Dynamics of protein damage in yeast frataxin mutant exposed to oxidative stress.

Oxidative stress and protein carbonylation is implicated in aging and various diseases such as neurodegenerative disorders, diabetes, and cancer. Therefore, the accurate identification and quantification of protein carbonylation may lead to the discovery of new biomarkers. We have developed a new method that combines avidin affinity selection of carbonylated proteins with iTRAQ labeling and LC fractionation of intact proteins. This simple LC-based workflow is an effective technique to reduce sample complexity, minimize technical variation, and enable simultaneous quantification of four samples. This method was used to determine protein oxidation in an iron accumulating mutant of Saccharomyces cerevisiae exposed to oxidative stress. Overall, 31 proteins were identified with 99% peptide confidence, and of those, 27 proteins were quantified. Most of the identified proteins were associated with energy metabolism (32.3%), and cellular defense, transport, and folding (38.7%), suggesting a drop in energy production and reducing power of the cells due to the damage of glycolytic enzymes and decrease in activity of enzymes involved in protein protection and regeneration. In addition, the oxidation sites of seven proteins were identified and their estimated position also indicated a potential impact on the enzymatic activities. Predicted 3D structures of peroxiredoxin (TSA1) and thioredoxin II (TRX2) revealed close proximity of all oxidized amino acid residues to the protein active sites.

[1]  Trong Khoa Pham,et al.  Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). , 2007, Journal of proteome research.

[2]  S. Lignon,et al.  Carbonylated Proteins Are Detectable Only in a Degradation-Resistant Aggregate State in Escherichia coli , 2008, Journal of bacteriology.

[3]  E. Cabiscol,et al.  Identification of the Major Oxidatively Damaged Proteins inEscherichia coli Cells Exposed to Oxidative Stress* , 1998, The Journal of Biological Chemistry.

[4]  N. Bykova,et al.  Identification of oxidised proteins in the matrix of rice leaf mitochondria by immunoprecipitation and two-dimensional liquid chromatography-tandem mass spectrometry. , 2004, Phytochemistry.

[5]  F. Regnier,et al.  Proteomic analysis of carbonylated proteins in two‐dimensional gel electrophoresis using avidin‐fluorescein affinity staining , 2004, Electrophoresis.

[6]  A. Sickmann,et al.  Identification of novel centrosomal proteins in Dictyostelium discoideum by comparative proteomic approaches. , 2006, Journal of proteome research.

[7]  George Perry,et al.  Oxidative stress and neurotoxicity. , 2008, Chemical research in toxicology.

[8]  D. Radisky,et al.  The Yeast Frataxin Homologue Mediates Mitochondrial Iron Efflux , 1999, The Journal of Biological Chemistry.

[9]  B. Biteau,et al.  Oxidative stress responses in yeast , 2003 .

[10]  Brad T. Sherman,et al.  DAVID: Database for Annotation, Visualization, and Integrated Discovery , 2003, Genome Biology.

[11]  David L Tabb,et al.  Efficient and specific trypsin digestion of microgram to nanogram quantities of proteins in organic-aqueous solvent systems. , 2006, Analytical chemistry.

[12]  中尾 光輝,et al.  KEGG(Kyoto Encyclopedia of Genes and Genomes)〔和文〕 (特集 ゲノム医学の現在と未来--基礎と臨床) -- (データベース) , 2000 .

[13]  Albert Sickmann,et al.  Precise protein quantification based on peptide quantification using iTRAQ™ , 2007, BMC Bioinformatics.

[14]  Trong Khoa Pham,et al.  Isobaric tags for relative and absolute quantitation (iTRAQ) reproducibility: Implication of multiple injections. , 2006, Journal of proteome research.

[15]  A. Schmidt,et al.  A novel strategy for quantitative proteomics using isotope‐coded protein labels , 2005, Proteomics.

[16]  E. Herrero,et al.  Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae. , 2000, The Journal of biological chemistry.

[17]  R. W. Gracy,et al.  A double stain for total and oxidized proteins from two-dimensional fingerprints. , 1998, Analytical biochemistry.

[18]  F. Foury,et al.  Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria , 1997, FEBS letters.

[19]  F. Regnier,et al.  Primary amine coding as a path to comparative proteomics , 2006, Proteomics.

[20]  T. Griffin,et al.  Gel‐free mass spectrometry‐based high throughput proteomics: Tools for studying biological response of proteins and proteomes , 2006, Proteomics.

[21]  D. Berg,et al.  Brain iron pathways and their relevance to Parkinson's disease. , 2001, Journal of neurochemistry.

[22]  H. Vinters,et al.  The Role of Oxidative Stress in the Pathophysiology of Cerebrovascular Lesions in Alzheimer's Disease , 2002, Brain pathology.

[23]  M. Mann,et al.  4. Proteomic Analysis of Posttranslational Modifications , 2013 .

