Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time.

WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 Å resolution has revealed that this domain is composed of a globular structure with five β strands, forming an antiparallel β-sheet. A novel zinc-binding site is situated at one end of the β-sheet, between strands β4 and β5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at β2 and β3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.

Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti(4+)-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold.

We identified the d‐galacturonic acid (GA)‐responsive transcriptional activator GaaR of the saprotrophic fungus, Aspergillus niger, which was found to be essential for growth on GA and polygalacturonic acid (PGA). Growth of the ΔgaaR strain was reduced on complex pectins. Genome‐wide expression analysis showed that GaaR is required for the expression of genes necessary to release GA from PGA and more complex pectins, to transport GA into the cell, and to induce the GA catabolic pathway. Residual growth of ΔgaaR on complex pectins is likely due to the expression of pectinases acting on rhamnogalacturonan and subsequent metabolism of the monosaccharides other than GA.

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Cytosolic NPR1 has been shown to be essential for the salicylic acid (SA)-mediated suppression of jasmonic acid (JA)-responsive gene expression. However, factors downstream of NPR1 in the cross-talk between SA and JA signaling are unclear. Here we show that Arabidopsis WRKY62, a member of WRKY group III transcription factors, was induced by methyl jasmonate (MeJA) and SA treatment. The presence of basal SA is required for the MeJA-induced WRKY62 expression, and both chemicals exhibit a synergistic effect on WRKY62 induction. In addition, upon treatment with an extremely low concentration of SA, cytosolic NPR1 controls the MeJA-induced expression of WRKY62. TGA transcription factors, which up-regulate SA-induced expression of WRKY62, are dispensable for the induction of WRKY62 in JA signaling. Genetic dissection of both wrky62 mutants and WRKY62-overexpressing plants indicated that WRKY62 down-regulates JA-responsive LOX2 and VSP2 expression. Our results demonstrate that WRKY62 acts downstream of cytosolic NPR1 and negatively regulates JA-responsive gene expression, suggesting that WRKY62 may be involved in the SA-mediated suppression of JA signaling.

Recently, body fluids have widely become an important target for proteomic research and proteomic study has produced more and more body fluid related protein data. A database is needed to collect and analyze these proteome data. Thus, we developed this web-based body fluid proteome database Sys-BodyFluid. It contains eleven kinds of body fluid proteomes, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, seminal fluid, human milk and amniotic fluid. Over 10 000 proteins are presented in the Sys-BodyFluid. Sys-BodyFluid provides the detailed protein annotations, including protein description, Gene Ontology, domain information, protein sequence and involved pathways. These proteome data can be retrieved by using protein name, protein accession number and sequence similarity. In addition, users can query between these different body fluids to get the different proteins identification information. Sys-BodyFluid database can facilitate the body fluid proteomics and disease proteomics research as a reference database. It is available at http://www.biosino.org/bodyfluid/.

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