Identification of novel target proteins of cyclic GMP signaling pathways using chemical proteomics

For deciphering the cyclic guanosine monophosphate (cGMP) signaling pathway, we employed chemical proteomics to identify the novel target molecules of cGMP. We used cGMP that was immobilized onto agarose beads with linkers directed at three different positions of cGMP. We performed a pull-down assay using the beads as baits on tissue lysates and identified 9 proteins by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry. Some of the identified proteins were previously known cGMP targets, including cGMP-dependent protein kinase and cGMP-stimulated phosphodiesterase. Surprisingly, some of the coprecipitated proteins were never formerly reported to associate with the cGMP signaling pathway. The competition binding assays showed that the interactions are not by nonspecific binding to either the linker or bead itself, but by specific binding to cGMP. Furthermore, we observed that the interactions are highly specific to cGMP against other nucleotides, such as cyclic adenosine monophosphate (cAMP) and 5\'-GMP, which are structurally similar to cGMP. As one of the identified targets, MAPK1 was confirmed by immunoblotting with an anti-MAPK1 antibody. For further proof, we observed that the membrane-permeable cGMP (8-bromo cyclic GMP) stimulated mitogen-activated protein kinase 1 signaling in the treated cells. Our present study suggests that chemical proteomics can be a very useful and powerful technique for identifying the target proteins of small bioactive molecules.     

Analysis of the soluble ATP-binding proteome of plant mitochondria identifies new proteins and nucleotide triphosphate interactions within the matrix

The interactions of ATP inside plant mitochondria were investigated by identifying the soluble nucleotide binding proteome captured using immobilized ATP. Selected proteins were separated by 1D SDS-PAGE and 2D IEF-SDS-PAGE and identified by ESI-Q-TOF MS/MS. A range of highly enriched proteins were identified from the mitochondrial proteome, including 14-3-3 proteins and RNA binding proteins, as well as proteins known to contain nucleotide binding domains and/or to be inhibited or stimulated by ATP.     

A Simplified Thermal Proteome Profiling Approach to Screen Protein Targets of a Ligand

Thermal proteome profiling is a powerful energetic‐based chemical proteomics method to reveal the ligand‐protein interaction. However, the costly multiplexed isotopic labeling reagent, mainly Multiplexed isobaric tandem mass tag (TMT), and the long mass spectrometric time limits the wide application of this method. Here a simple and cost‐effective strategy by using dimethyl labeling technique instead of TMT labeling is reported to quantify proteins and by using the peptides derived from the same protein to determine significantly changed proteins in one LC‐MS run. This method is validated by identifying the known targets of methotrexate and geldanamycin. In addition, several potential off‐targets involved in detoxification of reactive oxygen species pathway are also discovered for geldanamycin. This method is further applied to map the interactome of adenosine triphosphate (ATP) in the 293T cell lysate by using ATP analogue, adenylyl imidodiphosphate (AMP‐PNP), as the ligand. As a result, a total of 123 AMP‐PNP‐sensitive proteins are found, of which 59 proteins are stabilized by AMP‐PNP. Approximately 53% and 20% of these stabilized candidate protein targets are known as ATP and RNA binding proteins. Overall, above results demonstrated that this approach could be a valuable platform for the unbiased target proteins identification with reduced reagent cost and mass spectrometric time.     

Strategy for surveying the proteome using affinity proteomics and mass spectrometry

Antibody‐based microarrays is a rapidly evolving technology that has gone from the first proof‐of‐concept studies to more demanding proteome profiling applications, during the last years. Miniaturized microarrays can be printed with large number of antibodies harbouring predetermined specificities, capable of targeting high‐ as well as low‐abundant analytes in complex, nonfractionated proteomes. Consequently, the resolution of such proteome profiling efforts correlate directly to the number of antibodies included, which today is a key limiting factor. To overcome this bottleneck and to be able to perform in‐depth global proteome surveys, we propose to interface affinity proteomics with MS‐based read‐out, as outlined in this technical perspective. Briefly, we have defined a range of peptide motifs, each motif being present in 5–100 different proteins. In this manner, 100 antibodies, binding 100 different motifs commonly distributed among different proteins, would potentially target a protein cluster of 10⁴ individual molecules, i.e. around 50% of the nonredundant human proteome. Notably, these motif‐specific antibodies would be directly applicable to any proteome in a specie independent manner and not biased towards abundant proteins or certain protein classes. The biological sample is digested, exposed to these immobilized antibodies, whereby motif‐containing peptides are specifically captured, enriched and subsequently detected and identified using MS.     

