Proteomic Expression Changes Research Articles

Metataxonomic analysis and host proteome response in dairy cows with high and low somatic cell count: a quarter level investigation

Host response to invasive microbes in the bovine udder has an important role on the animal health and is essential to the dairy industry to ensure production of high-quality milk and reduce the mastitis incidence. To better understand the biology behind these host-microbiome interactions, we investigated the somatic cell proteomes at quarter level for four cows (collected before and after milking) using a shotgun proteomics approach. Simultaneously, we identified the quarter microbiota by amplicon sequencing to detect presence of mastitis pathogens or other commensal taxa. In total, 32 quarter milk samples were analyzed divided in two groups depending on the somatic cell count (SCC). The high SCC group (>100,000 cell/mL) included 10 samples and significant different proteome profiles were detected. Differential abundance analysis uncovers a specific expression pattern in high SCC samples revealing pathways involved in immune responses such as inflammation, activation of the complement system, migration of immune cells, and tight junctions. Interestingly, different proteome profiles were also identified in quarter samples containing one of the two mastitis pathogens, Staphylococcus aureus and Streptococcus uberis, indicating a different response of the host depending on the pathogen. Weighted correlation network analysis identified three modules of co-expressed proteins which were correlated with the SCC in the quarters. These modules contained proteins assigned to different aspects of the immune response, but also amino sugar and nucleotide sugar metabolism, and biosynthesis of amino acids. The results of this study provide deeper insights on how the proteome expression changes at quarter level in naturally infected cows and pinpoint potential interactions and important biological functions during host-microbe interaction.

Evaluating Cellular Viability by iTRAQ Proteomic Profiling.

Cellular health, functionality, response to environment, and other variables affecting cell, tissue, or organ viability are reflected in the cellular proteomes and metabolomes. These "omic" profiles are in constant flux even during normal cellular functioning, to maintain cellular homeostasis, in response to small environmental changes and maintenance of optimal cell viability. However proteomic "fingerprints" can also provide insight into cellular ageing, response to disease, adjustment to environmental changes, and other variables that impact cellular viability. A variety of proteomic methods can be used to determine qualitative and quantitative proteomic change. In this chapter, we will focus on a labeling method called isobaric tags for relative and absolute quantification (iTRAQ), which is frequently used to identify and quantify proteomic expression changes in cells and tissues.

Protein Kinase C Activation Drives a Differentiation Program in an Oligodendroglial Precursor Model through the Modulation of Specific Biological Networks.

Protein kinase C (PKC) activation induces cellular reprogramming and differentiation in various cell models. Although many effectors of PKC physiological actions have been elucidated, the molecular mechanisms regulating oligodendrocyte differentiation after PKC activation are still unclear. Here, we applied a liquid chromatography–mass spectrometry (LC–MS/MS) approach to provide a comprehensive analysis of the proteome expression changes in the MO3.13 oligodendroglial cell line after PKC activation. Our findings suggest that multiple networks that communicate and coordinate with each other may finally determine the fate of MO3.13 cells, thus identifying a modular and functional biological structure. In this work, we provide a detailed description of these networks and their participating components and interactions. Such assembly allows perturbing each module, thus describing its physiological significance in the differentiation program. We applied this approach by targeting the Rho-associated protein kinase (ROCK) in PKC-activated cells. Overall, our findings provide a resource for elucidating the PKC-mediated network modules that contribute to a more robust knowledge of the molecular dynamics leading to this cell fate transition.

Identification of Cyclophilin A as a Potential Anticancer Target of Novel Nargenicin A1 Analog in AGS Gastric Cancer Cells.

