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

Abstract

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. 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. The application of reductionism and experimental manipulation in the 20th century biological research has generated important insights into functional processes of life. Based on this successful paradigm, researchers rationally dissected multiple underlying molecular mechanisms of “living systems” and efficiently developed drugs. However, drugs or dietary interventions can interfere with numerous proteins in hundreds of different cell types in various tissues, not to mention potential crosstalk on various levels of biological organization. Not surprisingly, conventional in vitro and lengthy preclinical studies that target only specific marker molecules often missed out important but unexpected physiological effects of drug treatment. Although complex biological phenomena such as physiological outcomes of disease treatment depend on various individual molecules, they are based on in vivo network properties, which cannot be adequately described or explained by “parts of the sum” of mechanistic events. Soft-ionization mass spectrometry (MS) has been widely validated as a tool for precise quantitative analysis of biomolecules (1Domon B. Aebersold R. Mass spectrometry and protein analysis.Science. 2006; 312: 212-217Crossref PubMed Scopus (1619) Google Scholar, 2Sauer S. Kliem M. Mass spectrometry tools for the classification and identification of bacteria.Nat. Rev. Microbiol. 2010; 8: 74-82Crossref PubMed Scopus (358) Google Scholar), and isotope-labeling procedures were introduced to detect protein expression, primarily in cell culture models (3Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1317) Google Scholar, 4Ong S.E. Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).Nat. Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (684) Google Scholar). Previous attempts of using mass spectrometry for protein quantification in mammalian disease models were limited to analysis of a small number of usually abundant proteins, which made comprehensive pathway analysis and physiological outcome prediction impossible (5Millioni R. Puricelli L. Iori E. Arrigoni G. Tessari P. The effects of rosiglitazone and high glucose on protein expression in endothelial cells.J. Proteome Res. 2010; 9: 578-584Crossref PubMed Scopus (4) Google Scholar, 6Ahmed M. Neville M.J. Edelmann M.J. Kessler B.M. Karpe F. Proteomic Analysis of Human Adipose Tissue After Rosiglitazone Treatment Shows Coordinated Changes to Promote Glucose Uptake.Obesity. 2010; 18: 27-34Crossref PubMed Scopus (55) Google Scholar). Recent technical pilot studies provided extensive information on the protein inventories of different mouse tissues (7Villén J. Beausoleil S.A. Gerber S.A. Gygi S.P. Large-scale phosphorylation analysis of mouse liver.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1488-1493Crossref PubMed Scopus (628) Google Scholar, 8Huttlin E.L. Jedrychowski M.P. Elias J.E. Goswami T. Rad R. Beausoleil S.A. Villén J. Haas W. Sowa M.E. Gygi S.P. A tissue-specific atlas of mouse protein phosphorylation and expression.Cell. 2010; 143: 1174-1189Abstract Full Text Full Text PDF PubMed Scopus (1219) Google Scholar), and isotope-labeled mice have been introduced as a resource for accurate protein quantification (9Krüger M. Moser M. Ussar S. Thievessen I. Luber C.A. Forner F. Schmidt S. Zanivan S. Fässler R. Mann M. SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function.Cell. 2008; 134: 353-364Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). The development of diet-induced obesity and diabetes is a complex pathophysiological process involving a number of interacting organs, in which chronic hyperglycemia and hyperlipidemia lead to cumulative damaging effects on metabolic tissues such as skeletal muscle, liver, and adipose tissues. As we show here, disease processes and in particular physiological effects of drug treatment are largely determined by the actual cellular protein expression levels and post-translational modifications of proteins. Whereas analyses of single protein changes were mostly uninformative, quantitative protein set enrichment analysis was an efficient tool to monitor tissue-specific responses of anti-diabetic treatments. This approach allows for investigation of interacting molecular and physiological processes that occur on the pathway level, and enables sensitive, unbiased and robust diagnostic detection of treatments in vivo. In this pilot study, we compared the effects of the drug rosiglitazone (RSG) 1The abbreviations used are:A1Amorfrutin A1DIODiet-Induced ObesityHFDHigh-Fat DietLFDLow-Fat DietPDMProtein Distance MatrixPSEAProtein Set Enrichment AnalysisRSGRosiglitazoneSILACStable isotope labeling by amino acids in cell culture. 