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.

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.

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.

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.

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.

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 Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling.

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as ß-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.

Differential Tandem Mass Spectrometry-Based Cross-Linker: A New Approach for High Confidence in Identifying Protein Cross-Linking.

Chemical cross-linking and mass spectrometry are now widely used to analyze large-scale protein-protein interactions. The major challenge in cross-linking approaches is the complexity of the mass spectrometric data. New approaches are required that can identify cross-linked peptides with high-confidence and establish a user-friendly analysis protocol for the biomedical scientific community. Here, we introduce a novel cross-linker that can be selectively cleaved in the gas phase using two differential tandem mass-spectrometric fragmentation methods, such as collision-induced or electron transfer dissociation (CID and ETD). This technique produces two signature mass spectra of the same cross-linked peptide, thereby producing high confidence in identifying the sites of interaction. Further tandem mass spectrometry can also give additional confidence on the peptide sequences. We demonstrate a proof-of-concept for this method using standard peptides and proteins. Peptides and proteins were cross-linked and their fragmentation characteristics were analyzed using CID and ETD tandem mass spectrometry. Two sequential cleavages unambiguously identified cross-linked peptides. In addition, the labeling efficiency of the new cross-linker was evaluated in macrophage immune cells after stimulation with the microbial ligand lipopolysaccharide and subsequent pulldown experiments with biotin-avidin affinity chromatography. We believe this strategy will help advance insights into the structural biology and systems biology of cell signaling.

Mass spectrometry cleavable strategy for identification and differentiation of prenylated peptides.

Prenylation of protein (farnesylation and geranylgeranylation) is involved in several human cancers, such as pancreatic, colon, and acute myeloid leukemia as well as Hutchinson-Gilford progeria syndrome (HGPS), a genetic disease that is associated with premature aging for children. Current biochemical methods are not very efficient in identifying and differentiating large-scale prenylations in vivo or in vitro. There are limited methods available for large-scale detection of prenylated proteins using mass spectrometry and no methods currently available which can distinguish farnesylation and geranylgeranylation modification in a single experimental setup. In this study, a simple and novel method for detection and distinction of large-scale prenylated peptides using mass spectrometry-cleavable approaches was developed. The method utilizes simple chemistry on the prenyl group and cleavable properties of a sulfoxide group in the gas phase to produce a signature mass spectrum during tandem mass spectrometric events. The characteristic masses lost from the modified prenylated peptides distinguished the types of prenylation. We also introduced epoxy groups in the prenylation sites of the proteins to make them more hydrophilic and enrichable from complex samples. Stability of the epoxide group was also studied under liquid chromatography-mass spectrometry (LC-MS) conditions. The proof-of-concept of this method was established using prenylated peptides which mimicked the prenyl motifs in the proteins. We believe this method will advance the identification and differentiation of the types of prenylation in proteins in large-scale studies and will improve significantly our knowledge of the mechanism of cancer, cancer treatments, and diagnosis.

Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine-modified proteins by mass spectrometry.

RATIONALE: Proteolytic cleavages generate active precursor proteins by creating new N-termini in the proteins. A number of strategies have recently been published regarding the enrichment of original or newly formed N-terminal peptides using guanidination of lysine residues and amine-reactive reagents. For effective enrichment of N-terminal peptides, the efficiency of trypsin proteolysis on homoarginine (guanidinated) modified proteins must be understood and simple and versatile solid-phase N-terminal capture strategies should be developed. METHODS: We present here a mass spectrometry (MS)-based study to evaluate and optimize the trypsin proteolysis on a guanidinated-modified protein. Trypsin proteolysis was studied using different amounts of trypsin to modified protein ratios. To capture the original N-termini, after guanidination of proteins, original N-termini were acetylated and the proteins were digested with trypsin. The newly formed N-terminal tryptic peptides were captured with a new amine reactive acid-cleavable solid-phase reagent. The original N-terminal peptides were then collected from the supernatant of the solution. RESULTS: We demonstrated a detailed study of the efficiency of enzyme trypsin on homoarginine-modified proteins. We observed that the rate of hydrolysis of homoarginine residues compared to their lysine/arginine counterparts were slower but generally cleaved after an overnight digestion period depending on the protein to protease concentration ratios. Selectivity of the solid-phase N-terminal reagent was studied by enrichment of original N-terminal peptides from two standard proteins, ubiquitin and RNaseS. CONCLUSIONS: We found enzyme trypsin is active in the guanidinated form of the protein depending on the enzyme to protein concentrations, time and the proximity of arginine residues in the sequence. The novel solid-phase capture reagent also successfully enriched N-terminal peptides from the standard protein mixtures. We believe this trypsin proteolysis study on homoarginine-modified proteins and our simple and versatile solid-phase capture strategy could be very useful for enrichment and sequence determination of proteins N-termini by MS.

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.

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.

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.

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.

Xlink-identifier: an automated data analysis platform for confident identifications of chemically cross-linked peptides using tandem mass spectrometry.

Chemical cross-linking combined with mass spectrometry provides a powerful method for identifying protein-protein interactions and probing the structure of protein complexes. A number of strategies have been reported that take advantage of the high sensitivity and high resolution of modern mass spectrometers. Approaches typically include synthesis of novel cross-linking compounds, and/or isotopic labeling of the cross-linking reagent and/or protein, and label-free methods. We report Xlink-Identifier, a comprehensive data analysis platform that has been developed to support label-free analyses. It can identify interpeptide, intrapeptide, and deadend cross-links as well as underivatized peptides. The software streamlines data preprocessing, peptide scoring, and visualization and provides an overall data analysis strategy for studying protein-protein interactions and protein structure using mass spectrometry. The software has been evaluated using a custom synthesized cross-linking reagent that features an enrichment tag. Xlink-Identifier offers the potential to perform large-scale identifications of protein-protein interactions using tandem mass spectrometry.

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.

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).

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.