Protein-Protein Interaction Network Mapping by Affinity Purification Cross-Linking Mass Spectrometry (AP-XL-MS) based Proteomics.

Protein-protein interactions (PPIs) are the physical interactions formed among proteins. These interactions are primarily functional, i.e., they arise from specific biomolecular events, and each interaction interface serves a specific purpose. A significant number of methods have been developed for protein interactions in the field of proteomics in the last decade. Advanced mass spectrometry technology significantly contributed to the development of these methods. The rapid advancement of groundbreaking MS technology has greatly aided the mapping of protein interaction from large-data sets comprehensively. This chapter describes the affinity purification (AP) mass spectrometry (MS)-based methods combined with chemical cross-linking (XL) of protein complexes. This chapter includes sample preparation methods involving cell culture, cell treatments with ligands, drugs, and cross-linkers, protein extractions, affinity purification, sodium dodecyl sulfate (SDS) polyacrylamide gel separation, in-solution or in-gel digestion, liquid-chromatography, and mass spectrometry analysis of samples (LC-MS/MS). Application of a cleavable cross-linker, dual cleavable cross-linking technology (DUCCT) in combination with the affinity purification (AP) method has also been described. Methods for data analysis using unmodified and cross-linked peptide analysis are discussed.

The prohibitin complex regulates macrophage fatty acid composition, plasma membrane packing, and lipid raft-mediated inflammatory signaling.

Prohibitins (PHB1 and PHB2) are ubiquitously expressed proteins which play critical roles in multiple biological processes, and together form the ring-like PHB complex found in phospholipid-rich cellular compartments including lipid rafts. Recent studies have implicated PHB1 as a mediator of fatty acid transport as well as a membrane scaffold mediating B lymphocyte and mast cell signal transduction. However, the specific role of PHBs in the macrophage have not been characterized, including their role in fatty acid uptake and lipid raft-mediated inflammatory signaling. We hypothesized that the PHB complex regulates macrophage inflammatory signaling through the formation of lipid rafts. To evaluate our hypothesis, RAW 264.7 macrophages were transduced with shRNA against PHB1, PHB2, or scrambled control (Scr), and then stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-α), which activate lipid raft-dependent receptor signaling (CD14/TLR4 and TNFR1, respectively). PHB1 knockdown was lethal, whereas PHB2 knockdown (PHB2kd), which also resulted in decreased PHB1 expression, led to attenuated nuclear factor-kappa-B (NF-κB) activation and subsequent cytokine and chemokine production. PHB2kd macrophages also had decreased cell surface TNFR1, CD14, TLR4, and lipid raft marker ganglioside GM1 at baseline and post-stimuli. Post-LPS, PHB2kd macrophages did not increase the concentration of cellular saturated, monounsaturated, and polyunsaturated fatty acids. This was accompanied by decreased lipid raft formation and modified plasma membrane molecular packing, further supporting the PHB complex's importance in lipid raft formation. Taken together, these data suggest a critical role for PHBs in regulating macrophage inflammatory signaling via maintenance of fatty acid composition and lipid raft structure. SUMMARY: Prohibitins are proteins found in phospholipid-rich cellular compartments, including lipid rafts, that play important roles in signaling, transcription, and multiple other cell functions. Macrophages are key cells in the innate immune response and the presence of membrane lipid rafts is integral to signal transduction, but the role of prohibitins in macrophage lipid rafts and associated signaling is unknown. To address this question, prohibitin knockdown macrophages were generated and responses to lipopolysaccharide and tumor necrosis factor-alpha, which act through lipid raft-dependent receptors, were analyzed. Prohibitin knockdown macrophages had significantly decreased cytokine and chemokine production, transcription factor activation, receptor expression, lipid raft assembly and membrane packing, and altered fatty acid remodeling. These data indicate a novel role for prohibitins in macrophage inflammatory signaling through regulation of fatty acid composition and lipid raft formation.

Lipopolysaccharide and statin-mediated immune-responsive protein networks revealed in macrophages through affinity purification spacer-arm controlled cross-linking (AP-SPACC) proteomics.

