Proteomic Characterization of the Oral Pathogen Filifactor alocis Reveals Key Inter-Protein Interactions of Its RTX Toxin: FtxA.

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod that has been isolated from a variety of oral infections including periodontitis, peri-implantitis, and odontogenic abscesses. As a newly emerging pathogen, its type strain has been investigated for pathogenic properties, yet little is known about its virulence variations among strains. We previously screened the whole genome of nine clinical oral isolates and a reference strain of F. alocis, and they expressed a novel RTX toxin, FtxA. In the present study, we aimed to use label-free quantification proteomics to characterize the full proteome of those ten F. alocis strains. A total of 872 proteins were quantified, and 97 among them were differentially expressed in FtxA-positive strains compared with the negative strains. In addition, 44 of these differentially expressed proteins formed 66 pairs of associations based on their predicted functions, which included clusters of proteins with DNA repair/mediated transformation and catalytic activity-related function, indicating different biosynthetic activities among strains. FtxA displayed specific interactions with another six intracellular proteins, forming a functional cluster that could discriminate between FtxA-producing and non-producing strains. Among them were FtxB and FtxD, predicted to be encoded by the same operon as FtxA. While revealing the broader qualitative and quantitative proteomic landscape of F. alocis, this study also sheds light on the deeper functional inter-relationships of FtxA, thus placing this RTX family member into context as a major virulence factor of this species.

Mass Spectrometry in Proteomics: Technologies, Methods, and Research Applications for the Life Sciences.

Mass spectrometry is a powerful tool in the hand of life science researchers, who constantly develop and apply new methods for the investigation of biomolecules, such as proteins, peptides, metabolites, lipids, and glycans. In this review, we will discuss the importance of mass spectrometry for the life science sector, with a special focus on the most relevant current applications in the field of proteomics. Moreover, we will comment on the factors that research groups should consider when setting up a mass spectrometry laboratory, and on the fundamental role played by academic core facilities and industrial service providers.

Gas-Phase Fragmentation of ADP-Ribosylated Peptides: Arginine-Specific Side-Chain Losses and Their Implication in Database Searches.

ADP-ribosylation is a reversible post-translational modification of proteins that has been linked to many biological processes. The identification of ADP-ribosylated proteins and particularly of their acceptor amino acids remains a major challenge. The attachment sites of the modification are difficult to localize by mass spectrometry (MS) because of the labile nature of the linkage and the complex fragmentation pattern of the ADP-ribose in MS/MS experiments. In this study we performed a comprehensive analysis of higher-energy collisional dissociation (HCD) spectra acquired from ADP-ribosylated peptides which were modified on arginine, serine, glutamic acid, aspartic acid, tyrosine, or lysine residues. In addition to the fragmentation of the peptide backbone, various cleavages of the ADP-ribosylated amino acid side chains were investigated. We focused on gas-phase fragmentations that were specific either to ADP-ribosylated arginine or to ADP-ribosylated serine and other O-linked ADP-ribosylations. The O-glycosidic linkage between ADP-ribose and serine, glutamic acid, or aspartic acid was the major cleavage site, making localization of these modification sites difficult. In contrast, the bond between ADP-ribose and arginine was relatively stable. The main cleavage site was the inner bond of the guanidine group, which resulted in the formation of ADP-ribosylated carbodiimide and of ornithine in place of modified arginine. Taking peptide fragment ions resulting from this specific cleavage into account, a considerably larger number of peptides containing ADP-ribosylated arginine were identified in database searches. Furthermore, the presence of diagnostic ions and of losses of fragments from peptide ions allowed us, in most cases, to distinguish between ADP-ribosylated arginine and serine residues.

A rice Serine/Threonine receptor-like kinase regulates arbuscular mycorrhizal symbiosis at the peri-arbuscular membrane.

