain and in the attachment position at the nucleobase (8, 9). This approach, however, suffers from the relatively large amount of protein input required to obtain significant data, precluding e.g. the profiling of the target proteins in samples of limited abundance. Furthermore, it has not been demonstrated yet that affinity-based enrichment of cAMP-binding proteins is suitable for cAMP-binding membrane proteins that are known to be difficult to access. On the other hand, soluble cAMP- and cGMP-binding proteins along with their interaction partners were robustly identified with this methodology. Another approach described in the literature used a cyclic guanosine monophosphate analogue immobilized on a Biacore chip to isolate cGMP- and cAMP-binding proteins from a cell lysate, estimate the quantity of the material, and elute proteins for proteolysis and identification by LC-MS/MS. In addition, for single purified proteins, binding constants can be measured (10). The applicability of this approach to transmembrane cGMP/cAMP-binding proteins, however, has yet to be determined.Here we describe the synthesis and application of a trifunctional Capture Compound^TM (CC) (see Fig. 1A) as a novel approach for the functional isolation of cAMP-binding proteins from complex protein mixtures using low amounts of protein input. In contrast to current pulldown approaches, the CC enables the covalent linkage to the target proteins by a photoactivatable reactivity group in addition to the reversible binding of target proteins by the selectivity group. The Capture Compound-protein conjugate can be isolated from the complex protein mixture via the sorting function (a biotin moiety) of the Capture Compound by means of streptavidin-coated magnetic beads (see Fig. 1, B and C) (11). The cAMP-binding protein-selective Capture Compound described here was successfully applied to the isolation of cAMP-binding proteins from E. coli lysate and cultured eukaryotic HepG2 cells, respectively. Furthermore, we report the applicability of the CCMS approach for the capturing of cAMP-binding HCN channel proteins from rat brain synaptosome preparations as well. To our knowledge, this has not yet been achieved by any cAMP affinity bead approach. In addition, the ion channels, which by antibody- and in situ hybridization-based techniques have been shown to be located in neuronal tissues at synaptic sites (12, 13), have also escaped detection in many detailed proteomics profiling studies conducted to establish the protein complements of synaptic structures (see e.g. Refs. 14–17). Our data suggest that the cAMP-CC approach is uniquely efficient and sensitive for the identification and profiling of cAMP-binding proteins in complex protein mixtures.Open in a separate windowFig. 1.A, schematic design of a CC. Three functionalities are coupled to a core. The selectivity function (red), e.g. modified cAMP, for target recognition; the reactivity function (orange), e.g. diazirines, for covalent cross-linking; and the sorting function (yellow), e.g. biotin, for pullout of captured proteins; and a variable linker (green) that can modify the hydrophilicity of the system are shown. B, structure of 8-AHA-cAMP-CC (7c), which represents one of several cAMP-CCs that are available. C, flow chart of the CCMS technology.     

利用质谱技术结合EST库搜索鉴定厚壳贻贝新型足丝蛋白

贻贝利用足丝粘附于水下各种基质表面.作为一种具有优异粘附性能的生物材料,贻贝足丝蛋白在新型水下粘附剂及表面保护涂层的研制与开发中具有重要的仿生学意义.目前,已报道的贻贝足丝蛋白分子达11种,但是仍然有更多的足丝蛋白分子不为人知.为进一步探讨贻贝足丝蛋白的分子多样性,并从中筛选具有特殊生物学功能的足丝蛋白分子,本文采用鸟枪法-液相色谱-质谱/质谱技术（shotgun-LC-MS/MS）对厚壳贻贝足丝蛋白进行了蛋白质组学分析.将厚壳贻贝足丝分为足丝纤维和足丝盘两部分,每一部分均采用醋酸-尿素溶液,以及醋酸-盐酸胍溶液进行蛋白质抽提;抽提后的足丝蛋白经胰蛋白酶酶解,利用线性离子阱四级杆质谱（LTQ）进行鸟枪法质谱分析.二级质谱图（MS/MS）用以搜索公共数据库中的贻贝表达序列标签（expressed sequence tag,EST）数据库.采用上述方法,获得14种贻贝新型足丝蛋白的高可信度结果及其所匹配的部分或全长cDNA序列;经结构域分析,发现上述新型贻贝足丝蛋白分子的序列中多数包含各种类型的结构域,包括胶原蛋白结构域、C1Q结构域、C1Q结合结构域、微管蛋白辅助折叠结构域、蛋白酶拮抗结构域、VWA结构域、几丁质酶结构域等.在此基础上,对上述新型足丝蛋白在贻贝足丝形成以及粘附方面的功能进行了推测.上述结果对进一步了解贻贝足丝的分子组成以及粘附机理奠定了基础.     

Sensitive and Specific Peak Detection for SELDI-TOF Mass Spectrometry Using a Wavelet/Neural-Network Based Approach

SELDI-TOF mass spectrometer''s compact size and automated, high throughput design have been attractive to clinical researchers, and the platform has seen steady-use in biomarker studies. Despite new algorithms and preprocessing pipelines that have been developed to address reproducibility issues, visual inspection of the results of SELDI spectra preprocessing by the best algorithms still shows miscalled peaks and systematic sources of error. This suggests that there continues to be problems with SELDI preprocessing. In this work, we study the preprocessing of SELDI in detail and introduce improvements. While many algorithms, including the vendor supplied software, can identify peak clusters of specific mass (or m/z) in groups of spectra with high specificity and low false discover rate (FDR), the algorithms tend to underperform estimating the exact prevalence and intensity of peaks in those clusters. Thus group differences that at first appear very strong are shown, after careful and laborious hand inspection of the spectra, to be less than significant. Here we introduce a wavelet/neural network based algorithm which mimics what a team of expert, human users would call for peaks in each of several hundred spectra in a typical SELDI clinical study. The wavelet denoising part of the algorithm optimally smoothes the signal in each spectrum according to an improved suite of signal processing algorithms previously reported (the LibSELDI toolbox under development). The neural network part of the algorithm combines those results with the raw signal and a training dataset of expertly called peaks, to call peaks in a test set of spectra with approximately 95% accuracy. The new method was applied to data collected from a study of cervical mucus for the early detection of cervical cancer in HPV infected women. The method shows promise in addressing the ongoing SELDI reproducibility issues.     

Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform

Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25⁺⁴⁰ isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25⁺⁴⁰ as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.     

双向电泳结合质谱分析长双歧杆菌XY01分泌蛋白质图谱

菌体的分泌蛋白质在宿主和菌体的相互作用之间起着重要的作用. 本研究采用双向凝胶电泳的方法建立了长双歧杆菌XY01分泌蛋白质图谱,通过MALDI-TOF/TOF质 谱鉴定和数据库搜索,对鉴定到的分泌蛋白进行了分析. 共检测到21个蛋白质点, 成功鉴定18个蛋白质点,分别代表14个不同的蛋白质,等电点分布在4.5～7.0之间 ,分子质量分布在20 ～65 kD之间;通过COGs分类和功能分析,信号肽和细胞定位及KEGG代谢通路分析. 结果表明,这些蛋白质对菌体细胞壁/膜的形成、生物信号传导和物质代谢等起着重要作用. 研究结果为长双歧杆菌蛋白质组学和基因组学的研究提供了参考.