Toll-like receptor 4 (TLR4), a pattern recognition receptor, is activated by lipopolysaccharides (LPS) and induces the MyD88 pathway, which subsequently produces pro-inflammatory cytokines through activation of transcriptional nuclear factor (NF)-κB. Statins have been widely prescribed to reduce cholesterol synthesis for patients with cardiovascular disease. Statins may have pleiotropic effects, which include anti- and pro-inflammatory effects on cells. The molecular mechanism of the sequential influence of LPS and statin on the innate immune system remains unknown. We employed affinity purification-spacer-arm controlled cross-linking (AP-SPACC) MS-based proteomics analysis to identify the LPS- and statin-LPS-responsive proteins and their networks. LPS-stimulated RAW 264.7 macrophage cells singly and combined with the drug statin used in this study. Two chemical cross-linkers with different spacer chain lengths were utilized to stabilize the weak and transient interactors. Proteomic analysis identified 1631 differentially expressed proteins. We identified 151 immune-response proteins through functional enrichment analysis and visualized their interaction networks. Selected candidate protein-coding genes were validated, specifically squamous cell carcinoma antigens recognized by T cells 3, sphingosine-1-phosphate lyase 1, Ras-related protein Rab-35, and tumor protein D52 protein-coding genes through transcript-level expression analysis. The expressions of those genes were significantly increased upon statin treatment and decreased in LPS-stimulated macrophage cells. Therefore, we presumed that the expression changes of genes occurred due to immune response during activation of inflammation. These results highlight the immune-responsive proteins network, providing a new platform for novel investigations and discovering future therapeutic targets for inflammatory diseases.

Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics.

Toll-like receptor 4 (TLR4) is a receptor on an immune cell that can recognize the invasion of bacteria through their attachment with bacterial lipopolysaccharides (LPS). Hence, LPS is a pro-immune response stimulus. On the other hand, statins are lipid-lowering drugs and can also lower immune cell responses. We used human embryonic kidney (HEK 293) cells engineered to express HA-tagged TLR-4 upon treatment with LPS, statin, and both statin and LPS to understand the effect of pro- and anti-inflammatory responses. We performed a monoclonal antibody (mAb) directed co-immunoprecipitation (CO-IP) of HA-tagged TLR4 and its interacting proteins in the HEK 293 extracted proteins. We utilized an ETD cleavable chemical cross-linker to capture weak and transient interactions with TLR4 protein. We tryptic digested immunoprecipitated and cross-linked proteins on beads, followed by liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the peptides. Thus, we utilized the label-free quantitation technique to measure the relative expression of proteins between treated and untreated samples. We identified 712 proteins across treated and untreated samples and performed protein network analysis using Ingenuity Pathway Analysis (IPA) software to reveal their protein networks. After filtering and evaluating protein expression, we identified macrophage myristoylated alanine-rich C kinase substrate (MARCKSL1) and creatine kinase proteins as a potential part of the inflammatory networks of TLR4. The results assumed that MARCKSL1 and creatine kinase proteins might be associated with a statin-induced anti-inflammatory response due to possible interaction with the TLR4.

Identification of an integrated stress and growth response signaling switch that directs vertebrate intestinal regeneration.

BACKGROUND: Snakes exhibit extreme intestinal regeneration following months-long fasts that involves unparalleled increases in metabolism, function, and tissue growth, but the specific molecular control of this process is unknown. Understanding the mechanisms that coordinate these regenerative phenotypes provides valuable opportunities to understand critical pathways that may control vertebrate regeneration and novel perspectives on vertebrate regenerative capacities. RESULTS: Here, we integrate a comprehensive set of phenotypic, transcriptomic, proteomic, and phosphoproteomic data from boa constrictors to identify the mechanisms that orchestrate shifts in metabolism, nutrient uptake, and cellular stress to direct phases of the regenerative response. We identify specific temporal patterns of metabolic, stress response, and growth pathway activation that direct regeneration and provide evidence for multiple key central regulatory molecules kinases that integrate these signals, including major conserved pathways like mTOR signaling and the unfolded protein response. CONCLUSION: Collectively, our results identify a novel switch-like role of stress responses in intestinal regeneration that forms a primary regulatory hub facilitating organ regeneration and could point to potential pathways to understand regenerative capacity in vertebrates.

Mass Spectrometry-Cleavable Protein N-Terminal Tagging Strategy for System-Level Protease Activity Profiling.