1The abbreviations used are:A1Amorfrutin A1DIODiet-Induced ObesityHFDHigh-Fat DietLFDLow-Fat DietPDMProtein Distance MatrixPSEAProtein Set Enrichment AnalysisRSGRosiglitazoneSILACStable isotope labeling by amino acids in cell culture., which has been associated with a number of undesirable side effects (10Kahn B.B. McGraw T.E. Rosiglitazone, PPARgamma, and type 2 diabetes.N. Engl. J. Med. 2010; 363: 2667-2669Crossref PubMed Scopus (53) Google Scholar), and the plant-derived amorfrutin A1 (A1) (11Weidner C. de Groot J.C. Prasad A. Freiwald A. Quedenau C. Kliem M. Witzke A. Kodelja V. Han C.T. Giegold S. Baumann M. Klebl B. Siems K. Müller-Kuhrt L. Schürmann A. Schüler R. Pfeiffer A.F. Schroeder F.C. Büssow K. Sauer S. Amorfrutins are potent antidiabetic dietary natural products.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7257-7262Crossref PubMed Scopus (163) Google Scholar) in diet-induced obesity (DIO) mice. Both compounds' antidiabetic effects appear to be derived from activation of the peroxisome proliferator-activated receptor gamma (PPARγ). Amorfrutin A1 Diet-Induced Obesity High-Fat Diet Low-Fat Diet Protein Distance Matrix Protein Set Enrichment Analysis Rosiglitazone Stable isotope labeling by amino acids in cell culture. Amorfrutin A1 Diet-Induced Obesity High-Fat Diet Low-Fat Diet Protein Distance Matrix Protein Set Enrichment Analysis Rosiglitazone Stable isotope labeling by amino acids in cell culture. Animal studies were carried out according to internationally approved standards as described recently (11Weidner C. de Groot J.C. Prasad A. Freiwald A. Quedenau C. Kliem M. Witzke A. Kodelja V. Han C.T. Giegold S. Baumann M. Klebl B. Siems K. Müller-Kuhrt L. Schürmann A. Schüler R. Pfeiffer A.F. Schroeder F.C. Büssow K. Sauer S. Amorfrutins are potent antidiabetic dietary natural products.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7257-7262Crossref PubMed Scopus (163) Google Scholar), and have been validated and approved by the State Office of Health and Social Affairs Berlin (LAGeSo). The animals were maintained one per cage under temperature-, humidity- and light-controlled conditions (22 °C, 50% humidity, 12 h light/12 h dark-cycle). The health status and behavior of mice were examined daily. Mice had ad libitum access to food and water. Mice and food were to changes in and food diet was diet was the study, we mice to a treatment. mice were with a for 12 The mice were and to mice were with or A1 or A number of physiological such as glucose or were as described Mice of only with were as and tissues were and the A1 study, mice were and to treatment. the mice were with or with of A1 A number of physiological were as described recently (11Weidner C. de Groot J.C. Prasad A. Freiwald A. Quedenau C. Kliem M. Witzke A. Kodelja V. Han C.T. Giegold S. Baumann M. Klebl B. Siems K. Müller-Kuhrt L. Schürmann A. Schüler R. Pfeiffer A.F. Schroeder F.C. Büssow K. Sauer S. Amorfrutins are potent antidiabetic dietary natural products.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7257-7262Crossref PubMed Scopus (163) Google Scholar). After of mice were by and tissues were and changes in expression of important metabolic peripheral target tissues such as and we used of mice per treatment were in and in of cell culture we tissues to tissues with and mice we not The only was the for the for which we used and from As this can a number of with a M. K. Mann M. Large-scale quantification in tissues by a SILAC 8: PubMed Scopus Google Scholar). from mice were in and a was In we tissue proteins in were with a for to SILAC mouse tissues were in the The of and mouse tissues under investigation allows for quantitative protein as tissues types are to were for the and for were to protein protein were with the SILAC and protein was a After the from high to molecular were and into small of The was as described P. L. with mass spectrometry to of protein Biol. 2008; PubMed Scopus Google Scholar). was in and of were used for was in the liver of the were and with and SILAC were with of protein from liver tissues of or a of of proteins and in were and in with was for h by a in 2 were with The of the were on a and was according to J. Gygi S.P. The enrichment approach for phosphorylation analysis by mass Protoc. 