Functional proteomic analysis of long-term growth factor stimulation and receptor tyrosine kinase coactivation in Swiss 3T3 fibroblasts.

In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after (35)S- and (33)P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated.

Distinct cell surface proteome profiling by biotin labeling and glycoprotein capturing.

We performed here MS-based cell surface proteome profiling of HCT-116 cells by two distinct methods based on biotin labeling and glycoprotein capturing. In total, 742 biotinylated and 219 glycosylated proteins were identified by the biotin labeling and glycoprotein capturing, of which 224 and 138 proteins known to be located on plasma membrane were included, respectively, according to ingenuity pathway analysis. Although 104 plasma membrane proteins were identified by both methods, the rest of 154 were identified only by one.

Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways.

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant.

Integration of proteomic analyses to monitor the activity status of phosphorylation signaling.

We performed here MS-based phosphoproteomics using both metal oxide affinity chromatography (pSTY proteomics) and anti-phosphotyrosine antibody (pY proteomics). The former method identified mainly phospho-serine and -threonine of nuclear or cytoplasmic proteins, whereas the latter did phosphotyrosine including more plasma membrane proteins and kinases. The overlap between these two methods was limited (24 tyrosine phosphorylation sites out of 325) and, by combining the two, coverage of the signaling molecules was enhanced as exemplified by Erk signaling.

A software program for more reliable precursor ion assignation from LC-MS analysis using LTQ ultra zoom scan.

We developed a software program (titled Precursor Ion Calibration software for LTQ or, in short, PICsL) that increases the reliability of precursor ion assignations from LC-MS analysis using ultra zoom scanning of LTQ linear ion trap MS and automatically corrects the assignations. Although existing software calculates the theoretical isotopic distribution according to m/z with a computational algorithm, our method simply searches for ions close to the theoretical mass value using both MS/MS raw data and Mascot search result files, followed by a second database search that identifies the proteins using the regenerated peak list files. Our software program mimics the manual inspection of the spectral data of precursor ions and is expected to be applicable not only for low resolution MS, such as LTQ, but also for a wide variety of MS instruments.

Phosphoproteomic analysis of distinct tumor cell lines in response to nocodazole treatment.

Here, we report for the first time a comparative phosphoproteomic analysis of distinct tumor cell lines in the presence or absence of the microtubule-interfering agent nocodazole. In total, 1525 phosphorylation sites assigned to 726 phosphoproteins were identified using LC-MS-based technology following phosphopeptide enrichment. Analysis of the amino acid composition surrounding the identified in vivo phosphorylation sites revealed that they could be classified into two motif groups: pSer-Pro and pSer-Asp/Glu.

Cell surface biomarkers of embryonic stem cells.

Embryonic stem cells (ESCs) can give rise to any adult cell type and thus offer enormous potential for regenerative medicine and drug discovery. Molecular biomarkers serve as valuable tools to classify and isolate ESCs and to monitor their differentiation state by antibody-based techniques. A number of biomarkers, such as certain cell surface antigens, are used to assign pluripotent ESCs; however, accumulating evidence suggests that ESCs are heterogeneous in morphology, phenotype and function, and are thereby classified into subpopulations characterized by multiple sets of molecular biomarkers.

PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E3-ligase MIR.

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome.

Cell surface labeling and mass spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells.

Although interactions between cell surface proteins and extracellular ligands are key to initiating embryonic stem cell differentiation to specific cell lineages, the plasma membrane protein components of these cells are largely unknown. We describe here a group of proteins expressed on the surface of the undifferentiated mouse embryonic stem cell line D3. These proteins were identified using a combination of cell surface labeling with biotin, subcellular fractionation of plasma membranes, and mass spectrometry-based protein identification technology.

Large-scale identification of proteins expressed in mouse embryonic stem cells.

A protein subset expressed in the mouse embryonic stem (ES) cell line, E14-1, was characterized by mass spectrometry-based protein identification technology and data analysis. In total, 1790 proteins including 365 potential nuclear and 260 membrane proteins were identified from tryptic digests of total cell lysates. The subset contained a variety of proteins in terms of physicochemical characteristics, subcellular localization, and biological function as defined by Gene Ontology annotation groups.

Differential protein synthesis and expression levels in normal and neoplastic human prostate cells and their regulation by type I and II interferons.

Protein expression and de novo synthesis in normal and prostate cancer cell lines derived from the same patient were compared by proteomic analysis, and the effects of INFalpha and INFgamma (INF=interferon) determined. The expressions of several INF-inducible proteins, including MxA, Nmi, PA28a and IFP53, were downregulated in the cancer cells. INFgamma induced a more than twofold increase or decrease in the synthesis rates of almost twice as many proteins in the cancer cell line.