In terrestrial ecosystems most plant species live in mutualistic symbioses with nutrient-delivering arbuscular mycorrhizal (AM) fungi. Establishment of AM symbioses includes transient, intracellular formation of fungal feeding structures, the arbuscules. A plant-derived peri-arbuscular membrane (PAM) surrounds the arbuscules, mediating reciprocal nutrient exchange. Signaling at the PAM must be well coordinated to achieve this dynamic cellular intimacy. Here, we identify the PAM-specific Arbuscular Receptor-like Kinase 1 (ARK1) from maize and rice to condition sustained AM symbiosis. Mutation of rice ARK1 causes a significant reduction in vesicles, the fungal storage structures, and a concomitant reduction in overall root colonization by the AM fungus Rhizophagus irregularis. Arbuscules, although less frequent in the ark1 mutant, are morphologically normal. Co-cultivation with wild-type plants restores vesicle and spore formation, suggesting ARK1 function is required for the completion of the fungal life-cycle, thereby defining a functional stage, post arbuscule development.

Phosphoproteomic-based kinase profiling early in influenza virus infection identifies GRK2 as antiviral drug target.

Although annual influenza epidemics affect around 10% of the global population, current treatment options are limited and development of new antivirals is needed. Here, using quantitative phosphoproteomics, we reveal the unique phosphoproteome dynamics that occur in the host cell within minutes of influenza A virus (IAV) infection. We uncover cellular kinases required for the observed signaling pattern and find that inhibition of selected candidates, such as the G protein-coupled receptor kinase 2 (GRK2), leads to decreased IAV replication. As GRK2 has emerged as drug target in heart disease, we focus on its role in IAV infection and show that it is required for viral uncoating. Replication of seasonal and pandemic IAVs is severely decreased by specific GRK2 inhibitors in primary human airway cultures and in mice. Our study reveals the IAV-induced changes to the cellular phosphoproteome and identifies GRK2 as crucial node of the kinase network that enables IAV replication.

Proteomic Characterization of the Heart and Skeletal Muscle Reveals Widespread Arginine ADP-Ribosylation by the ARTC1 Ectoenzyme.

The clostridium-like ecto-ADP-ribosyltransferase ARTC1 is expressed in a highly restricted manner in skeletal muscle and heart tissue. Although ARTC1 is well studied, the identification of ARTC1 targets in vivo and subsequent characterization of ARTC1-regulated cellular processes on the proteome level have been challenging and only a few ARTC1-ADP-ribosylated targets are known. Applying our recently developed mass spectrometry-based workflow to C2C12 myotubes and to skeletal muscle and heart tissues from wild-type mice, we identify hundreds of ARTC1-ADP-ribosylated proteins whose modifications are absent in the ADP-ribosylome of ARTC1-deficient mice. These proteins are ADP-ribosylated on arginine residues and mainly located on the cell surface or in the extracellular space. They are associated with signal transduction, transmembrane transport, and muscle function. Validation of hemopexin (HPX) as a ARTC1-target protein confirmed the functional importance of ARTC1-mediated extracellular arginine ADP-ribosylation at the systems level.

Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography-Tandem Mass Spectrometry.

Protein ADP-ribosylation is a covalent, reversible posttranslational modification (PTM) catalyzed by ADP-ribosyltransferases (ARTs). Proteins can be either mono- or poly-ADP-ribosylated under a variety of physiological and pathological conditions. To understand the functional contribution of protein ADP-ribosylation to normal and disease/stress states, modified protein and corresponding ADP-ribose acceptor site identification is crucial. Since ADP-ribosylation is a transient and relatively low abundant PTM, systematic and accurate identification of ADP-ribose acceptor sites has only recently become feasible. This is due to the development of specific ADP-ribosylated protein/peptide enrichment methodologies, as well as technical advances in high-accuracy liquid chromatography-tandem mass spectrometry (LC-MS/MS). The standardized protocol described here allows the identification of ADP-ribose acceptor sites in in vitro ADP-ribosylated proteins and will, thus, contribute to the functional characterization of this important PTM.

