Article

Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis.

Departments of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Journal of Proteome Research (Impact Factor: 5). 04/2008; 7(3):1346-51. DOI: 10.1021/pr0705441
Source: PubMed

ABSTRACT 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.

1 Follower
 · 
122 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS) is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST) through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Runx2 is the master switch controlling osteoblast differentiation and formation of the mineralized skeleton. The post-translational modification (PTM) of Runx2 by phosphorylation, ubiquitinylation, and acetylation modulates its activity, stability, and interactions with transcriptional co-regulators and chromatin remodeling proteins downstream of osteogenic signals. Characterization of Runx2 by electron transfer dissociation tandem mass spectrometry revealed sites of O-linked N-acetylglucosamine (O-GlcNAc) modification, a nutrient-responsive PTM which modulates the action of numerous transcriptional effectors. O-GlcNAc modification occurs in close proximity to phosphorylated residues and novel sites of arginine methylation within regions known to regulate Runx2 transactivation. An interaction between Runx2 and the O-GlcNAcylated, O-GlcNAc transferase enzyme was also detected. Pharmacological inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc from Ser/Thr residues, enhanced basal (39.9%) and BMP2/7-induced (43.3%) Runx2 transcriptional activity in MC3T3-E1 preosteoblasts. In bone marrow-derived mesenchymal stem cells (BMMSCs) differentiated for 6 days in osteogenic media, inhibition of OGA resulted in elevated expression (24.3%) and activity (65.8%) of alkaline phosphatase (ALP) an early marker of bone formation and a transcriptional target of Runx2. Osteogenic differentiation of BMMSCs in the presence of BMP2/7 for 8 days culminated in decreased OGA activity (39.0%) and an increase in the abundance of O-GlcNAcylated Runx2, as compared to unstimulated cells. Furthermore, BMP2/7-induced ALP activity was enhanced by 35.6% in BMMSCs differentiated in the presence of the OGA inhibitor, demonstrating that direct or BMP2/7-induced inhibition of OGA is associated with increased ALP activity. Altogether these findings link O-GlcNAc cycling to the Runx2-dependent regulation of the early ALP marker under osteoblast differentiation conditions.
    Molecular &amp Cellular Proteomics 09/2014; 13(12). DOI:10.1074/mcp.M114.040691 · 7.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Class IIa histone deacetylases (HDACs4, -5, -7, and -9) modulate the physiology of the human cardiovascular, musculoskeletal, nervous and immune systems. The regulatory capacity of this family of enzymes stems from their ability to shuttle between nuclear and cytoplasmic compartments in response to signal-driven post-translational modification. Here, we review the current knowledge of modifications that control spatial and temporal HDAC functions by regulating subcellular localization, transcriptional functions, and cell cycle-dependent activity, ultimately impacting on human disease. We discuss the contribution of these modifications to cardiac and vascular hypertrophy, myoblast differentiation, neuronal cell survival, and neurodegenerative disorders. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Molecular &amp Cellular Proteomics 01/2015; DOI:10.1074/mcp.O114.046565 · 7.25 Impact Factor