We recently discovered a novel nargenicin A1 analog, 23-demethyl 8,13-deoxynargenicin (compound 9), with potential anti-cancer and anti-angiogenic activities against human gastric adenocarcinoma (AGS) cells. To identify the key molecular targets of compound 9, that are responsible for its biological activities, the changes in proteome expression in AGS cells following compound 9 treatment were analyzed using two-dimensional gel electrophoresis (2-DE), followed by MALDI/TOF/MS. Analyses using chemical proteomics and western blotting revealed that compound 9 treatment significantly suppressed the expression of cyclophilin A (CypA), a member of the immunophilin family. Furthermore, compound 9 downregulated CD147-mediated mitogen-activated protein kinase (MAPK) signaling pathway, including c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) by inhibiting the expression of CD147, the cellular receptor of CypA. Notably, the responses of AGS cells to CypA knockdown were significantly correlated with the anticancer and antiangiogenic effects of compound 9. CypA siRNAs reduced the expression of CD147 and phosphorylation of JNK and ERK1/2. In addition, the suppressive effects of CypA siRNAs on proliferation, migration, invasion, and angiogenesis induction of AGS cells were associated with G2/M cell cycle arrest, caspase-mediated apoptosis, inhibition of MMP-9 and MMP-2 expression, inactivation of PI3K/AKT/mTOR pathway, and inhibition of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression. The specific interaction between compound 9 and CypA was also confirmed using the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) approaches. Moreover, in silico docking analysis revealed that the structure of compound 9 was a good fit for the cyclosporin A binding cavity of CypA. Collectively, these findings provide a novel molecular basis for compound 9-mediated suppression of gastric cancer progression through the targeting of CypA.

Proteomics unravels new candidate genes for Meloidogyne resistance in wild Arachis

Arachis stenosperma is a wild peanut relative exclusive to South America that harbors high levels of resistance against several pathogens, including the peanut root-knot nematode (RKN) Meloidogyne arenaria. In this study, a proteomic survey of A. stenosperma-M. arenaria interaction using 2-DE and LC-MS/MS identified approximately 1400 proteins, out of which 222 were differentially abundant (DAPs) when RKN inoculated root samples were compared to the control. Most of these DAPs were assigned to functional categories related to plant responses to pathogens including stress, glycolysis, redox and tricarboxylic acid cycle. The comparison between the transcriptome (RNA-Seq) and proteome expression changes, showed that almost 55% of these DAPs encode genes with a similar expression trend to their protein counterparts. Most of these genes were induced during RKN infection and some were related to plant defense, such as MLP-like protein 34 (MLP34), cinnamoyl-CoA reductase 1 (CCR1), enolase (ENO), alcohol dehydrogenase (ADH) and eukaryotic translation initiation factor 5A (eIF5A). The overexpression of AsMLP34 in Agrobacterium rhizogenes transgenic roots in a susceptible peanut cultivar showed a reduction in the number of M. arenaria galls and egg masses, indicating that AsMLP34 is a promising candidate gene to be exploited in breeding programs for RKN control in peanut. SignificanceThe use of an integrated approach to compare plant-nematode transcriptional and translational data enabled the identification of a new gene, AsMLP34, for Meloidogyne resistance.

THE ROLE OF OSTEOMACS IN REGULATING STEM CELL FUNCTION AND THE HEMATOPOIETIC NICHE

Networking between hematopoietic stem cells (HSC) and cells of the hematopoietic niche is not only critical for the maintenance of stem cell function, but also for the competence of the hematopoietic niche. Among the key cellular components of the niche are a group of specialized bone-resident macrophages known as osteomacs (OM). We previously documented that close interactions between OM, osteoblasts (OB) and megakaryocytes (MK) are critical for sustaining in vitro and in vivo stem and progenitor cell function. To understand the importance of OM-OB-MK interactions within the niche, we performed 3D tissue cytometry on fixed and stained unperturbed bone marrow sections. This approach identified the spatial relationships between OM, OB, MK and HSC within the niche and defined parameters, under which these cell types coexist in undamaged bone marrow. Next, we performed single cell mRNAseq and CyTOF to assess genetic and proteomic expression changes in OM following their interaction with MK. These studies revealed the upregulation of CD166 and embigin on OM via MK interactions. We then examined how these molecules impact hematopoietic function in clonogenic assays. When these assays were initiated with CD166 KO OM or treated with anti-embigin blocking antibodies, we established that loss of these surface molecules on OM caused a decline in the normal OM-mediated hematopoietic enhancing activity. Conversely, recombinant CD166 and embigin partially substituted for OM activity thus identifying potential mediators through which OM maintain hematopoietic function. Our data, for the first time, reveal intimate spatial interactions between OM, OB, MK and HSC in the hematopoietic niche. They also illustrate the importance of crosstalk between OM, OB and MK and reveal novel mediators such as CD166 and embigin that cooperate with other elements of the niche to support HSC function.