The extracellular proteome of two Bifidobacterium species reveals different adaptation strategies to low iron conditions.

BACKGROUND: Bifidobacteria are among the first anaerobic bacteria colonizing the gut. Bifidobacteria require iron for growth and their iron-sequestration mechanisms are important for their fitness and possibly inhibit enteropathogens. Here we used combined genomic and proteomic analyses to characterize adaptations to low iron conditions of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2, 2 strains isolated from the feces of iron-deficient African infants and selected for their high iron-sequestering ability. RESULTS: Analyses of the genome contents revealed evolutionary adaptation to low iron conditions. A ferric and a ferrous iron operon encoding binding proteins and transporters were found in both strains. Remarkably, the ferric iron operon of B. pseudolongum PV8-2 is not found in other B. pseudolongum strains and likely acquired via horizontal gene transfer. The genome B. kashiwanohense PV20-2 harbors a unique region encoding genes putatively involved in siderophore production. Additionally, the secretomes of the two strains grown under low-iron conditions were analyzed using a combined genomic-proteomic approach. A ferric iron transporter was found in the secretome of B. pseudolongum PV8-2, while ferrous binding proteins were detected in the secretome of B. kashiwanohense PV20-2, suggesting different strategies to take up iron in the strains. In addition, proteins such as elongation factors, a glyceraldehyde-3-phosphate dehydrogenase, and the stress proteins GroEL and DnaK were identified in both secretomes. These proteins have been previously associated with adhesion of lactobacilli to epithelial cells. CONCLUSION: Analyses of the genome and secretome of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2 revealed different adaptations to low iron conditions and identified extracellular proteins for iron transport. The identified extracellular proteins might be involved in competition for iron in the gastrointestinal tract.

Basement membrane protein ladinin-1 and the MIF-CD44-ß1 integrin signaling axis are implicated in laryngeal cancer metastasis.

Laryngeal squamous cell carcinoma (LSCC) is the most common form of malignant disease in the head and neck region characterized by frequent occurrence of metastases in the neck lymph nodes early in the disease onset. In the presented study, we performed quantitative proteomic profiling of patient-matched primary tumor and adjacent non-tumorous tissues derived from metastatic LSCC as to identify new protein candidates with potential diagnostic and therapeutic significance. Obtained results revealed for the first time involvement of the basement membrane protein ladinin-1 in laryngeal cancer metastases. Alterations in the cellular microenvironment that propel metastatic events in laryngeal cancer include activation of MIF-CD44-ß1 integrin signal transduction pathway and induction of downstream signaling mediated by NF-κB and Src tyrosine kinase, which ultimately impinge on cytoskeletal dynamics and architecture resulting in increased cellular motility and invasiveness. In this context, particularly interesting finding is upregulation of several actin-binding proteins novel to laryngeal cancer pathogenesis including coronin-1C and plastin-2, whose functional significance in laryngeal carcinogenesis has yet to be established. We also detected for the first time a complete loss of afamin in metastatic laryngeal cancer tissues, which warrants further studies into its use as a possible marker for monitoring disease progression and/or treatment outcome.

Protein adsorption steers blood contact activation on engineered cobalt chromium alloy oxide layers.

