A Comparative Proteomic Strategy for Subcellular Proteome Research

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of “contaminating” proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these “contaminants” represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM >1.0, while another 79 proteins have an ICAT ratio of PM:CM <1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM >1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM <1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM >1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.

[1]  M. Ünlü,et al.  Difference gel electrophoresis. A single gel method for detecting changes in protein extracts , 1997, Electrophoresis.

[2]  Ventsislava Y Petrova,et al.  Dual targeting of yeast catalase A to peroxisomes and mitochondria. , 2004, The Biochemical journal.

[3]  Bradford W. Gibson,et al.  Characterization of the human heart mitochondrial proteome , 2003, Nature Biotechnology.

[4]  G. Attardi.  Role of mitochondrial DNA in human aging. , 2002, Mitochondrion.

[5]  I. Weissman,et al.  Cloning and characterization of cyclophilin C-associated protein: a candidate natural cellular ligand for cyclophilin C. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[6]  D. Hochstrasser,et al.  Proteomics meets cell biology: The establishment of subcellular proteomes , 2000, Electrophoresis.

[7]  Hanno Langen,et al.  The rat liver mitochondrial proteins , 2002, Electrophoresis.

[8]  V. Trézéguet,et al.  The human mitochondrial ADP/ATP carriers: kinetic properties and biogenesis of wild-type and mutant proteins in the yeast S. cerevisiae. , 2002, Biochemistry.

[9]  Guoyao Wu,et al.  Compartmentation and kinetics of urea cycle enzymes in porcine enterocytes. , 1998, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[10]  B Chance,et al.  Hydroperoxide metabolism in mammalian organs. , 1979, Physiological reviews.

[11]  J. Garin,et al.  A survey of the plant mitochondrial proteome in relation to development , 2002, Proteomics.

[12]  J. García-Sancho,et al.  Calcium Influx through Receptor-operated Channel Induces Mitochondria-triggered Paraptotic Cell Death* 210 , 2003, The Journal of Biological Chemistry.

[13]  P. Maeng,et al.  Identification of a cryptic N-terminal signal in Saccharomyces cerevisiae peroxisomal citrate synthase that functions in both peroxisomal and mitochondrial targeting. , 2000, Journal of Biochemistry (Tokyo).

[14]  L. Huber,et al.  Organelle Proteomics Implications for Subcellular Fractionation in Proteomics , 2003 .

[15]  Marjan S. Bolouri,et al.  Integrated Analysis of Protein Composition, Tissue Diversity, and Gene Regulation in Mouse Mitochondria , 2003, Cell.

[16]  K. Davies,et al.  Protein degradation in mitochondria: implications for oxidative stress, aging and disease: a novel etiological classification of mitochondrial proteolytic disorders. , 2001, Mitochondrion.

[17]  N Howell,et al.  Clinical mitochondrial genetics , 1999, Journal of medical genetics.

[18]  Etienne Gagnon,et al.  Organelle proteomics: looking at less to see more. , 2003, Trends in cell biology.

[19]  M. Pandolfo.  Iron metabolism and mitochondrial abnormalities in Friedreich ataxia. , 2002, Blood cells, molecules & diseases.

[20]  Kathryn S Lilley,et al.  Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. , 2002, Current opinion in chemical biology.

[21]  Albert Sickmann,et al.  The proteome of Saccharomyces cerevisiae mitochondria , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A. Paetau,et al.  Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer , 2002, Nature Genetics.

[23]  R. Terkeltaub,et al.  Invited review: the mitochondrion in osteoarthritis. , 2002, Mitochondrion.

[24]  Rod B. Watson,et al.  The use of isotope-coded affinity tags (ICAT) to study organelle proteomes in Arabidopsis thaliana. , 2004, Biochemical Society transactions.

[25]  Steven W. Taylor,et al.  Global organellar proteomics. , 2003, Trends in biotechnology.

[26]  K. Nakai,et al.  PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. , 1999, Trends in biochemical sciences.

[27]  J. Maassen.  Mitochondrial diabetes: pathophysiology, clinical presentation, and genetic analysis. , 2002, American journal of medical genetics.

[28]  S. Gygi,et al.  Quantitative analysis of complex protein mixtures using isotope-coded affinity tags , 1999, Nature Biotechnology.

[29]  Pei Hao,et al.  A High-throughput Approach for Subcellular Proteome , 2004, Molecular & Cellular Proteomics.

[30]  T. Sherer,et al.  Environment, Mitochondria, and Parkinson's Disease , 2002, The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry.

[31]  S. Kish,et al.  Mitochondria in Alzheimer's disease. , 2002, International review of neurobiology.

[32]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[33]  K. Murayama,et al.  One‐step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing‐thawing and two‐dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles , 2001, Electrophoresis.

[34]  R. Aebersold,et al.  Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry , 2001, Nature Biotechnology.

[35]  C. Ponting,et al.  Evidence that peroxiredoxins are novel members of the thioredoxin fold superfamily , 1998, Protein science : a publication of the Protein Society.