Proteome study of embryogenic competence acquisition in the callus cultures of Nothapodytes nimmoniana (J. Graham) Mabberly

Among the alternative plant sources of camptothecin (CPT), Nothapodytes nimmoniana is regarded as the most convenient source for large-scale isolation of the monoterpenoid indole anticancer alkaloid. As a result, CPT annual trade value has grossed over billion US Dollars in recent years. Somatic embryogenesis (SE) offers potential application in the rapid clonal propagation of the tree and production of the alkaloid, so as to mitigate indiscriminate harvest of its endangered natural population to meet industrial demand. However, response to the production of embryogenic callus (EC) in the in vitro cultures of N. nimmoniana is poor to scant. In the present study, two-dimensional electrophoresis (2-DE) and mass spectrometry (MaSp/MaSp) were employed in studying proteome expression changes between EC and non-embryogenic callus (NEC) of the forest tree. The results of the study showed higher metabolic and physiological processes associated with embryogenic competence acquisition in the callus cultures; high cellular oxidative stress, energy metabolism, protein synthesis, and other metabolic processes played a key role in upregulated expression of the identified proteins in EC over NEC. Putative role of the expressed proteins during embryogenic competence acquisition by N. nimmoniana callus cultures has provided some insight into the physiology of the competence acquisition through cellular roles played by oxidative stress and metabolic processes. Further studies on metabolic physiological processes associated with EC production could have application in optimizing culture conditions for mass propagation through the SE, so as to mitigate the indiscriminate harvest of endangered N. Nimmoniana natural population for CPT.

Proteome study of somatic embryogenesis in Nothapodytes nimmoniana (J. Graham) Mabberly.

Somatic embryogenesis (SE) is the most suitable biotechnological tool for the rapid clonal propagation of endangered woody plants, but many bottlenecks limit understanding its molecular and physiological processes in Nothapodytes nimmoniana. Combinations of two-dimensional electrophoresis (2-DE) and mass spectrometry (MaSp) were used to study proteomic expression changes during SE of the forest tree. Callus was induced from mature seed embryos, and embryogenic callus (EC) obtained at very low frequency after about 6month culture. Globular embryos were induced from the seed embryo-derived EC and the subsequent stages of the SE. Analysis of the extracted proteins from globular, heart/torpedo-shaped, and maturing embryo stages resolved in the 2-DE gels showed increased protein expression across developmental stages of the somatic embryos. The mass spectrometric analysis with database search aided identification of 55 out of 100 and 54 selected protein spots. Identified proteins classified by the cellular role which they perform are involved in aspects of stress responses, energy metabolism, carbon fixation, secondary metabolism, and other metabolic functions, while three proteins are of unknown cellular role. The putative role of the expressed proteins during SE provided insight into the physiology of somatic embryo development in N. nimmoniana.

Protein Palmitoylation Plays an Important Role in Trichomonas vaginalis Adherence

The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide. As an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host and a better understanding of this process is a prerequisite for the development of therapies to combat infection. In this sense, recent work has shown S-acylation as a key modification that regulates pathogenesis in different protozoan parasites. However, there are no reports indicating whether this post-translational modification is a mechanism operating in T. vaginalis In order to study the extent and function of S-acylation in T. vaginalis biology, we undertook a proteomic study to profile the full scope of S-acylated proteins in this parasite and reported the identification of 363 proteins involved in a variety of biological processes such as protein transport, pathogenesis related and signaling, among others. Importantly, treatment of parasites with the palmitoylation inhibitor 2-bromopalmitate causes a significant decrease in parasite: parasite aggregation as well as adherence to host cells suggesting that palmitoylation could be modifying proteins that are key regulators of Trichomonas vaginalis pathogenesis.

