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Briefings in Functional Genomics and Proteomics Advance Access published online on May 10, 2006
Briefings in Functional Genomics and Proteomics, doi:10.1093/bfgp/ell021
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© Oxford University Press, 2006, All rights reserved. For permissions please email: journals.permissions@oxfordjournals.org

Original Article

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

Martin E. Barrios-Llerena, Poh Kuan Chong, Chee Sian Gan, Ambrosius P. L. Snijders, Kenneth F. Reardon, and Phillip C. Wright *

* To whom correspondence should be addressed.
Phillip C. Wright, E-mail: P.C.Wright{at}Sheffield.ac.uk


  Abstract

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 N2 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.

Keywords: Shotgun proteomics; cyanobacteria; membrane; insoluble; abundance; Anabaena variabilis.

Martin Barrios-Llerena is a graduate student at The University of Sheffield.

Poh Kuan Chong is a graduate student at The University of Sheffield.

Chee Sian Gan is a graduate student at The University of Sheffield.

Ambrosius ‘Bram’ Snijders is a graduate student at The University of Sheffield.

Kenneth F. Reardon is a professor of chemical and biological engineering at Colorado State University, USA. He and his research group are involved in projects that use proteomics as a tool to solve problems in bioprocessing, environmental biotechnology and biomedical engineering.

Phillip Wright is a professor of systems biology in the Department of Chemical and Process Engineering, The University of Sheffield, UK. His laboratory develops generic tools to aid the understanding of biological systems for chemical, environmental and medical applications.


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