In Central Obesity,Weight Loss Restores Platelet Sensitivity to Nitric Oxide and Prostacyclin

Central obesity shows impaired platelet responses to the antiaggregating effects of nitric oxide (NO), prostacyclin, and their effectors—guanosine 3′,5′‐cyclic monophosphate (cGMP) and adenosine 3′,5′‐cyclic monophosphate (cAMP). The influence of weight loss on these alterations is not known. To evaluate whether a diet‐induced body‐weight reduction restores platelet sensitivity to the physiological antiaggregating agents and reduces platelet activation in subjects affected by central obesity, we studied 20 centrally obese subjects before and after a 6‐month diet intervention aiming at reducing body weight by 10%, by measuring (i) insulin sensitivity (homeostasis model assessment of insulin resistance (HOMA_IR)); (ii) plasma lipids; (iii) circulating markers of inflammation of adipose tissue and endothelial dysfunction, and of platelet activation (i.e., soluble CD‐40 ligand (sCD‐40L) and soluble P‐selectin (sP‐selectin)); (iv) ability of the NO donor sodium nitroprusside (SNP), the prostacyclin analog Iloprost and the cyclic nucleotide analogs 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) and 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) to reduce platelet aggregation in response to adenosine‐5‐diphosphate (ADP); and (v) ability of SNP and Iloprost to increase cGMP and cAMP. The 10 subjects who reached the body‐weight target showed significant reductions of insulin resistance, adipose tissue, endothelial dysfunction, and platelet activation, and a significant increase of the ability of SNP, Iloprost, 8‐Br‐cGMP, and 8‐Br‐cAMP to reduce ADP‐induced platelet aggregation and of the ability of SNP and Iloprost to increase cyclic nucleotide concentrations. No change was observed in the 10 subjects who did not reach the body‐weight target. Changes of platelet function correlated with changes of HOMA_IR. Thus, in central obesity, diet‐induced weight loss reduces platelet activation and restores the sensitivity to the physiological antiaggregating agents, with a correlation with improvements in insulin sensitivity.     

Affinity prefractionation for MS‐based plasma proteomics

The plasma proteome has proven to be one of the most challenging proteomes to profile using currently available proteomics technologies. A plethora of methodologies have been used to profile human plasma in order to discover potential biomarkers for disease and for therapy optimization. Affinity‐based prefractionation coupled to MS has been shown to be one of the most successful ways to dig deeper into the plasma proteome. Depletion of high abundant plasma proteins is becoming an initial method of choice in any plasma profiling project. However, several other affinity‐based enrichment methods have been published in recent years. Here we review both protein and peptide affinity prefractionation methods coupled with MS‐based proteomics. Analysis of the proportion of cellular and extracellular annotated proteins of publicly available MS plasma proteomics data is performed to estimate the analytical depth of various prefractionation methods.     

Enhanced recovery of lyophilized peptides in shotgun proteomics by using an LC‐ESI‐MS compatible surfactant

LC‐ESI/MS/MS‐based shotgun proteomics is currently the most commonly used approach for the identification and quantification of proteins in large‐scale studies of biomarker discovery. In the past several years, the shotgun proteomics technologies have been refined toward further enhancement of proteome coverage. In the complex series of protocols involved in shotgun proteomics, however, loss of proteolytic peptides during the lyophilization step prior to the LC/MS/MS injection has been relatively neglected despite the fact that the dissolution of the hydrophobic peptides in lyophilized samples is difficult in 0.05–0.1% TFA or formic acid, causing substantial loss of precious peptide samples. In order to prevent the loss of peptide samples during this step, we devised a new protocol using Invitrosol (IVS), a commercially available surfactant compatible with ESI‐MS; by dissolving the lyophilized peptides in IVS, we show improved recovery of hydrophobic peptides, leading to enhanced coverage of proteome. Thus, the use of IVS in the recovery step of lyophilized peptides will help the shotgun proteomics analysis by expanding the proteome coverage, which would significantly promote the discovery and development of new diagnostic markers and therapeutic targets.     

The human oligodendrocyte proteome

Myelination of the CNS is performed by oligodendrocytes (OLs), which have been implicated in brain disorders, such as multiple sclerosis and schizophrenia. We have used the human oligodendroglial cell line MO3.13 to establish an OL reference proteome database. Proteins were prefractionationated by SDS‐PAGE and after in‐gel digestion subjected to nanoflow LC‐MS analysis. Approximately 11 600 unique peptides were identified and, after stringent filtering, resulted in 2290 proteins representing nine distinct biological processes and various molecular classes and functions. OL‐specific proteins, such as myelin basic protein (MBP) and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), as well as other proteins involved in multiple sclerosis and schizophrenia were also identified and are discussed. Proteins of this dataset have also been classified according to their chromosomal origin for providing useful data to the Chromosome‐centric Human Proteome Project (C‐HPP). Given the importance of OLs in the etiology of demyelinating and oligodendrogial disorders, the MO3.13 proteome database is a valuable data resource. The MS proteomics data have been deposited to the ProteomeXchange with identifier PXD000263 ( http://proteomecentral.proteomexchange.org/dataset/PXD000263 ).     

