A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

<p>Abstract</p> <p>Background</p> <p>The p38α mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear.</p> <p>Methods</p> <p>To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant <it>in vivo </it>kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used <it>in vitro </it>to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms.</p> <p>Results</p> <p>Applying the technique to p38α resulted in the identification of seven <it>in vivo </it>phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An <it>in vitro </it>comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation.</p> <p>Conclusion</p> <p>Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.</p

MiCroKit 3.0: an integrated database of midbody, centrosome and kinetochore

During cell division/mitosis, a specific subset of proteins is spatially and temporally assembled into protein super complexes in three distinct regions, i.e. centrosome/spindle pole, kinetochore/centromere and midbody/cleavage furrow/phragmoplast/bud neck, and modulates cell division process faithfully. Although many experimental efforts have been carried out to investigate the characteristics of these proteins, no integrated database was available. Here, we present the MiCroKit database (http://microkit.biocuckoo.org) of proteins that localize in midbody, centrosome and/or kinetochore. We collected into the MiCroKit database experimentally verified microkit proteins from the scientific literature that have unambiguous supportive evidence for subcellular localization under fluorescent microscope. The current version of MiCroKit 3.0 provides detailed information for 1489 microkit proteins from seven model organisms, including Saccharomyces cerevisiae, Schizasaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Mus musculus and Homo sapiens. Moreover, the orthologous information was provided for these microkit proteins, and could be a useful resource for further experimental identification. The online service of MiCroKit database was implemented in PHP + MySQL + JavaScript, while the local packages were developed in JAVA 1.5 (J2SE 5.0)

Detergent-Assisted Glycoprotein Capture: A Versatile Tool for In-Depth N-Glycoproteome Analysis

Large-scale N-glycoproteome studies have been hindered by poor solubility of hydrophobic membrane proteins and the complexity of proteome samples. Herein, we developed a detergent-assisted glycoprotein capture method to reduce these issues by conducting hydrazide chemistry-based glycoprotein capture in the presence of strong detergents such as sodium dodecyl sulfate and Triton X-100. The strong detergents helped to solubilize hydrophobic membrane proteins and then increased the access of hydrazide groups to oxidized glycoproteins, thus increasing the coverage of the N-glycoproteome. Compared with the conventional glycopeptide capture method, the detergent-assisted glycoprotein capture approach nearly doubled the number of N-glycosylation sites identified from HEK 293T cells with improved specificity. Application of this approach in the larger scale N-glycoproteomics analysis of the HEK 293T cell membrane led to the identification of 2253 unique N-glycosites from 953 proteins. Furthermore, the application of this approach to human serum resulted in the identification of 850 N-glycosylation sites without any immunodepletion or fractionation. Overall, the detergent-assisted glycoprotein capture method simplified the capture process, and it increased the number of sites observed on both hydrophobic membrane proteins and hydrophilic secreted proteins

Recent advances in mass spectrometry-based peptidome analysis

The peptidome, which is the low-molecular-weight subset of the proteome, has attracted increasing attention in recent years. However, with the interference of high-abundance protein components in complex biological mixtures (e.g., serum), selective extraction of endogenous peptides is the first and most important step before analyzing the peptidome. A number of methods and technologies have now been developed for the selective enrichment, fractionation, quantitative analysis of the endogenous peptides and their application in the potential biomarker discovery. This review will cover the methods and technologies developed in recent years for the peptidome analysis on the selective extraction, multidimensional separation and quantitative analysis, as well as their application for clinical diagnosis and biomarker discovery. The future prospects of the peptidome are also discussed