2008; PubMed Scopus Google Scholar), enrichment for according to M. K. Mann M. Large-scale quantification in tissues by a SILAC 8: PubMed Scopus Google Scholar), and according to A. Mann M. of and allows analysis of the Proteome Res. 8: PubMed Scopus Google Scholar). The of allows to the in a SILAC mouse tissues are the from culture not the protein of in example, as detected by mass spectrometry, is in liver but only to a limited in for quantitative analysis mass spectrometry was carried out by to a mass the was using a with were using a from 2 to a of A was for A of one mass of was by in the with for were for for were to for J. I. M. M. Mann M. A practical to the for quantitative Protoc. PubMed Scopus Google Scholar), based on the liver with liver we used based on The mouse from was used for The was set to to be the As SILAC we used but in the of the we and modifications were as modifications during protein and for the and was used as or was set with a of two missed Mass for and was and and and are in was with two Protein were as the of the of the by were set and were SILAC protein were determined as the of to the analysis of treatment using the derived from two in high of the as was observed in studies F. R. G. Mann M. Large-scale proteomics analysis of the 8: Full Text Full Text PDF PubMed Scopus Google Scholar). In SILAC were for adipose which were was used to of of the liver Protein expression changes were using this treatment treatment 2 treatment treatment and including can be of pathways we protein set enrichment analysis using the tool A. P. S. A. set enrichment a approach for expression Natl. Acad. Sci. U.S.A. 2005; PubMed Scopus Google Scholar, J. M. E. S. M. Mann M. analysis of protein tissue and regulation in mouse Full Text Full Text PDF PubMed Scopus Google Scholar), to an a set of proteins and two diet or treatments. the were not protein set enrichment set of and with expression and protein SILAC were using the from the regulated pathways only as the was were carried out with F. S. 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The using to small molecules, and 2006; PubMed Scopus Google Scholar), which is a of expression from with small molecules in with a used a protein of the pathway and detected by to a for the This was for with expression of small drugs have been to we a of drugs that are to or from the M. M. I. P. A side resource to effects of Biol. 2010; PubMed Scopus Google Scholar). enrichment analysis A. A. enrichment PubMed Scopus Google Scholar, The of PubMed Scopus Google Scholar) was using a tool with an underlying to of mammalian proteins with the that several to enrichment based on the of in the compared with to be associated with a of proteins. using of regulated with one phosphorylation from mass spectrometry-based we and of pathways in the of we and proteins with the Analysis was and using with of the according to the were and in with for was on using the was determined by the was generated from the the was The was on were according to the were in biological expression analyses were carried out using were and the were for significant detection and expression treatment according to the and were according to the of the expression were in to the was as the of the that were regulated in the by and protein A of 50% was as only by analyses and protein expression on single level, or proteins that were in and were for with or treatment. analyses on the pathway level, we compared the pathway regulation determined by for and for protein expression, and only regulated pathways with for or analyses were for treatment and of was with the was determined by quantitative as described recently (11Weidner C. de Groot J.C. Prasad A. Freiwald A. Quedenau C. Kliem M. Witzke A. Kodelja V. Han C.T. Giegold S. Baumann M. Klebl B. Siems K. Müller-Kuhrt L. Schürmann A. Schüler R. Pfeiffer A.F. Schroeder F.C. Büssow K. Sauer S. Amorfrutins are potent antidiabetic dietary natural products.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7257-7262Crossref PubMed Scopus (163) Google Scholar). was by the and with of the were and and liver tissues were and a of in was with for After for °C, the were and in the in tissues were and a of in was with for After for °C, the were and using the according to the levels in were in a quantification and were by as by of liver tissues were and a of in using the to for and for and of were to of or with and for 2 were with of and glucose was with a glucose of in liver, tissues were and a of in a tissue and using with for were for and °C, and were in a and with the was quantitatively using a quantification according to the tissues from mice a high-fat diet or a rosiglitazone were with a tissue in 2 and with a The was for The the was for and from the were were with and developed with the analysis was with a the protein A mouse was as were using of the was determined according to A. 