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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: TuP-208
Session: Proteomics: New Methods
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

Development of Shotgun Proteomics Approaches to Investigate the Function of Bacterial Signal Molecules

James Ault1, David Sumpton1, Joao Rodrigues1, Jerry Thomas1, Clara Diaz2, Herman Spaink2, Jane Thomas-Oates1

1 University of York, York, United Kingdom
2 Universiteit Leiden, Leiden, Netherlands

Correspondence address: James R. Ault, University of York, Department of Chemistry, Heslington Road, York, YO10 5DD United Kingdom.

Keywords: Chromatography, Liquid -, Nanoscale Capillary; Digests, Tryptic; Electrophoresis, 2D; Proteomic.

Novel aspect: Shotgun Proteomics approaches have been developed and applied to investigate legume response to purified nodulation factors and bacterially secreted proteins. The results have been compared with a classical two dimensional electrophoresis approach.

 

Biological nitrogen fixation occurs as a result of a highly species specific interaction between legumes and rhizobial bacteria. Specificity is mediated both by the structures of bacterially derived Nodulation Factors (NFs) that bear a variety of species dependent substitutions, as well as via bacterial secreted proteins (Nop proteins). The mechanisms by which the plant root perceives and responds to the structures of NFs, and the role of the secreted proteins are both poorly understood. To better characterise these systems, we have been exploring the use of various qualitative and quantitative proteomic workflows. We have identified novel bacterially-derived proteins and plant proteins that are differentially regulated in soybean roots after exposure to purified NFs from symbiotic and non-symbiotic bacteria.

2D gel electrophoresis (2-DE) has been employed for differential proteomics of three proteins extracts: proteins that have been extracted from roots exposed to purified NFs from 1) the symbiont Sinorhizbium fredii HH103, 2) the non-symbiont Rhizobium leguminosarum bv. viciae, and 3) roots not exposed to NFs. Comparison of the gel images of the three extracts revealed a total of 177 spots that showed up- or down- regulation by at least two-fold.

This ‘classical’ proteomic approach using 2-DE has proved problematic due to the high salt content and the low protein amounts. As a result, peptide mass fingerprint analysis by MALDI-MS(/MS) failed to generate any protein identities. To overcome this, we have been developing and investigating a variety of alternative, LC-based, proteomic workflows for routine analysis of our biological systems.

Monolithic stationary phases provide rapid, high-efficiency separations of the peptide mixtures. We have been evaluating their performance within a nanoLC-ES-MS/MS workflow. This application has allowed rapid and sensitive analysis of the tryptic peptides from proteins taken from D gels of the NF exposed roots. 25 gel spots from the comparison of the two NF exposed gels have been analysed and 18 proteins have been identified, whereas none could be identified using MALDI-MS(/MS) alone.

A gel-C-MS/MS approach has been used for identification of the proteins secreted by symbiotic bacteria. Monolithic LC-MS/MS with both MALDI and ES in this approach yielded complementary data and we have identified 11 proteins.

Due to the difficulties experienced using 2-DE of plant extracts, we are exploring the use of gel-free approaches for quantitative differential proteomics. Relative intensities of LC/MS signals for intact proteins or peptides in each of the samples are compared. Owing to the low protein amounts, we are particularly interested in strategies that do not involve chemical labelling of the peptides or proteins. Using the 2-DE data as a bench mark, we are assessing the capabilities of these novel label-free approaches.