Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli.

Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins.

Proteome study of the phloem sap of pumpkin using multidimensional protein identification technology.

The phloem is the major transport route for both small substances and large molecules, such as proteins and RNAs, from their sources to sink tissues. To investigate the proteins present in pumpkin phloem sap, proteome analysis using multidimensional protein identification technology was carried out. Pumpkin phloem peptides obtained by liquid chromatography/mass spectrometry/mass spectrometry were searched against pumpkin protein data derived from the National Center for Biotechnology Information. A total of 47 pumpkin phloem proteins were identified. The identified proteins mainly corresponded to enzymes involved in gibberellin biosynthesis, antioxidation processes, or defense mechanisms. Interestingly, seven enzymes required for gibberellin biosynthesis were identified for the first time by this proteomics approach. In summary, the new phloem proteins identified in this study provide strong evidence for stress and defense signaling and new insights regarding the role of gibberellin in the developmental programming of higher plants through the phloem.

Comprehensive proteome analysis of lettuce latex using multidimensional protein-identification technology.

Commercially, lettuce (Lactuca sativa) is one of the most important leafy vegetables. Lettuce produces a milky latex of variable chemical compositions within its laticifers. As a step toward understanding the main physiological roles of this latex in higher plants, we embarked on its proteomic analysis. We investigated 587 latex proteins that were identified from the lettuce latex using multidimensional protein-identification technology. A bioinformatics analysis showed that the most frequently encountered proteins in the latex were organellar proteins from plastids and mitochondria, followed by nucleic and cytoplasmic proteins. Functional classification of the identified proteins showed that proteins related to metabolism, cell rescue, defense, and virulence were the most abundant in lettuce latex. Furthermore, numerous resistance proteins of lettuce and viral proteins were present in the latex suggesting for the first time a possible function of the lettuce latex in defense or pathogenesis. To the knowledge of the authors, this is the first large-scale proteome analysis of lettuce latex.

Proteomic analysis of the secretome of rice calli.

The cell wall and extracellular matrix in higher plants include secreted proteins that play critical roles in a wide range of cellular processes, such as structural integrity and biogenesis. Compared with the intensive cell wall proteomic studies in Arabidopsis, the list of cell wall proteins identified in monocot species is lacking. Therefore, we conducted a large-scale proteomic analysis of secreted proteins from rice. Highly purified secreted rice proteins were obtained from the medium of a suspension of callus culture and were analyzed with multidimensional protein identification technology (MudPIT). As a result, we could detect a total of 555 rice proteins by MudPIT analysis. Based on bioinformatic analyses, 27.7% (154 proteins) of the identified proteins are considered to be secreted proteins because they possess a signal peptide for the secretory pathway. Among the 154 identified proteins, 27% were functionally categorized as stress response proteins, followed by metabolic proteins (26%) and factors involved in protein modification (24%). Comparative analysis of cell wall proteins from Arabidopsis and rice revealed that one third of the secreted rice proteins overlapped with those of Arabidopsis. Furthermore, 25 novel rice-specific secreted proteins were found. This work presents the large scale of the rice secretory proteome from culture medium, which contributes to a deeper understanding of the rice secretome.

Proteomics of weakly bound cell wall proteins in rice calli.

In the present work, we present a proteomic analysis of weakly bound cell wall proteins (CWPs) in rice. CWPs from rice calli were extracted with mannitol/CaCl(2), followed by back extraction with water-saturated phenol. The isolated CWPs were evaluated for contamination by cytosolic proteins by measuring the enzymatic activity of an intracellular marker (glucose-6-phosphate dehydrogenase). This revealed the presence of low levels of intracellular proteins and a significant enrichment of CWPs, as compared to the total extract. Protein samples were digested in gels with trypsin and analyzed using the multidimensional protein identification technology (MudPIT). A total of 292 proteins were identified, which included numerous classical CWPs and antioxidant proteins. Bioinformatics analysis showed that 72.6% of these proteins possessed a signal peptide, and a total of 198 proteins were determined to be CWPs in rice. Functional classification divided the extracellular proteins into different groups, including glycosyl hydrolases (23%), antioxidant proteins (12%), cell wall structure-related proteins (6%), metabolic pathways (9%), protein modifications (4%), defense (4%), and protease inhibitors (3%). Furthermore, comparative analysis of our identified rice CWPs with known Arabidopsis CWPs revealed 25 novel rice-specific CWPs. The study described here is an unprecedented large-scale analysis of CWPs in rice.

Improved detection of multi-phosphorylated peptides by LC-MS/MS without phosphopeptide enrichment.

Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi- phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.