ИСТИНА

The sequencing of the moss Physcomitrella patens genome has facilitated studies of the plant proteome. To develop a proteome reference map based on the genome sequence, we conducted 2D electrophoreses of proteins extracted from moss protoplasts, protonemata, and gametophores grown under standard conditions on Petri dishes. On silver_stained gels, depending on the developmental stage of the moss, we resolved from 500 to 600 protein spots that were then excised and digested by trypsin, and 212 proteins were identified by PMF-MALDI-TOF. To enhance the proteome coverage, we performed 1D SDS-PAGE with subsequent separation of tryptic peptides derived from digested gel band slices by LC-ESI-MS/MS. The proposed approach allowed us to identify 186 proteins had not been determined by 2D PMF-MALDI-TOF. Proteins identified by both methods were categorized using a system of clusterization of orthologous genes as metabolism (26%), cellular processes and signaling (16%), and information storage and processing (7%). Proteome analysis by differential gel electrophoresis revealed moderate differences between filamentous protonemata and leafy shoots. Surprisingly, protoplasts isolated from protonema filaments displayed significant differences in protein composition compared with both protonemata. We used moss protoplasts as an ideal source of nuclei, chloroplasts and mitochondria for organelle proteome analysis. Chloroplasts were fractionated additionally for thylakoids and stroma. Chloroplast proteins were separated by 1D SDS-PAGE with subsequent identification of tryptic peptides by LC-ESI-MS/MS. Finally, we have identified 636 unique proteins and 436 of them were annotated as chloroplast resident proteins. Amongst the last ones photosynthetic proteins make up 21%. The same portion was composed by proteins involved in protein import, folding, post-translational modification and degradation.