Efficient peptide mapping and its application to identify embryo proteins in rice proteome analysis

Abstract

Using direct N-terminal analysis, only 31 N-terminally unblocked proteins out of 100 rice embryo proteins could be identified. To obtain protein sequence information for the remaining 69 blocked proteins, we developed a simple, efficient and rapid method. Using this method, we determined the peptide maps of 20 proteins per day in 10 pmol amounts. Applying this method to rice proteome analysis, we determined the internal sequences of all 69 blocked proteins. A total of 28 proteins out of 100 analyzed showed sequence similarity to the proteins with known functions in the SWISS-PROT and NCBI databases. Alternatively, we also used peptide mass fingerprinting determined by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to identify the rice proteins separated by two-dimensional electrophoresis (2-DE). Although peptide-mass fingerprinting is a high-throughput method, we could not easily identify all the rice proteins or genes by this method, because the complete database information on rice, is not yet available and many proteins are post-translationally modified. Therefore, at present, the improved peptide mapping method as we report here is considered to be very useful in rice proteome analysis, especially for blocked proteins.

Rice proteomics: Current status and future perspectives

“…Their recent well-conducted study using an efficient peptide mapping technique resulted in the detailed characterization of embryo (derived from seeds) proteins [38]. On a single 2-DE gel 700 proteins were visualized by CBB staining, and more than 1000 proteins could be displayed by silver staining.…”

“…One hundred embryo proteins were selected for analysis, and 31 N-terminally unblocked proteins could be identified. The remaining 69 blocked proteins were internally sequenced by using a simple, efficient, rapid and improved Cleveland peptide mapping method; a total of 28 proteins of the original 100 showed sequence similarity with known proteins in the databases [38]. Although they also analyzed a total of 150 proteins separated by 2-DE by peptide mass fingerprinting using MALDI-TOF-MS, only 46 proteins could be unambiguously identified.…”

“…Henceforth, a major interest for rice researchers will be the assigning of gene function for most, if not all, of the rice genes, where proteomics will definitely play a major role. Indeed, the last decade has seen a considerable progress in developing the field of rice proteomics, where several papers describing the separation and characterization of proteins from various tissues (where one of the main aims was datafile construction) and environmental stimuli (search for markers) have appeared [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]. However, there is no review that brings together all the studies conducted to date in rice at one platform.…”

Proteome Analysis of Wheat Lemma

“…9) After cen trifugation at 15,000~g for 5 min, proteins in the supernatant (100 ml) were separated by twodimen sional (2D) PAGE as described previously. 10) In parallel, 2D SDS PAGE Standards (BioRad) were also separated to estimate the M r and pI of unknown proteins by the replicate 2D gel. As shown in Fig.…”

“…11,12) The internal amino acid sequences of 8 protein spots (spot number #2, #3, #5, #11, #15, #18, #26, and #32) were disco vered by sequence analysis of PVDF membrane blotted polypeptides following the Cleveland peptide mapping with Staphylococcus aureus V8 protease. 10) The amino acid sequences of the above 56 protein spots were compared to the EST database of T. aesti vum using the TBLASTN program at DDBJ. Out of these 56 lemma protein spots, 41 spots (73z) could be assigned to corresponding ESTs the translated sequences of which perfectly match the amino acid sequences found.…”