Medicago truncatula proteomics for systems biology: novel rapid shotgun LC-MS approach for relative quantification based on full-scan selective peptide extraction (Selpex).

Medicago truncatula has become the focus of systems biology research for improved legume crop breeding. In plant systems biology, several comparative studies have been carried out using liquid chromatography shotgun mass spectrometry (LC-MS/MS) and database-dependent protein identification analyses in combination with the spectral count for relative quantification. In order to receive optimal protein identification rates and spectral count quantification, data-dependent tandem mass spectrometry with LC separation of more than 1 h is required. Thus LC-MS/MS analyses time is the bottleneck for high-throughput research of experiments with high sample number.We describe a novel method, called full-scan (FS) selective peptide extraction, that allows for comparative quantification of target peptides combined with a significant reduction in LC-MS analysis time. In future, it will be a useful tool to detect (15)N-labeled selected peptide patterns for the targeted analysis of protein turnover and synthesis. We provide a first reference library of selected target peptides generated for M. truncatula leaf tissue. These peptides are also suitable candidates for selective reaction monitoring approaches.

Two-dimensional electrophoresis based proteomic analysis of the pea (Pisum sativum) in response to Mycosphaerella pinodes.

Responses to Mycosphaerella pinodes in pea were studied by using a proteomics approach. Two-dimensional electrophoresis (2-DE) was used in order to compare the leaf proteome of two pea cultivars displaying different phenotypes (susceptible and partial resistance to the fungus), as well as in response to the inoculation. Multivariate statistical analysis identified 84 differential protein spots under the experimental conditions (cultivars/treatments). All of these 84 protein spots were subjected to MALDI-TOF/TOF mass spectrometry to deduce their possible functions. A total of 31 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of the identified proteins corresponded to enzymes belonging to photosynthesis, metabolism, transcription/translation and defense and stress categories. Results are discussed in terms of responses to pathogens.