Shotgun proteomics of cyanobacteria--applications of experimental and data-mining techniques.

Cyanobacteria are photosynthetic bacteria notable for their ability to produce hydrogen and a variety of interesting secondary metabolites. As a result of the growing number of completed cyanobacterial genome projects, the development of post-genomics analysis for this important group has been accelerating. DNA microarrays and classical two-dimensional gel electrophoresis (2DE) were the first technologies applied in such analyses. In many other systems, 'shotgun' proteomics employing multi-dimensional liquid chromatography and tandem mass spectrometry has proven to be a powerful tool. However, this approach has been relatively under-utilized in cyanobacteria. This study assesses progress in cyanobacterial shotgun proteomics to date, and adds a new perspective by developing a protocol for the shotgun proteomic analysis of the filamentous cyanobacterium Anabaena variabilis ATCC 29413, a model for N(2) fixation. Using approaches for enhanced protein extraction, 646 proteins were identified, which is more than double the previous results obtained using 2DE. Notably, the improved extraction method and shotgun approach resulted in a significantly higher representation of basic and hydrophobic proteins. The use of protein bioinformatics tools to further mine these shotgun data is illustrated through the application of PSORTb for localization, the grand average hydropathy (GRAVY) index for hydrophobicity, LipoP for lipoproteins and the exponentially modified protein abundance index (emPAI) for abundance. The results are compared with the most well-studied cyanobacterium, Synechocystis sp. PCC 6803. Some general issues in shotgun proteome identification and quantification are then addressed.

[1]  T. Rabilloud.  Membrane proteins ride shotgun , 2003, Nature Biotechnology.

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

[3]  O. Ohara,et al.  Towards a proteome project of cyanobacterium Synechocystis sp. strain PCC6803: Linking 130 protein spots with their respective genes , 1997, Electrophoresis.

[4]  Jeroen Krijgsveld,et al.  Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics , 2003, Nature Biotechnology.

[5]  M. Molloy,et al.  Membrane proteins and proteomics: Un amour impossible? , 2000, Electrophoresis.

[6]  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.

[7]  M. Hagemann,et al.  Proteomic screening of salt‐stress‐induced changes in plasma membranes of Synechocystis sp. strain PCC 6803 , 2006, Proteomics.

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

[9]  J. Yates,et al.  An automated multidimensional protein identification technology for shotgun proteomics. , 2001, Analytical chemistry.

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

[11]  Phillip C Wright,et al.  Identification and characterization of the Sulfolobus solfataricus P2 proteome. , 2005, Journal of proteome research.

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

[13]  F. Cross,et al.  Accurate quantitation of protein expression and site-specific phosphorylation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J. Betton,et al.  Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli , 2001, Molecular microbiology.

[15]  H. Pakrasi,et al.  2D‐isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803 , 1998, FEBS letters.

[16]  M. Mann,et al.  Exponentially Modified Protein Abundance Index (emPAI) for Estimation of Absolute Protein Amount in Proteomics by the Number of Sequenced Peptides per Protein*S , 2005, Molecular & Cellular Proteomics.

[17]  R. Aebersold,et al.  Mass spectrometry-based proteomics , 2003, Nature.

[18]  J. Yates,et al.  Direct analysis of protein complexes using mass spectrometry , 1999, Nature Biotechnology.

[19]  Robert J Beynon,et al.  Metabolic Labeling of Proteins for Proteomics* , 2005, Molecular & Cellular Proteomics.

[20]  Jun Sun,et al.  Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803 , 2000, Electrophoresis.

[21]  Proteomics of Synechocystis sp. Strain PCC 6803 , 2002, Molecular & Cellular Proteomics.

[22]  Julian P Whitelegge,et al.  Mass spectrometry for high throughput quantitative proteomics in plant research: lessons from thylakoid membranes. , 2004, Plant physiology and biochemistry : PPB.

[23]  M. Medina,et al.  Reversible hydrogenase of Anabaena variabilis ATCC 29413: catalytic properties and characterization of redox centres , 1996, FEBS letters.

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

[25]  Joshua E. Elias,et al.  Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. , 2003, Journal of proteome research.