Proteolysis is one of the most important protein post-translational modifications (PTMs) that influences the functions, activities, and structures of nearly all proteins during their lifetime. To facilitate the targeted identification of low-abundant proteolytic products, we devised a strategy incorporating a novel biotinylated reagent PFP (pentafluorophenyl)-Rink-biotin to specifically target, enrich and identify proteolytic N-termini. Within the PFP-Rink-biotin reagent, a mass spectrometry (MS)-cleavable feature was designed to assist in the unambiguous confirmation of the enriched proteolytic N-termini. The proof-of-concept study was performed with multiple standard proteins whose N-termini were successfully modified, enriched and identified by a signature ion (SI) in the MS/MS fragmentation, along with the determination of N-terminal peptide sequences by multistage tandem MS of the complementary fragment generated after the cleavage of MS-cleavable bond. For large-scale application, the enrichment and identification of protein N-termini from Escherichia coli cells were demonstrated, facilitated by an in-house developed NTermFinder bioinformatics workflow. We believe this approach will be beneficial in improving the confidence of identifying proteolytic substrates in a native cellular environment.

Dual-Stage Neutral Loss Tandem Mass Spectrometric Strategy for Confident Identification of Protein Prenylation.

Protein prenylation is an important post-translational modification that regulates protein interactions, localizations, and signaling pathways in normal functioning of eukaryotic cells. It is also a critical step in the oncogenic developments of various cancers. Direct identification of native protein prenylation by mass spectrometry (MS) has been challenging due to high hydrophobicity and the lack of an efficient enrichment technique. Prior MS studies of prenylation revealed that prenyl peptides readily generate high-intensity fragments after neutral loss of the prenyl group (R group), and more recent investigation of oxidized prenyl peptides discovered more consistent neutral loss of the oxidized prenyl group (RSOH group). Here, a dual-stage neutral loss MS3 (DS-NLMS3)-based strategy is therefore developed by combining both gas-phase cleavable properties of the prenyl thioether bond and mono-oxidized thioether to improve the large-scale identification of prenylation. Both neutral losses can individually and distinctively confirm the prenylation type in MS2 and the sequence of the prenyl peptide upon targeted MS3 fragmentation. This dual-faceted NLMS3 strategy significantly improves the confidence in the identification of protein prenylation from large-scale samples, which enables the unambiguous identification of prenylated sites of the spiked low-abundance farnesyl peptide and native prenyl proteins from mouse macrophage cells, even without prior enrichment during sample preparation. The ease of incorporating this strategy into the prenylation study workflow and minimum disruption to the biological lipidome are advantageous for unraveling unknown native protein prenylation and further developments in profiling and quantifying prenylome.

Affinity and chemical enrichment strategies for mapping low-abundance protein modifications and protein-interaction networks.

Protein post-translational modifications and protein interactions are the central research areas in mass-spectrometry-based proteomics. Protein post-translational modifications affect protein structures, stabilities, activities, and all cellular processes are achieved by interactions among proteins and protein complexes. With the continuing advancements of mass spectrometry instrumentations of better sensitivity, speed, and performance, selective enrichment of modifications/interactions of interest from complex cellular matrices during the sample preparation has become the overwhelming bottleneck in the proteomics workflow. Therefore, many strategies have been developed to address this issue by targeting specific modifications/interactions based on their physical properties or chemical reactivities, but only a few have been successfully applied for systematic proteome-wide study. In this review, we summarized the highlights of recent developments in the affinity enrichment methods focusing mainly on low stoichiometric protein lipidations. Besides, to identify potential glyoxal modified arginines, a small part was added for profiling reactive arginine sites using an enrichment reagent. A detailed section was provided for the enrichment of protein interactions by affinity purification and chemical cross-linking, to shed light on the potentials of different enrichment strategies, along with the unique challenges in investigating individual protein post-translational modification or protein interaction network.

Proteomics Network Analysis of Polarized Macrophages.

Macrophages play a critical role in innate immunity through Toll-like receptor (TLR) signaling. Lipopolysaccharides (LPS) are a ligand of microbial origin that can trigger cell signaling in macrophages through TLRs and production of pro-inflammatory cytokines. Statin, a hypercholesterolemia drug, on the contrary, can reduce inflammatory cytokine production, and inflammation at large. Discovery-based quantitative proteomics is a useful method for unraveling complex protein networks and inter-protein interactions. Here, we describe protocols for studying the inflammatory proteomics network in RAW 264.7 cells (a model murine macrophage cell line) with the singular or sequential treatment of LPS and statin. We provide detailed protocols, including a quantitative proteomic analysis by mass spectrometry data, a protein network analysis by bioinformatics, and a validation of target through biochemical methods (e.g., immunocytochemistry, immunoblotting, gene silencing, and real-time PCR).

