Proteome analysis of the moss Physcomitrella patens (Hedw.) B.S.G.
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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 and gametophores.
Key wordsproteomics 2D electrophoresis MALDI-TOF LC-ESI-MS/MS Physcomitrella patens protoplasts DiGE
differential gel electrophoresis
EuKaryotic Orthologous Groups
liquid chromatography-electrospray ionization-tandem mass spectrometry
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
polyacrylamide gel electrophoresis
peptide mass fingerprinting
ribulose bisphosphate carboxylase
sodium dodecyl sulfate
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