Biomaterials upon implantation are immediately covered by blood proteins which direct the subsequent blood activation. These early events determine the following cascade of biological reactions and consequently the long-term success of implants. The ability to modulate surface properties of biomaterials is therefore of considerable clinical significance. Goal of this study was an in-depth understanding of the biological response to cobalt chromium stent alloys with engineered surface oxide layers, which showed altered body reactions in vivo. We analyzed in vitro the biological events following initial blood contact on engineered cobalt chromium surfaces featuring said oxide layers. Surface-specific blood reactions were confirmed by scanning electron microscopy and the adsorbed protein layers were characterized by mass spectrometry. This powerful proteomics tool allowed the identification and quantification of over hundred surface-adhering proteins. Proteins associated with the coagulation cascade, platelet adhesion and neutrophil function correlated with the various blood surface activations observed. Furthermore, results of pre-coated surfaces with defined fibrinogen-albumin mixtures suggest that neutrophil adhesion was controlled by fibrinogen orientation and conformation rather than quantity. This study highlights the importance of controlling the biological response in the complex protein-implant surface interactions and the potential of the surface modifications to improve the clinical performance of medical implants. STATEMENT OF SIGNIFICANCE: The blood contact activation of CoCr alloys is determined by their surface oxide layer properties. Modifications of the oxide layer affected the total amount of adsorbed proteins and the composition of the adsorbed protein layer. Additionally fibrinogen coatings mediated the surface-dependent neutrophil adhesion in a concentration-independent manner, indicating the influence of conformation and/or orientation of the adsorbed protein. Despite the complexity of protein-implant interactions, this study highlights the importance of understanding and controlling mechanisms of protein adhesion in order to improve and steer the performance of medical implants. It shows that modification of the surface oxide layer is a very attractive strategy to directly functionalize metallic implant surfaces and optimize their blood interaction for the desired orthopedic or cardiovascular applications.

UV-induced retinal proteome changes in the rat model of age-related macular degeneration.

Age-related macular degeneration (AMD) is characterized by irreversible damage of photoreceptors in the central posterior part of the retina, called the macula and is the most common cause of vision loss in those aged over 50. A growing body of evidence shows that cumulative long-term exposure to UV radiation may be harmful to the retina and possibly leads to AMD irrespective of age. In spite of many research efforts, cellular and molecular mechanisms leading to UV-induced retinal damage and possibly retinal diseases such as AMD are not completely understood. In the present study we explored damage mechanisms accounting for UV-induced retinal phototoxicity in the rats exposed to UVA and UVB irradiation using a proteomics approach. Our study showed that UV irradiation induces profound changes in the retinal proteomes of the rats associated with the disruption of energy homeostasis, oxidative stress, DNA damage response and structural and functional impairments of the interphotoreceptor matrix components and their cell surface receptors such as galectins. Two small leucine-rich proteoglycans, biglycan and lumican, were identified as phototoxicity biomarkers associated with UV-induced disruption of interphotoreceptor matrix (IPM). In addition, UVB induced activation of Src kinase, which could account for cytoskeletal rearrangements in the retina was observed at the proteomics level. Pharmacological intervention either to target Src kinase with the aim of preventing cytoskeletal rearrangements in the retinal pigment epithelium (RPE) and neuronal retina or to help rebuild damaged IPM may provide fresh avenues of treatment for patients suffering from AMD.

PTM MarkerFinder, a software tool to detect and validate spectra from peptides carrying post-translational modifications.

Mass spectrometry (MS) analysis of peptides carrying post-translational modifications is challenging due to the instability of some modifications during MS analysis. However, glycopeptides as well as acetylated, methylated and other modified peptides release specific fragment ions during CID (collision-induced dissociation) and HCD (higher energy collisional dissociation) fragmentation. These fragment ions can be used to validate the presence of the PTM on the peptide. Here, we present PTM MarkerFinder, a software tool that takes advantage of such marker ions. PTM MarkerFinder screens the MS/MS spectra in the output of a database search (i.e., Mascot) for marker ions specific for selected PTMs. Moreover, it reports and annotates the HCD and the corresponding electron transfer dissociation (ETD) spectrum (when present), and summarizes information on the type, number, and ratios of marker ions found in the data set. In the present work, a sample containing enriched N-acetylhexosamine (HexNAc) glycopeptides from yeast has been analyzed by liquid chromatography-mass spectrometry on an LTQ Orbitrap Velos using both HCD and ETD fragmentation techniques. The identification result (Mascot .dat file) was submitted as input to PTM MarkerFinder and screened for HexNAc oxonium ions. The software output has been used for high-throughput validation of the identification results.

iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections.

