Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.
"I in early publication) phosphorylated proteins were essential for the elongation of body segments, production of hooded hooks, and preparation of juvenile tissues during larval metamorphosis (Chandramouli et al., 2011a). Protein enrichment methods have enabled the separation of proteins according to their PTM (e.g., phosphorylation) (Gilchrist et al., 2006; Puente et al., 2006; Cantin et al., 2008). This approach was used successfully by Chandramouli et al. (2011b) to improve the detection and identification of phosphoproteins during larval metamorphosis of P. vexillosa, revealing that competent larvae exhibit a higher degree of phosphorylation than precompetent larvae and juveniles. "
[Show abstract][Hide abstract] ABSTRACT: The transition in an animal from a pelagic larval stage to a sessile benthic juvenile typically requires major morphological and behavioral changes. Larval competency, attachment and initiation of metamorphosis are thought to be regulated by intrinsic chemical signals and specific sets of proteins. However, the molecular mechanisms that regulate larval attachment and metamorphosis in marine invertebrates have yet to be fully elucidated. Despite the many challenges associated with analysis of the larvae proteome, recent proteomic technologies have been used to address specific questions in larval developmental biology. These and other molecular studies have generated substantial amount of information of the proteins and molecular pathways involved in larval attachment and metamorphosis. Furthermore, the results of these studies have shown that systematic changes in protein expression patterns and post-translational modifications (PTM) are crucial for the transition from larva to juvenile. The degeneration of larval tissues is mediated by protein degradation, while the development of juvenile organs may require PTM. In terms of application, the identified proteins may serve as targets for antifouling compounds, and biomarkers for environmental stressors. In this review we highlight the strengths and limitations of proteomic tools in the context of the study of marine invertebrate larval biology.
"Since the initial release of P3DB, high-quality phosphorylation sites in this database have accumulated at a rapid pace due to improvements in enrichment techniques and mass spectrometry [Supplementary Figure S1a and b]. Most of the datasets in the database came from large-scale experiments (MS/MS) (3), although several smaller datasets were also deposited. To help users analyze the proteome-wide phosphorylation data more systematically, the new P3DB 3.0 provides more information and annotations about phosphoproteins such as gene ontology, homolog, 3D structures, kinase and phosphatase families, protein–protein interactions (PPIs) and protein domains, together with protein–protein networks, kinase-substrate or phosphatase-substrate networks and domain co-occurrence networks (4). "
[Show abstract][Hide abstract] ABSTRACT: In the past few years, the Plant Protein Phosphorylation Database (P(3)DB, http://p3db.org) has become one of the most significant in vivo data resources for studying plant phosphoproteomics. We have substantially updated P(3)DB with respect to format, new datasets and analytic tools. In the P(3)DB 3.0, there are altogether 47 923 phosphosites in 16 477 phosphoproteins curated across nine plant organisms from 32 studies, which have met our multiple quality standards for acquisition of in vivo phosphorylation site data. Centralized by these phosphorylation data, multiple related data and annotations are provided, including protein-protein interaction (PPI), gene ontology, protein tertiary structures, orthologous sequences, kinase/phosphatase classification and Kinase Client Assay (KiC Assay) data-all of which provides context for the phosphorylation event. In addition, P(3)DB 3.0 incorporates multiple network viewers for the above features, such as PPI network, kinase-substrate network, phosphatase-substrate network, and domain co-occurrence network to help study phosphorylation from a systems point of view. Furthermore, the new P(3)DB reflects a community-based design through which users can share datasets and automate data depository processes for publication purposes. Each of these new features supports the goal of making P(3)DB a comprehensive, systematic and interactive platform for phosphoproteomics research.
"Some isoforms decreased in abundance but new isoforms accumulated in the S phase samples (Figure 5C, compare lane 1 with lanes 2 and 3). Of note, the hnRNPA3 protein has been reported to be heavily phosphorylated, raising the possibility that the decrease observed by mass spectrometry was due to cell cycle regulated post-translational modifications , , , , , , , . Indeed, a number of hnRNPs, including hnRNPD0, were identified as Cyclin A/Cdk2 substrates . "
[Show abstract][Hide abstract] ABSTRACT: Cell proliferation involves dramatic changes in DNA metabolism and cell division, and control of DNA replication, mitosis, and cytokinesis have received the greatest attention in the cell cycle field. To catalogue a wider range of cell cycle-regulated processes, we employed quantitative proteomics of synchronized HeLa cells. We quantified changes in protein abundance as cells actively progress from G1 to S phase and from S to G2 phase. We also describe a cohort of proteins whose abundance changes in response to pharmacological inhibition of the proteasome. Our analysis reveals not only the expected changes in proteins required for DNA replication and mitosis but also cell cycle-associated changes in proteins required for biological processes not known to be cell-cycle regulated. For example, many pre-mRNA alternative splicing proteins are down-regulated in S phase. Comparison of this dataset to several other proteomic datasets sheds light on global mechanisms of cell cycle phase transitions and underscores the importance of both phosphorylation and ubiquitination in cell cycle changes.
PLoS ONE 03/2013; 8(3):e58456. DOI:10.1371/journal.pone.0058456 · 3.23 Impact Factor
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