Differential Proteomic Analysis of Date Palm Leaves Infested with the Red Palm Weevil (Coleoptera: Curculionidae)

The red palm weevil, Rhynchophorus ferrugineus (Oliv.) (Coleoptera: Curculionidae), is a highly damaging pest of palm trees worldwide. The infestation is highly concealed in nature. Hence, a highly sensitive and reliable early detection technique needs to be applied in the field for identification and treatment of the infested date palms to curtail further infestation. We have recently reported the differential proteomic analysis of the date palm stem tissues associated with the red palm weevil infestation. In this study, we examine the response of date palm infested with red palm weevil based on the leaf proteome expression changes detected using two-dimensional differential gel electrophoresis (2D-DIGE) followed by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF). We observed qualitative and quantitative proteome differences between the control and weevil-infested date palm samples. The red palm weevil infestation induced specific responses attributable to weevil feeding, relative to artificially wounded trees (which were used as a control). Differential proteomics led to the identification of 32 red palm weevil infestation-specific protein spots (P ≤ 0.05 having ≥ 1.5-fold modulation) that were further subjected to mass spectrometer analysis for identification and characterization. Proteins involved in plant stress and plant defense, photosynthesis, carbohydrate utilization, and protein degradation were affected in infested plants. The differentially expressed red palm weevil infestation-specific peptides can be used as biomarkers for the identification of early infestation with this insect in date palm trees. Moreover, our study demonstrates the potential use of proteomic strategies in diagnosing phyto-infestation caused by insect pests, diseases, and perhaps even for variety selection.

Proteomic Expression Changes in Large Cerebral Arteries After Experimental Subarachnoid Hemorrhage in Rat Are Regulated by the MEK-ERK1/2 Pathway

Subarachnoid hemorrhage (SAH) is a serious clinical condition where leakage of blood into the subarachnoid space causes an acute rise in intracranial pressure and reduces cerebral blood flow, which may lead to delayed cerebral ischemia and poor outcome. In experimental SAH, we have previously shown that the outcome can be significantly improved by early inhibition of the MAPK/ERK kinase/extracellular signal-regulated kinase (MEK/ERK1/2) pathway. The aim of this study was to apply mass spectrometry to investigate the overall late effects of experimental SAH on cerebrovascular protein expression. SAH was induced in rats that were treated with the MEK1/2 inhibitor U0126 or vehicle. Neurological outcome was assessed using a battery of behavioral tests. Specific protein expression of large cerebral arteries was analyzed quantitatively with high-throughput tandem mass spectrometry. SAH resulted in a marked reduction of neurological scores, which was counteracted by U0126 treatment. Mass spectrometry analysis demonstrated regulation of 184 proteins after SAH, regulations that were in part prevented by U0126 treatment. Network analysis identified several protein networks including a strong structural network centered around 14-3-3. Additionally, protein networks with functions in mRNA metabolism and protein folding were identified. Treatment with U0126 inhibited cerebral vessel wall pERK1/2 expression and significantly improved outcome of the rats. In conclusion, we show that SAH induces a broad array of specific changes in the overall protein networks in cerebral artery smooth muscle cells and suggest that this is essential for understanding the vascular pathophysiology after SAH.

Nutrient restriction of glucose or serum results in similar proteomic expression changes in 3D colon cancer cell cultures

Nutrient restriction, also known as caloric restriction, has been extensively examined for its positive impact on lifespan, immune system boost, and aging. In addition, nutrient restriction is implicated in decreasing cancer initiation and progression. Given the phenotypic changes associated with nutrient restriction, we hypothesized significant protein expression alterations must be associated with caloric restriction. To compare the molecular and phenotypic changes caused by glucose restriction and fetal bovine serum restriction there is need for an efficient model system. We establish 3-dimensional cell culture models, known as spheroids, in the HCT 116 colorectal cancer cell line as a high throughput model for studying the proteomic changes associated with nutrient restriction. Flow cytometry was used to assess apoptosis and autophagy levels in the spheroids under nutrient restriction. Isobaric tags for relative and absolute quantification and liquid chromatography tandem mass spectrometry were used to determine differential protein abundances between the nutrient restriction conditions. We identified specific proteins that have implications in cancer progression and metastasis that are differentially regulated by restriction of either glucose or serum. These proteins include the up-regulation of sirtuin 1 and protein inhibitor of activated STAT 1 and down-regulation of multi-drug resistance protein and Zinc finger and BTB domain-containing protein 7A. The results indicate nutrient restriction causes lower apoptotic and higher autophagy rates in HCT 116 spheroids. In addition, proteins shown to be differentially regulated by both glucose and serum restriction were similarly regulated.