Both isobaric tags for relative and absolute quantitation (iTRAQ) and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24 h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or label-free methods were used, and the resulting samples were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). To reduce a possible bias from quantitation software, we applied several software packages for each procedure. ProteinPilot and Scaffold Q+ software were used for iTRAQ-labeled samples, while Progenesis LC-MS and ProgenesisF-T2PQ/T3PQ were employed for label-free analyses. R (2) correlation coefficients correlated well between two software packages applied to the same datasets with values between 0.48 and 0.78 for iTRAQ-label-based quantitations and 0.5 and 0.86 for label-free quantitations. Analyses of label-free samples showed higher levels of protein up- or downregulation in comparison to iTRAQ-labeled samples. The concentration differences were further evaluated by Western blotting for four downregulated proteins. These data suggested that the label-free method was more accurate than the iTRAQ method.

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J & C. Presl) F.K. Meyer.

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state-of-the-art quantitative 2D liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size-exclusion chromatography/inductively coupled plasma mass spectrometer (SEC-ICP-MS) method, in order to identify the Zn-binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S-transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn-nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn-nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type-specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.

A bacterial-fungal metaproteomic analysis enlightens an intriguing multicomponent interaction in the rhizosphere of Lactuca sativa.

Fusarium oxysporum MSA 35 [wild-type (WT) strain] is an antagonistic isolate that protects plants against pathogenic Fusaria. This strain lives in association with ectosymbiotic bacteria. When cured of the prokaryotic symbionts [cured (CU) form], the fungus is pathogenic, causing wilt symptoms similar to those of F. oxysporum f.sp. lactucae. The aim of this study was to understand if and how the host plant Lactuca sativa contributes to the expression of the antagonistic/pathogenic behaviors of MSA 35 strains. A time-course comparative analysis of the proteomic profiles of WT and CU strains was performed. Fungal proteins expressed during the early stages of plant-fungus interaction were involved in stress defense, energy metabolism, and virulence and were equally induced in both strains. In the late phase of the interkingdom interaction, only CU strain continued the production of virulence- and energy-related proteins. The expression analysis of lettuce genes coding for proteins involved in resistance-related processes corroborated proteomic data by showing that, at the beginning of the interaction, both fungi are perceived by the plant as pathogen. On the contrary, after 8 days, only the CU strain is able to induce plant gene expression. For the first time, it was demonstrated that an antagonistic F. oxysporum behaves initially as pathogen, showing an interesting similarity with other beneficial organisms such as mychorrizae.

Insights into the gene expression profile of uncultivable hemotrophic Mycoplasma suis during acute infection, obtained using proteome analysis.

Hemotrophic mycoplasmas, bacteria without cell walls whose niche is the erythrocytes of their hosts, have never been cultivated in vitro. Therefore, knowledge of their pathogenesis is fundamental. Mycoplasma suis infects pigs, causing either acute fatal hemolytic anemia or chronic low-grade anemia, growth retardation, and immune suppression. Recently, the complete genomes of two hemotrophic mycoplasma species, M. suis and M. haemofelis, were sequenced, offering new strategies for the analysis of their pathogenesis. In this study we implemented a proteomic approach to identify M. suis proteins during acute infection by using tandem mass spectrometry. Twenty-two percent of the predicted proteins encoded in M. suis strain KI_3806 were identified. These included nearly all encoded proteins of glycolysis and nucleotide metabolism. The proteins for lipid metabolism, however, were underrepresented. A high proportion of the detected proteins are involved in information storage and processing (72.6%). In addition, several proteins of different functionalities, i.e., posttranslational modification, membrane genesis, signal transduction, intracellular trafficking, inorganic ion transport, and defense mechanisms, were identified. In its reduced genome, M. suis harbors 65.3% (strain Illinois) and 65.9% (strain KI_3806) of the genes encode hypothetical proteins. Of these, only 6.3% were identified at the proteome level. All proteins identified in this study are present in both M. suis strains and are encoded in more highly conserved regions of the genome sequence. In conclusion, our proteome approach is a further step toward the elucidation of the pathogenesis and life cycle of M. suis as well as the establishment of an in vitro cultivation system.