[26]  J. Yates,et al.  Large-scale analysis of the yeast proteome by multidimensional protein identification technology , 2001, Nature Biotechnology.

[27]  Paula Tamagnini,et al.  Hydrogenases and Hydrogen Metabolism of Cyanobacteria , 2002, Microbiology and Molecular Biology Reviews.

[28]  Martin Ester,et al.  Sequence analysis PSORTb v . 2 . 0 : Expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis , 2004 .

[29]  T. Jansén,et al.  Proteomic analysis of heterotrophy in Synechocystis sp. PCC 6803 , 2006, Proteomics.

[30]  Andrew Emili,et al.  In silico proteome analysis to facilitate proteomics experiments using mass spectrometry , 2003, Proteome Science.

[31]  H. Bothe,et al.  Transcriptional Analysis of Hydrogenase Genes in the Cyanobacteria Anacystis nidulans and Anabaena variabilis Monitored by RT-PCR , 2000, Current Microbiology.

[32]  Phillip C Wright,et al.  Novel approach for peptide quantitation and sequencing based on 15N and 13C metabolic labeling. , 2005, Journal of proteome research.

[33]  Ronald J. Moore,et al.  Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry. , 2002, Journal of proteome research.

[34]  A. Krogh,et al.  Prediction of lipoprotein signal peptides in Gram‐negative bacteria , 2003, Protein science : a publication of the Protein Society.

[35]  H. Pakrasi,et al.  Proteomics of Synechocystis sp. Strain PCC 6803 , 2000, Molecular & Cellular Proteomics.

[36]  B. Whitton.  Diversity, Ecology, and Taxonomy of the Cyanobacteria , 1992 .

[37]  Phillip C Wright,et al.  Cyanobacterial postgenomic research and systems biology. , 2003, Trends in biotechnology.

[38]  R D Appel,et al.  Protein identification and analysis tools in the ExPASy server. , 1999, Methods in molecular biology.

[39]  Paulette M. Vignais,et al.  Sustained Photoevolution of Molecular Hydrogen in a Mutant of Synechocystis sp. Strain PCC 6803 Deficient in the Type I NADPH-Dehydrogenase Complex , 2004, Journal of bacteriology.

[40]  Y. Nakamura,et al.  Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions (supplement). , 1996, DNA research : an international journal for rapid publication of reports on genes and genomes.

[41]  Jeffrey C. Smith,et al.  Robust method for proteome analysis by MS/MS using an entire translated genome: demonstration on the ciliome of Tetrahymena thermophila. , 2005, Journal of proteome research.

[42]  I. Suzuki,et al.  Proteomic study of the soluble proteins from the unicellular cyanobacterium Synechocystis sp. PCC6803 using automated matrix‐assisted laser desorption/ionization‐time of flight peptide mass fingerprinting , 2002, Proteomics.

[43]  Ilan Beer,et al.  Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins , 2005, Proteomics.

[44]  E. Schleiff,et al.  Proteomic analysis of the outer membrane of Anabaena sp. strain PCC 7120. , 2005, Journal of proteome research.

[45]  Richard D. Smith,et al.  Global analysis of the membrane subproteome of Pseudomonas aeruginosa using liquid chromatography-tandem mass spectrometry. , 2004, Journal of proteome research.

[46]  O. Ohara,et al.  Cyano2Dbase updated: Linkage of 234 protein spots to corresponding genes through N‐terminal microsequencing , 1999 .

[47]  X. Yao,et al.  Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. , 2001, Analytical chemistry.

[48]  D. Hall,et al.  Hydrogen production by Anabaena variabilis PK84 under simulated outdoor conditions. , 2000, Biotechnology and bioengineering.

[49]  Phillip C. Wright,et al.  Marine cyanobacteria—a prolific source of natural products , 2001 .

[50]  T. Kieselbach,et al.  Isolation of Outer Membrane of Synechocystis sp. PCC 6803 and Its Proteomic Characterization* , 2004, Molecular & Cellular Proteomics.

[51]  P. Wright,et al.  Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803 , 2005, Proteomics.