2Biology Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-1268
3Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, pr. Nauki 6, 142290 Pushchino, Moscow Region, Russia; E-mail: firstname.lastname@example.org
4Scientific Research Institute of Physicochemical Medicine, ul. Malaya Pirogovskaya 1a, 119435 Moscow, Russia; fax: (495) 246-4401; E-mail: email@example.com
* To whom correspondence should be addressed.
Received April 30, 2008; Revision received July 3, 2008
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 WORDS: proteomics, 2D electrophoresis, MALDI-TOF, LC-ESI-MS/MS, Physcomitrella patens, protoplasts, DiGE
Supplemental TABLE (MS Excel)