Identification of distinct amino acids as ADP-ribose acceptor sites by mass spectrometry.

ADP-ribosylation is a well-known post-translational protein modification, which regulates a variety of -cellular processes. The proteins able to catalyze mono- or poly ADP-ribosylation of proteins belong to the family of ADP-ribosyltransferases. A variety of nuclear proteins has been described to be ADP-ribosylated, including ARTD1 itself and histone proteins. Despite intensive research during the last 40 years, the acceptor amino acids in ARTD1 or histone proteins could be identified and confirmed only recently by MS/MS and by site-directed mutagenesis. The establishment of a standardized protocol including the specific enrichment of ADP-ribosylated proteins and peptides and subsequent mass spectrometric analysis allows the identification of ADP-ribose acceptor sites of modified proteins and to address the functional contribution of ADP-ribosylation in vitro as well as in vivo.

Nematotoxicity of Marasmius oreades agglutinin (MOA) depends on glycolipid binding and cysteine protease activity.

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Galα1,3Gal/GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca(2+) concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.

iTRAQ-based analysis of changes in the cassava root proteome reveals pathways associated with post-harvest physiological deterioration.

The short storage life of harvested cassava roots is an important constraint that limits the full potential of cassava as a commercial food crop in developing countries. We investigated the molecular changes during physiological deterioration of cassava root after harvesting using isobaric tags for relative and absolute quantification (iTRAQ) of proteins in soluble and non-soluble fractions prepared during a 96 h post-harvest time course. Combining bioinformatic approaches to reduce information redundancy for unsequenced or partially sequenced plant species, we established a comprehensive proteome map of the cassava root and identified quantitatively regulated proteins. Up-regulation of several key proteins confirmed that physiological deterioration of cassava root after harvesting is an active process, with 67 and 170 proteins, respectively, being up-regulated early and later after harvesting. This included regulated proteins that had not previously been associated with physiological deterioration after harvesting, such as linamarase, glutamic acid-rich protein, hydroxycinnamoyl transferase, glycine-rich RNA binding protein, ß-1,3-glucanase, pectin methylesterase, maturase K, dehydroascorbate reductase, allene oxide cyclase, and proteins involved in signal pathways. To confirm the regulation of these proteins, activity assays were performed for selected enzymes. Together, our results show that physiological deterioration after harvesting is a highly regulated complex process involving proteins that are potential candidates for biotechnology approaches to reduce such deterioration.

Extracellular hemoglobin polarizes the macrophage proteome toward Hb-clearance, enhanced antioxidant capacity and suppressed HLA class 2 expression.

Peripheral blood monocytes and macrophages are the only cell population with a proven hemoglobin (Hb) clearance capacity through the CD163 scavenger receptor pathway. Hb detoxification and related adaptive cellular responses are assumed to be essential processes to maintaining tissue homeostasis and promoting wound healing in injured tissues. Using a dual platform mass spectrometry analysis with MALDI-TOF/TOF and LTQ-Orbitrap instruments combined with isobaric tag for relative and absolute quantitation (iTRAQ), we analyzed how Hb exposure could modulate the macrophage phenotype on a proteome level. We identified and relatively quantified 3691 macrophage proteins, representing the largest human macrophage proteome published to date. The Hb polarized macrophage phenotype was characterized by an induced Hb:Hp-CD163-HO1-ferritin pathway and enhanced antioxidant enzymes while suppression of HLA class 2 was the most prominent effect. Enhanced Hb clearance and antioxidant capacity together with reduced antigen presentation might therefore be essential qualities of Hb polarized macrophages in wounded tissues and hemorrhage or atherosclerotic plaques.