Evaluating the performance of an ETD-cleavable cross-linking strategy for elucidating protein structures.

Chemical cross-linking is a powerful strategy for elucidating the structures of protein or protein complexes. The distance constraints obtained from cross-linked peptides represent the three-dimensional structures of the protein complexes. Unfortunately, structural analysis using cross-linking approach demands a significant amount of data to elucidate protein structures. This requires the development of several cleavable cross-linkers with different range of spacer chains. An Electron Transfer Dissociation (ETD) tandem mass spectrometry cleavable bond hydrazone was reported. Its fragmentation with conjugated peptides showed promise for the development of a new ETD cleavable cross-linker. However, no cross-linker was developed utilizing this ETD cleavable bond. For the first time, we attempted to develop an ETD cleavable cross-linker utilizing a hydrazone bond. We overcome the pitfall for the synthesis of this cross-linker and an easy synthesis scheme is reported. In this report, we evaluated the performance of this cross-linker called Hydrazone Incorporated ETD cleavable cross-linker (HI-ETD-XL) in model peptides and proteins. The characteristic fragmentation behavior of HI-ETD-XL during electron transfer dissociation and subsequent sequence identification of the peptide fragment ions by tandem mass spectrometry allowed the identification of cross-linked peptides unambiguously. We believe the availability of this ETD cleavable cross-linker will advance structural proteomics research significantly. SIGNIFICANCE: Many cellular processes rely on the structural dynamics of protein complexes. The detailed knowledge of the structure and dynamics of protein complexes is crucial for understanding their biological functions and regulations. However, most of the structure of these multiprotein entities remain uncharacterized and sometimes is very challenging to reveal with biophysical techniques alone. Chemical cross-linking combined with mass spectrometry (MS) has proven to be a dependable strategy in structural proteomics field. However, data complexity and false identifications are significant hindrances for unambiguous identification of cross-linked peptides. Confident identifications demand structural studies with cross-linkers with different properties and variable spacer chain lengths. This new ETD cleavable cross-linking workflow will provide additional confidence to overcome these drawbacks and allow us to pinpoint cross-linked peptides confidently.

Improved in-solution trypsin digestion method for methanol-chloroform precipitated cellular proteomics sample.

Methanol-chloroform based protein precipitation is an essential step in many liquid chromatography-tandem mass spectrometry-based cellular proteomics applications. However, re-solubilization of the total protein precipitate is difficult using regular in-solution digestion protocol. Sodium deoxycholate is reported as an efficient surfactant for re-solubilization of membrane fractions. In this study, we demonstrated an application combining methanol-chloroform based protein precipitations and deoxycholic acid assisted re-solubilization of pellets to evaluate the improvement of protein identifications in mass spectrometry-based bottom-up proteomics. We evaluated the modified method using an equal amount of Raw 264.7 mouse macrophage cell lysate. Detailed in-solution trypsin digestion studies were presented on methanol-chloroform precipitated samples with or without deoxycholic acid treatments and compared with popular sample digestion methods. A mass spectrometric analysis confirmed an 82% increase in protein identification in deoxycholic acid-treated samples compared to other established methods. Furthermore, liquid chromatography-tandem mass spectrometry analysis of an equal amount of proteins from methanol-chloroform precipitated, and methanol-chloroform/deoxycholic acid-treated macrophage cell lysate showed a 14% increase and 27% unique protein identifications. We believe this improved digestion method could be a complementary or alternative method for mammalian cell sample preparations where sodium dodecyl sulfate based lysis buffer is frequently used.

High Confidence Identification of Cross-Linked Peptides by an Enrichment-Based Dual Cleavable Cross-Linking Technology and Data Analysis tool Cleave-XL.

