6533b86ffe1ef96bd12cd909

RESEARCH PRODUCT

Technical improvements for analysis of recalcitrant proteins by LC-MS : the mycorrhiza responsive membrane proteome as a case study

subject

description

Arbuscular mycorrhizas (AM) are widespread symbiotic associations between plant roots and AM fungi. Deep membrane alterations are the foremost morphological changes occurring in the host plant in response to AM symbiosis. Two-dimensional gel electrophoresis (2-DE) is the workhorse method in AM proteomics. Membrane proteins are under-represented in 2-DE because of their hydrophobicity, low abundance, and precipitation at their isoelectric point, thereby few are the identified membrane proteins involved in sustaining the AM symbiosis. Membrane proteomics is still challenging due to 2-DE related shortcomings, however latest trends and advancements in mass spectrometry (MS)-based quantitative proteomics offer enormous potential to monitor membrane protein change in abundance in large scale experiments. In the current work microsomal proteins of Medicago truncatula roots inoculated with Rhizophagus irregularis were, for the first time, scrutinised by state-of-the-art MS-based proteomic approaches iTRAQ-OFFGEL-LC-MS/MS and label-free 1-DE-LC-MS/MS. The applied workflows combine two novel proteomic procedures, label-based and -free, targeting an insight view on the membrane proteome changes in AM symbiosis. A subcellular fractionation method is herein described to access the total membrane-associated proteins with sufficient recovery and purity for their subsequent in-depth analysis. In addition to the biological gain by shedding the light on candidate AM-related membrane proteins, a methodological approach was carried out in the present work in order to elucidate the iTRAQ labelling impact on peptide isoelectric points