[36]  K. Nakai,et al.  Review: prediction of in vivo fates of proteins in the era of genomics and proteomics. , 2001, Journal of structural biology.

[37]  Y. Maeda,et al.  71-Kilodalton Heat Shock Cognate Protein Acts as a Cellular Receptor for Syncytium Formation Induced by Human T-Cell Lymphotropic Virus Type 1 , 1998, Journal of Virology.

[38]  A. Millar,et al.  Analysis of the Arabidopsis mitochondrial proteome. , 2001, Plant physiology.

[39]  Zhi-Ping Feng,et al.  An overview on predicting the subcellular location of a protein , 2002, Silico Biol..

[40]  T. Meitinger,et al.  Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free‐flow electrophoresis , 2003, Proteomics.

[41]  S. Brunak,et al.  Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. , 2000, Journal of molecular biology.

[42]  Ruedi Aebersold,et al.  Complementary Analysis of the Mycobacterium tuberculosis Proteome by Two-dimensional Electrophoresis and Isotope-coded Affinity Tag Technology * , 2004, Molecular & Cellular Proteomics.

[43]  M. Dreger.  Proteome analysis at the level of subcellular structures. , 2003, European journal of biochemistry.

[44]  M. Consalvo,et al.  Cytochrome c-induced cytosolic nicotinamide adenine dinucleotide oxidation, mitochondrial permeability transition, and apoptosis. , 2003, Archives of biochemistry and biophysics.

[45]  Ruedi Aebersold,et al.  Quantitative Proteome Analysis by Solid-phase Isotope Tagging and Mass Spectrometry Beads Photocleavable Linker Isotope Tag Reactive Group , 2022 .

[46]  J. Crapo,et al.  Detection of catalase in rat heart mitochondria. , 1991, The Journal of biological chemistry.

[47]  Naoya Hatano,et al.  Proteomic Analysis of Rat Liver Peroxisome , 2004, Journal of Biological Chemistry.

[48]  J. Tower,et al.  Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster. , 2002, Genetics.

[49]  C. Falany,et al.  Rat liver bile acid CoA:amino acid N-acyltransferase: expression, characterization, and peroxisomal localization. , 2003, Journal of lipid research.

[50]  P. Chinnery.  Inheritance of mitochondrial disorders. , 2002, Mitochondrion.

[51]  Paolo Gasparini,et al.  Ethylmalonic encephalopathy is caused by mutations in ETHE1, a gene encoding a mitochondrial matrix protein. , 2004, American journal of human genetics.

[52]  G von Heijne,et al.  Prediction of organellar targeting signals. , 2001, Biochimica et biophysica acta.

[53]  M. Mann,et al.  Large-scale Proteomic Analysis of the Human Spliceosome References , 2006 .

[54]  Shengbing Huang,et al.  Functional expression and processing of rat choline dehydrogenase precursor. , 2003, Biochemical and biophysical research communications.

[55]  D. Wallace.  Mitochondrial diseases in man and mouse. , 1999, Science.

[56]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[57]  C. Florentz,et al.  Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders. , 2002, Biochemistry.

[58]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.

[59]  Mathias Dreger,et al.  Subcellular proteomics , 2021, Nature Reviews Methods Primers.

[60]  Matthias Mann,et al.  Mass spectrometric-based approaches in quantitative proteomics. , 2003, Methods.

[61]  Zhirong Sun,et al.  Support vector machine approach for protein subcellular localization prediction , 2001, Bioinform..

[62]  A. Kastaniotis,et al.  The Yeast Mitochondrial Proteome, a Study of Fermentative and Respiratory Growth* , 2004, Journal of Biological Chemistry.

[63]  P Vincens,et al.  Computational method to predict mitochondrially imported proteins and their targeting sequences. , 1996, European journal of biochemistry.

[64]  A. Millar,et al.  Towards an Analysis of the Rice Mitochondrial Proteome1 , 2003, Plant Physiology.

[65]  Robert F. Murphy,et al.  Towards a Systematics for Protein Subcellular Location: Quantitative Description of Protein Localization Patterns and Automated Analysis of Fluorescence Microscope Images , 2000, ISMB.

[66]  Bertrand Séraphin,et al.  Recent developments in the analysis of protein complexes 1 , 2004, FEBS letters.

[67]  R. Moots,et al.  The Mitochondrial Network of Human Neutrophils: Role in Chemotaxis, Phagocytosis, Respiratory Burst Activation, and Commitment to Apoptosis 1 , 2003, The Journal of Immunology.

[68]  Wayne F. Patton,et al.  Two-dimensional gel electrophoresis; better than a poke in the ICAT? , 2002, Current opinion in biotechnology.

[69]  E. Crivellato,et al.  Mitochondrial localization of APE/Ref-1 in thyroid cells. , 2001, Mutation research.

[70]  Ruedi Aebersold,et al.  Quantitative proteomic analysis of Myc oncoprotein function , 2002, The EMBO journal.