Cleavable cross-linking technology requires further MS/MS of the cleavable fragments for unambiguous identification of cross-linked peptides. These spectra are sometimes very ambiguous due to the sensitivity and complex fragmentation pattern of the peptides with the cross-linked residues. We recently reported a dual cleavable cross-linking technology (DUCCT), which can enhance the confidence in the identification of cross-linked peptides. The heart of this strategy is a novel dual mass spectrometry cleavable cross linker that can be cleaved preferentially by two differential tandem mass spectrometry methods, collision induced dissociation and electron transfer dissociation (CID and ETD). Different signature ions from two different mass spectra for the same cross-linked peptide helped identify the cross-linked peptides with high confidence. In this study, we developed an enrichment-based photocleavable DUCCT (PC-DUCCT-biotin), where cross-linked products were enriched from biological samples using affinity purification, and subsequently, two sequential tandem (CID and ETD) mass spectrometry processes were utilized. Furthermore, we developed a prototype software called Cleave-XL to analyze cross-linked products generated by DUCCT. Photocleavable DUCCT was demonstrated in standard peptides and proteins. Efficiency of the software tools to search and compare CID and ETD data of photocleavable DUCCT biotin in standard peptides and proteins as well as regular DUCCT in protein complexes from immune cells were tested. The software is efficient in pinpointing cross-linked sites using CID and ETD cross-linking data. We believe this new DUCCT and associated software tool Cleave-XL will advance high confidence identification of protein cross-linking sites and automated identification of low-resolution protein structures.

Multi-species comparisons of snakes identify coordinated signalling networks underlying post-feeding intestinal regeneration.

Several snake species that feed infrequently in nature have evolved the ability to massively upregulate intestinal form and function with each meal. While fasting, these snakes downregulate intestinal form and function, and upon feeding restore intestinal structure and function through major increases in cell growth and proliferation, metabolism and upregulation of digestive function. Previous studies have identified changes in gene expression that underlie this regenerative growth of the python intestine, but the unique features that differentiate this extreme regenerative growth from non-regenerative post-feeding responses exhibited by snakes that feed more frequently remain unclear. Here, we leveraged variation in regenerative capacity across three snake species-two distantly related lineages ( Crotalus and Python) that experience regenerative growth, and one ( Nerodia) that does not-to infer molecular mechanisms underlying intestinal regeneration using transcriptomic and proteomic approaches. Using a comparative approach, we identify a suite of growth, stress response and DNA damage response signalling pathways with inferred activity specifically in regenerating species, and propose a hypothesis model of interactivity between these pathways that may drive regenerative intestinal growth in snakes.

Proteomic Investigation of a Diseased Gorgonian Coral Indicates Disruption of Essential Cell Function and Investment in Inflammatory and Other Immune Processes.

As scleractinian coral cover declines in the face of increased frequency in disease outbreaks, future reefs may become dominated by octocorals. Understanding octocoral disease responses and consequences is therefore necessary if we are to gain insight into the future of ecosystem services provided by coral reefs. In Florida, populations of the octocoral Eunicea calyculata infected with Eunicea black disease (EBD) were observed in the field in the fall of 2011. This disease was recognized by a stark, black pigmentation caused by heavy melanization. Histological preparations of E. calyculata infected with EBD demonstrated granular amoebocyte (GA) mobilization, melanin granules in much of the GA population, and the presence of fungal hyphae penetrating coral tissue. Previous transcriptomic analysis also identified immune trade-offs evidenced by increased immune investment at the expense of growth. Our investigation utilized proteogenomic techniques to reveal decreased investment in general cell signaling while increasing energy production for immune responses. Inflammation was also prominent in diseased E. calyculata and sheds light on factors driving the extreme phenotype observed with EBD. With disease outbreaks continuing to increase in frequency, our results highlight new targets within the cnidarian immune system and provide a framework for understanding transcriptomics in the context of an organismal disease phenotype and its protein expression.

Cross-linking Proteomics Indicates Effects of Simvastatin on the TLR2 Interactome and Reveals ACTR1A as a Novel Regulator of the TLR2 Signal Cascade.