A predictive disease-specific protein biomarker fingerprint for clinical relapses and glucocorticoids treatment of multiple sclerosis

Abstract Despite extensive research, multiple sclerosis (MS) remains a disease that lacks a definitive prognostic test to predict imminent disease relapses. Thus, patients may undergo years of unnecessary treatments. Additionally, current treatments for MS vary significantly in efficacy between individual patients, and thus there is a critical need to develop biomarkers for treatment efficacy and resistance. To address these issues, we recently developed a high-throughput quantitative proteomics method to measure changes in proteome expression over the course of the preclinical experimental autoimmune encephalomyelitis (EAE) model. Interestingly, using the EAE model we revealed characteristic CNS-specific protein expression waves prior to the onset of clinical symptoms. Moreover, we have identified key proteins with altered expression that correlated with the therapeutic efficacy of glucocorticoid treatment. Bioinformatics analysis revealed candidate protein biomarkers to predict treatment efficacy and clinical disease course. Importantly, these proteins could be detected in serum and expression trajectories analysis identified a strong correlation of the CNS proteome to their levels in serum. Prospective studies in the EAE model using these candidate protein biomarkers showed their effectiveness in predicting clinical disease and treatment responses. Our studies suggest the utility for establishing homologous protein biomarkers in human MS patients. Finally, our work investigating the CNS proteome over the course of EAE may provide novel insights and molecular targets for disease mechanisms and treatments of MS.

Global protein expression dataset acquired during isoniazid-induced cytoprotection against H2O2 challenge in HL-60 cells.

Isoniazid (INH) is one of the first-line anti-tuberculosis drugs. Its effect on oxidative stress, however, is unknown. Here we used a model of oxidative stress by employing glucose/glucose oxidase (GOx), which (based on the availability of glucose and oxygen) is known to produce H2O2. This reaction induces oxidative stress culminating in necrotic cell death in HL-60 cells (a human promyelocytic leukemia cell line). The changes in protein levels have been quantified using global proteome expression changes through stable isotope labeling by amino acids in cell culture (SILAC) followed by LC–MS/MS analysis. A total of 1459 and 1712 proteins were identified in forward and reverse experiments, respectively. However, only 390 proteins were reproducibly identified in both samples. These 390 proteins were taken into account for further analysis which has been described in “Cytoprotective effect of isoniazid against H2O2 derived injury in HL-60 cells” [1].

2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima

The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves.

Epigenetic and Proteomic Expression Changes Promoted by Eating Addictive-Like Behavior.

An increasing perspective conceptualizes obesity and overeating as disorders related to addictive-like processes that could share common neurobiological mechanisms. In the present study, we aimed at validating an animal model of eating addictive-like behavior in mice, based on the DSM-5 substance use disorder criteria, using operant conditioning maintained by highly palatable chocolate-flavored pellets. For this purpose, we evaluated persistence of food-seeking during a period of non-availability of food, motivation for food, and perseverance of responding when the reward was associated with a punishment. This model has allowed identifying extreme subpopulations of mice related to addictive-like behavior. We investigated in these subpopulations the epigenetic and proteomic changes. A significant decrease in DNA methylation of CNR1 gene promoter was revealed in the prefrontal cortex of addict-like mice, which was associated with an upregulation of CB1 protein expression in the same brain area. The pharmacological blockade (rimonabant 3 mg/kg; i.p.) of CB1 receptor during the late training period reduced the percentage of mice that accomplished addiction criteria, which is in agreement with the reduced performance of CB1 knockout mice in this operant training. Proteomic studies have identified proteins differentially expressed in mice vulnerable or not to addictive-like behavior in the hippocampus, striatum, and prefrontal cortex. These changes included proteins involved in impulsivity-like behavior, synaptic plasticity, and cannabinoid signaling modulation, such as alpha-synuclein, phosphatase 1-alpha, doublecortin-like kinase 2, and diacylglycerol kinase zeta, and were validated by immunoblotting. This model provides an excellent tool to investigate the neurobiological substrate underlying the vulnerability to develop eating addictive-like behavior.