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R. and of liver in 2010; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). As is for the various treatments in a of detected proteins was regulated to only proteins were or molecular pathways from protein expression of regulated individual proteins, we protein set enrichment analysis R. R. A. A. analysis of using and enrichment 2010; PubMed Scopus Google Scholar, S. T. In analysis of using by protein set enrichment analysis and 2010; 9: Full Text Full Text PDF PubMed Scopus Google Scholar), an of set enrichment analysis A. P. S. A. set enrichment a approach for expression Natl. Acad. Sci. U.S.A. 2005; PubMed Scopus Google Scholar). 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The using to small molecules, and 2006; PubMed Scopus Google Scholar) we compared the regulation of the protein pathway with expression of drugs with side effects as or we from analysis of mice to only of treatment with the for and of drugs were to protein expression from the In tissue pathway which were for of and and of pathways were the expression protein set analysis in the was for potential cardiovascular of treatment an preclinical and can be used as a for drug the natural A1 changes of the and in the obesity usually to liver of of in K. A. to in 2012; PubMed Scopus Google Scholar). In the of we observed of proteins in phosphorylation and and Whereas and A1 treatment significant on protein expression, application of A1 during feeding the of metabolic obesity to an of proteins and of proteins in and liver as observed in P. M. W. 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PubMed Scopus Google of feeding and treatment on different of proteins in phosphorylation in liver treatment of mice with diet or high-fat diet with rosiglitazone or amorfrutin A1 feeding or amorfrutin A1 during feeding as in of feeding and treatment on liver are as S.E. of feeding and treatment on of feeding and treatment on liver protein of feeding and treatment on liver are as S.E. processes in the liver, we the liver of and and phosphorylation in to proteins. observed mostly phosphorylation by and In the of amino phosphorylation were and phosphorylation of liver proteins was by treatment with A1 enrichment analysis phosphorylation of that are to be in of and is by phosphorylation pathways and in particular A1 this protein with of liver compared with a from to in the to regulation of phosphorylation pathways is for the protein network in and for phosphorylation of the factor which was efficiently by A1 treatment of with and cell E. J. J. a for and and cell Full Text PDF PubMed Scopus Google Scholar, A. L. A. M.E. proteins and to by Full Text Full Text PDF PubMed Scopus Google Scholar). effects of A1 treatment by phosphorylation pathways and cell In in liver and A1 feeding to mice had significant but A1 the liver from set enrichment analysis is based on the that changes in expression the of or interacting This functional to be as is based on a of biological organization. or as are important the individual is and the is small which is the in robust or physiological or in common disease In complex from in the expression of of multiple or proteins. as in this study, an to protein changes a of organization. in with quantitative mass spectrometry is an tool to functional or of complex physiological crosstalk in an in vivo the unbiased insights to to pathways underlying physiological The of multiple proteins in diagnostic for preclinical of drug candidates. In to has the to detect expression changes on the protein level, which in information with to functional as in study, protein expression analyses can be by analyses of post-translational to regulated The observed expression of proteins, and the of and protein expression on the but not on the individual protein level, the that physiological effects as a of the of various biomolecules in vivo R. C. Kodelja V. S. Haas S. M. B. A. Sauer S. analysis of activation new networks in Res. PubMed Scopus Google Scholar). the detected of regulation on the of protein the to physiological changes unbiased and robust proteins or information on the of particular proteins or protein set analysis including comprehensive protein expression and post-translational physiological pathways in mice and significant in the outcomes of treatment with two different the and the mice with for which was for potential cardiovascular In side effects several of treatment. of and detection of mass spectrometry analysis using for new mass for and analyses A. E. A. A. J. Mann M. S. Mass spectrometry-based proteomics using a mass Full Text Full Text PDF Scopus (628) Google Scholar), proteomics L. P. R. M. Aebersold R. and for 8: PubMed Scopus Google Scholar), into the actual in the of a S. B.M. J. L. in functional and Rev. 2005; PubMed Scopus Google Scholar). Using or quantitative mass spectrometry protein set analysis can be for including disease and treatment monitoring in