Toll-like receptor 2 (TLR2) is a pattern recognition receptor that, upon ligation by microbial molecules, interacts with other proteins to initiate pro-inflammatory responses by the cell. Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors), drugs widely prescribed to reduce hypercholesterolemia, are reported to have both pro- and anti-inflammatory effects upon cells. Some of these responses are presumed to be driven by effects on signaling proteins at the plasma membrane, but the underlying mechanisms remain obscure. We reasoned that profiling the effect of statins on the repertoire of TLR2-interacting proteins might provide novel insights into the mechanisms by which statins impact inflammation. In order to study the TLR2 interactome, we designed a coimmunoprecipitation (IP)-based cross-linking proteomics study. A hemagglutinin (HA)-tagged-TLR2 transfected HEK293 cell line was used to precipitate the TLR2 interactome upon cell exposure to the TLR2 agonist Pam3CSK4 and simvastatin, singly and in combination. To stabilize protein interactors, we used two different chemical cross-linkers with different spacer chain lengths. Proteomic analysis revealed important combinatorial effects of simvastatin and Pam3CSK4 on the TLR2 interactome. After stringent data filtering, we identified alpha-centractin (ACTR1A), an actin-related protein and subunit of the dynactin complex, as a potential interactor of TLR2. The interaction was validated using biochemical methods. RNA interference studies revealed an important role for ACTR1A in induction of pro-inflammatory cytokines. Taken together, we report that statins remodel the TLR2 interactome, and we identify ACTR1A, a part of the dynactin complex, as a novel regulator of TLR2-mediated immune signaling pathways.

Evaluation of Different Stationary Phases in the Separation of Inter-Cross-Linked Peptides.

Chemical cross-linking coupled with mass spectrometry (MS) is becoming a routinely and widely used technique for depicting and constructing protein structures and protein interaction networks. One major challenge for cross-linking/MS is the determination of informative low-abundant inter-cross-linked products, generated within a sample of high complexity. A C18 stationary phase is the conventional means for reversed-phase (RP) separation of inter-cross-linked peptides. Various RP stationary phases, which provide different selectivities and retentions, have been developed as alternatives to C18 stationary phases. In this study, two phenyl-based columns, biphenyl and fluorophenyl, were investigated and compared with a C18 phase for separating BS3 (bis(sulfosuccinimidyl)suberate) cross-linked bovine serum albumin (BSA) and myoglobin by bottom-up proteomics. Fractions from the three columns were collected and analyzed in a linear ion trap (LIT) mass spectrometer for improving detection of low abundant inter-cross-linked peptides. Among these three columns, the fluorophenyl column provides additional ion-exchange interaction and exhibits unique retention in separating the cross-linked peptides. The fractioned data was analyzed in pLink, showing the fluorophenyl column consistently obtained more inter-cross-linked peptide identifications than both C18 and biphenyl columns. For the BSA cross-linked sample, the identified inter-cross-linked peptide numbers of the fluorophenyl to C18 column are 136 to 102 in "low confident" results and 11 to 6 in "high confident" results. The fluorophenyl column could potentially be a better alternative for targeting the low stoichiometric inter-cross-linked peptides.

Comparative and network-based proteomic analysis of low dose ethanol- and lipopolysaccharide-induced macrophages.

Macrophages are specialized phagocytes that play an essential role in inflammation, immunity, and tissue repair. Profiling the global proteomic response of macrophages to microbial molecules such as bacterial lipopolysaccharide is key to understanding fundamental mechanisms of inflammatory disease. Ethanol is a widely abused substance that has complex effects on inflammation. Reports have indicated that ethanol can activate or inhibit the lipopolysaccharide receptor, Toll-like Receptor 4, in different settings, with important consequences for liver and neurologic inflammation, but the underlying mechanisms are poorly understood. To profile the sequential effect of low dose ethanol and lipopolysaccharide on macrophages, a gel-free proteomic technique was applied to RAW 264.7 macrophages. Five hundred four differentially expressed proteins were identified and quantified with high confidence using ≥ 5 peptide spectral matches. Among these, 319 proteins were shared across all treatment conditions, and 69 proteins were exclusively identified in ethanol-treated or lipopolysaccharide-stimulated cells. The interactive impact of ethanol and lipopolysaccharide on the macrophage proteome was evaluated using bioinformatics tools, enabling identification of differentially responsive proteins, protein interaction networks, disease- and function-based networks, canonical pathways, and upstream regulators. Five candidate protein coding genes (PGM2, ISYNA1, PARP1, and PSAP) were further validated by qRT-PCR that mostly related to glucose metabolism and fatty acid synthesis pathways. Taken together, this study describes for the first time at a systems level the interaction between ethanol and lipopolysaccharide in the proteomic programming of macrophages, and offers new mechanistic insights into the biology that may underlie the impact of ethanol on infectious and inflammatory disease in humans.

Inflammatory Proteomic Network Analysis of Statin-treated and Lipopolysaccharide-activated Macrophages.