Identification of predictive protein biomarkers for treatment efficacy and clinical relapses of multiple sclerosis (THER7P.950)

Abstract Despite extensive research, MS remains a disease that lacks a definitive prognostic test to predict imminent disease relapses. Thus, patients may undergo years of unnecessary treatments. Additionally, current treatments for MS can produce dramatically different outcomes in different individuals and therefore there is a critical need to develop biomarkers for treatment efficacy and resistance. We have recently developed a novel quantitative proteomics method to measure changes in proteome expression over the course of experimental autoimmune encephalomyelitis (EAE). Our statistical analyses indicate a strong correlation to EAE severity and/or clinical-phase. Interestingly, we revealed characteristic CNS-specific protein expression waves prior to the onset of clinical symptoms. We are currently testing whether these protein expression waves allow us to predict the onset of clinical episodes and forecast the severity of the disease to guide treatment strategies. Additionally, we have identified changes in the CNS proteome that can be measured in serum during EAE that correlate with the therapeutic efficacy of glucocorticoid treatment. Our studies will provide proof-of-principle for developing homologous human biomarkers that may be useful to predict disease onset and treatment efficacy. Finally, the detected changes in the CNS proteome may provide insights into key mechanisms that contribute to the disease pathology and may be useful to develop new therapeutic targets for MS.

Identification of candidate predictive protein biomarkers by M2 proteomics for clinical onset and treatment efficacy of multiple sclerosis

Despite extensive research, multiple sclerosis (MS) remains a disease that lacks a definitive diagnostic test to predict imminent disease relapses. Thus, patients may undergo years of unnecessary treatments. Additionally, current treatments for MS can produce dramatically different outcomes in different individuals and therefore there is a critical need to develop biomarkers for treatment efficacy and resistance. We have recently developed a novel quantitative Microwave & Magnetic (M2) proteomics method to quantitatively measure changes in proteome expression over the course of experimental autoimmune encephalomyelitis (EAE), the standard murine animal model of MS.

The Equine CD4+ Lymphocyte Proteome

CD4+ T cells are key players in immunology and disease pathology, including relapsing autoimmune uveitis. Equine recurrent uveitis is the only spontaneous animal model for this disease in man. Knowledge about the CD4+ cell proteome is crucial for studies on possible changes in proteome expression of CD4+ effector cells in disease. For this purpose, we generated a reference dataset of the equine CD4+ cell proteome by sorting equine CD4+ lymphocytes followed by analysis of whole cell lysate as well as membrane protein fraction using mass spectrometry.

Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

Gaining understanding of common complex diseases and their treatments are the main drivers for life sciences. As we show here, comprehensive protein set analyses offer new opportunities to decipher functional molecular networks of diseases and assess the efficacy and side-effects of treatments in vivo. Using mass spectrometry, we quantitatively detected several thousands of proteins and observed significant changes in protein pathways that were (dys-) regulated in diet-induced obesity mice. Analysis of the expression and post-translational modifications of proteins in various peripheral metabolic target tissues including adipose, heart, and liver tissue generated functional insights in the regulation of cell and tissue homeostasis during high-fat diet feeding and medication with two antidiabetic compounds. Protein set analyses singled out pathways for functional characterization, and indicated, for example, early-on potential cardiovascular complication of the diabetes drug rosiglitazone. In vivo protein set detection can provide new avenues for monitoring complex disease processes, and for evaluating preclinical drug candidates.