[24]  Steven C Hall,et al.  Assessing the effects of diurnal variation on the composition of human parotid saliva: quantitative analysis of native peptides using iTRAQ reagents. , 2005, Analytical chemistry.

[25]  T. Cotter,et al.  Identification of carbonylated proteins by MALDI-TOF mass spectroscopy reveals susceptibility of ER. , 2004, Biochemical and biophysical research communications.

[26]  M. Mann,et al.  Proteomic analysis of post-translational modifications , 2003, Nature Biotechnology.

[27]  O. Jensen Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. , 2004, Current opinion in chemical biology.

[28]  R. Levine Carbonyl modified proteins in cellular regulation, aging, and disease. , 2002, Free radical biology & medicine.

[29]  Jeffrey S. Miller,et al.  iTRAQ is a useful method to screen for membrane-bound proteins differentially expressed in human natural killer cell types. , 2007, Journal of proteome research.

[30]  Kai A. Reidegeld,et al.  Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research , 2007, Proteomics.

[31]  L. Benson,et al.  Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia. , 2000, American journal of human genetics.

[32]  D. Moinier,et al.  Existence of Abnormal Protein Aggregates in Healthy Escherichia coli Cells , 2007, Journal of bacteriology.

[33]  C. Maier,et al.  New role for an old probe: affinity labeling of oxylipid protein conjugates by N'-aminooxymethylcarbonylhydrazino d-biotin. , 2006, Analytical chemistry.

[34]  Stefani N. Thomas,et al.  High-Throughput Proteomic-Based Identification of Oxidatively Induced Protein Carbonylation in Mouse Brain , 2003, Pharmaceutical Research.

[35]  M. Pandolfo,et al.  Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin. , 1997, Science.

[36]  P Bork,et al.  The phylogenetic distribution of frataxin indicates a role in iron-sulfur cluster protein assembly. , 2001, Human molecular genetics.

[37]  S. David,et al.  Age-Related Changes in Iron Homeostasis and Cell Death in the Cerebellum of Ceruloplasmin-Deficient Mice , 2006, The Journal of Neuroscience.

[38]  E. Skrzydlewska,et al.  [Oxidative modification of proteins during aging]. , 2001, Postepy higieny i medycyny doswiadczalnej.

[39]  K. Lilley,et al.  Comparative proteomics of clathrin-coated vesicles , 2006, The Journal of cell biology.

[40]  I. Alafuzoff,et al.  Proteomic analysis of protein oxidation in Alzheimer's disease brain , 2002, Electrophoresis.

[41]  Thomas Nyström,et al.  Role of oxidative carbonylation in protein quality control and senescence , 2005, The EMBO journal.

[42]  A. Vivancos,et al.  The peroxiredoxin Tpx1 is essential as a H2O2 scavenger during aerobic growth in fission yeast. , 2007, Molecular biology of the cell.

[43]  E. Cabiscol,et al.  Major targets of iron-induced protein oxidative damage in frataxin-deficient yeasts are magnesium-binding proteins. , 2008, Free radical biology & medicine.

[44]  J. Watkins,et al.  Diabetes, oxidative stress, and antioxidants: A review , 2003, Journal of biochemical and molecular toxicology.

[45]  K. Parker,et al.  Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents*S , 2004, Molecular & Cellular Proteomics.

[46]  H. Sies,et al.  Oxidative stress: oxidants and antioxidants , 1997, Experimental physiology.

[47]  Crispin J. Miller,et al.  Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells. , 2006, Blood.

[48]  D J Jamieson,et al.  Oxidative stress responses of the yeast Saccharomyces cerevisiae , 1998, Yeast.

[49]  Xiang Zhang,et al.  In-gel stable isotope labeling for relative quantification using mass spectrometry , 2006, Nature Protocols.

[50]  Kelvin H. Lee,et al.  Quantitative analysis of protein expression using amine‐specific isobaric tags in Escherichia coli cells expressing rhsA elements , 2005, Proteomics.

[51]  F. Regnier,et al.  Oxidative stress studies in yeast with a frataxin mutant: a proteomics perspective. , 2010, Journal of proteome research.

[52]  P. Patel,et al.  Friedreich's Ataxia: Autosomal Recessive Disease Caused by an Intronic GAA Triplet Repeat Expansion , 1996, Science.

[53]  Hamid Mirzaei,et al.  Affinity chromatographic selection of carbonylated proteins followed by identification of oxidation sites using tandem mass spectrometry. , 2005, Analytical chemistry.

[54]  E. Stadtman,et al.  Determination of carbonyl content in oxidatively modified proteins. , 1990, Methods in enzymology.

[55]  Stefani N. Thomas,et al.  Reduced neuronal expression of synaptic transmission modulator HNK‐1/neural cell adhesion molecule as a potential consequence of amyloid β‐mediated oxidative stress: a proteomic approach , 2005, Journal of neurochemistry.