A significant component of immune biology research is the investigation of protein encoding genes that play central roles in contributing inflammatory response. A gel-free quantitative bottom-up proteomics study was performed on immune cell macrophages after the combined treatment of lipopolysaccharide (LPS) and statin drugs using mass spectrometry and a detailed bioinformatics analyses were conducted. Systematic bioinformatics analysis was applied for discovering novel relationships among proteins and effects of statin and lipopolysaccharide in macrophage cells. Based on gene ontology, majority of protein encoding genes was involved in metabolic and cellular processes and are actively associated with binding, structural molecular, and catalytic activity. Notably, proteomic data analyzed by Ingenuity Pathway Analysis (IPA), discovered the plectin and prohibitin 2 protein interactions network and inflammatory-disease based protein networks. Two up-regulated proteins, plectin and prohibitin 2, were further validated by immunoblotting. Plectin was also cross-validated by immunocytochemistry, since its expression was highly modulated by statin but inhibited during LPS-stimulation. Collectively, the significant up-regulation of plectin due to the treatment of statin, suggests that statin has a significant impact on the cytoskeletal networks of cells. Plectin might have a significant role in the intermediate filament assembly and dynamics, and possibly stabilizing and crosslinking intermediate filament networks.

Arginine-Selective Chemical Labeling Approach for Identification and Enrichment of Reactive Arginine Residues in Proteins.

Modification of arginine residues using dicarbonyl compounds is a common method to identify functional or reactive arginine residues in proteins. Arginine undergoes several kinds of posttranslational modifications in these functional residues. Identifying these reactive residues confidently in a protein or large-scale samples is a very challenging task. Several dicarbonyl compounds have been utilized, and the most effective ones are phenylglyoxal and cyclohexanedione. However, tracking these reactive arginine residues in a protein or large-scale protein samples using a chemical labeling approach is very challenging. Thus, the enrichment of modified peptides will provide reduced sample complexity and confident mass-spectrometric data analysis. To pinpoint arginine-labeled peptide efficiently, we developed a novel arginine-selective enrichment reagent. For the first time, we conjugated an azide tag in a widely used dicarbonyl compound cyclohexanedione. This provided us the ability to enrich modified peptides using a bio-orthogonal click chemistry and the biotin-avidin affinity chromatography. We evaluated the reagent in several standard peptides and proteins. Three standard peptides, bradykinin, substance P, and neurotensin, were labeled with this cyclohexanedione-azide reagent. Click labeling of modified peptides was tested by spiking the peptides in a myoglobin protein digest. A protein, RNase A, was also labeled with the reagent, and after click chemistry and biotin-avidin affinity chromatography, we identified two selective arginine residues. We believe this strategy will be an efficient way for identifying functional and reactive arginine residues in a protein or protein mixtures.

Globular domain structure and function of restriction-like-endonuclease LINEs: similarities to eukaryotic splicing factor Prp8.

BACKGROUND: R2 elements are a clade of early branching Long Interspersed Elements (LINEs). LINEs are retrotransposable elements whose replication can have profound effects on the genomes in which they reside. No crystal or EM structures exist for the reverse transcriptase (RT) and linker regions of LINEs. RESULTS: Using limited proteolysis as a probe for globular domain structure, we show that the protein encoded by the Bombyx mori R2 element has two major globular domains: (1) a small globular domain consisting of the N-terminal zinc finger and Myb motifs, and (2) a large globular domain consisting of the RT, linker, and type II restriction-like endonuclease (RLE). Further digestion of the large globular domain occurred within the RT. Mapping these RT cleavages onto an updated model of the R2Bm RT indicated that the thumb of the RT was largely protected from proteolytic cleavage. The crystal structure of the large globular domain of Prp8, a eukaryotic splicing factor, was a major template used in building the R2Bm RT model, particularly the thumb region. The large fragment of Prp8 consists not only of a RT similar to R2Bm, but also an RLE and a linker connecting the two regions. The linker sequences adjacent to the RLE in LINEs and Prp8 share a set of two important α-helices and a (presumptive) knuckle/ßßα structural motif that are closely associated with the thumb. The RLEs of LINEs and Prp8 share a unique catalytic core residue spacing as well as other key residues. CONCLUSIONS: The protein encoded by RLE LINEs consists of two major globular domains. The larger of the two globular domain contains the RT, linker, and RLE and is similar to the large fragment of the spliceosomal protein Prp8. The similarities are suggestive of possible common ancestry.