Cyclic AMP and higher plants

Despite the evidence in support, the extent of which is outlined in this review, the occurrence of cyclic AMP in tissues of higher plants has been doubted by a number of previous reviewers. Recent MS and other evidence vindicates earlier identification of an adenosine nucleotide from plant tissues as adenosine 3′:5′-cyclic monophosphate. The additional demonstration of 3′: 5′-cyclic nucleotide phosphodiesterases in higher plants, together with adenylate cyclase, a specific cyclic AMP binding protein, and calmodulin, means that plants possess all the necessary components for a functional cyclic AMP-regulated system. Whether such a system does function in plants is considered as are also the reported physiological effects of exogenously supplied cyclic AMP on plant tissues.     

Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.     

Sequential protein extraction as an efficient method for improved proteome coverage in larvae of Atlantic salmon (Salmo salar)

Understanding diet‐ and environmentally induced physiological changes in fish larvae is a major goal for the aquaculture industry. Proteomic analysis of whole fish larvae comprising multiple tissues offers considerable potential but is challenging due to the very large dynamic range of protein abundance. To extend the coverage of the larval phase of the Atlantic salmon (Salmo salar) proteome, we applied a two‐step sequential extraction (SE) method, based on differential protein solubility, using a nondenaturing buffer containing 150 mM NaCl followed by a denaturing buffer containing 7 M urea and 2 M thiourea. Extracts prepared using SE and one‐step direct extraction were characterized via label‐free shotgun proteomics using nanoLC‐MS/MS (LTQ‐Orbitrap). SE partitioned the proteins into two fractions of approximately equal amounts, but with very distinct protein composition, leading to identification of ～40% more proteins than direct extraction. This fractionation strategy enabled the most detailed characterization of the salmon larval proteome to date and provides a platform for greater understanding of physiological changes in whole fish larvae. The MS data are available via the ProteomeXchange Consortium PRIDE partner repository, dataset PXD003366.     

Increased proteome coverage by combining PAGE and peptide isoelectric focusing: Comparative study of gel‐based separation approaches

The in‐depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC‐MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in‐gel IEF, prior to RP‐HPLC‐MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

Mouse models for neurodegenerative diseases and a theory of proteomics

Proteome analysis is usually performed by separating complex cellular protein extracts by two‐dimensional‐electrophoresis followed by protein identification using mass spectrometry. In this way proteins are compared from normal and diseased tissue in order to detect disease related protein changes. In a strict sense, however, this procedure cannot be called proteome analysis: the tools of proteomics are used just to detect some interesting proteins which are then investigated by protein chemistry as usual. Real proteome research would be studying the cellular proteome as a whole, its composition, organization and its kind of action. At present however, we have no idea how a proteome works as a whole; we have not even a theory about that. If we would know how the proteome of a cell type is arranged, we probably would alter our strategy to detect and analyze disease‐related proteins. I will present a theory of proteomics and show some results from our laboratory which support this theory. The results come from investigations of the mouse brain proteome and include mouse models for neurodegenerative diseases.     

Proteolysis approach without chemical modification for a simple and rapid analysis of disulfide bonds using thermostable protease‐immobilized microreactors

Disulfide bonds in proteins are important not only for the conformational stability of the protein but also for the regulation of oxidation–reduction in signal transduction. The conventional method for the assignment of disulfide bond by chemical cleavage and/or proteolysis is a time‐consuming multi‐step procedure. In this study, we report a simple and rapid analysis of disulfide bond from protein digests that were prepared by the thermostable protease‐immobilized microreactors. The feasibility and performance of this approach were evaluated by digesting lysozyme and BSA at several temperatures. The proteins which stabilize their conformations by disulfide bonds were thermally denatured during proteolysis and were efficiently digested by the immobilized protease but not by free protease. The digests were directly analyzed by ESI‐TOF MS without any purification or concentration step. All four disulfide bonds on lysozyme and 10 of 17 on BSA were assigned from the digests by the trypsin‐immobilized microreactor at 50°C. The procedure for proteolysis and the assignment were achieved within 2 h without any reduction and alkylation procedure. From the present results, the proteolysis approach by the thermostable protease‐immobilized microreactor provides a strategy for the high‐throughput analysis of disulfide bond in proteomics.     

A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages

Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid "baits" with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.     

On the control mechanism of bacterial growth by cyclic adenosine 3',5'-monophosphate

Inhibition of E. coli growth by cyclic adenosine monophosphate is observed in wild type strains cultured in glucose as carbon source, but not in a cyclic AMP receptor protein deficient mutant. A deletion mutant of the adenylate cyclase gene requires cyclic adenosine monophosphate for optimal growth. Using glucose as carbon source, 2 mM cyclic AMP promotes maximal rates of cell multiplication in this mutant; however higher concentrations of the nucleotide inhibit growth. Cell multiplication of wild type strains grown in glycerol is not affected by cyclic adenosine monophosphate. Nevertheless, in this carbon source the growth rate of the adenylate cyclase mutant is strongly inhibited by concentrations of this nucleotide beyond 0.1 mM. This suggests that growth inhibition by exogenous cyclic adenosine monophosphate is highly dependent on the intracellular levels of the nucleotide.     

Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry

Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.