Proteomic analysis of Drosophila CLOCK complexes identifies rhythmic interactions with SAGA and Tip60 complex component NIPPED-A.

Circadian clocks keep time via ~ 24 h transcriptional feedback loops. In Drosophila, CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors are feedback loop components whose transcriptional status varies over a circadian cycle. Although changes in the state of activators and repressors has been characterized, how their status is translated to transcriptional activity is not understood.

Discovering protein interactions and characterizing protein function using HaloTag technology.

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast. In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance.

Phosphorylation of the transcription activator CLOCK regulates progression through a ∼ 24-h feedback loop to influence the circadian period in Drosophila.

Circadian (≅ 24 h) clocks control daily rhythms in metabolism, physiology, and behavior in animals, plants, and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which clock-cycle (CLK-CYC) initiates transcription of period (per) and timeless (tim), accumulating levels of PER and TIM proteins feed back to inhibit CLK-CYC, and degradation of PER and TIM allows CLK-CYC to initiate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in PER and CLK phosphorylation, where PER and CLK phosphorylation is high during transcriptional repression.

Bottom-Up Mass Spectrometry-Based Proteomics as an Investigative Analytical Tool for Discovery and Quantification of Proteins in Biological Samples.

Objective: The objective of this overview is to introduce bottom-up mass spectrometry (MS)-based proteomics approaches and strategies, widely used in other biomedical research fields, to the wound-healing research community.

Approaches: TWO MAJOR PROTEOMICS WORKFLOWS ARE DISCUSSED: gel-based and gel-free chromatographic separation to reduce the complexity of the sample at protein and peptide level, respectively, prior to nano-liquid chromatography-tandem mass spectrometry analysis. Other strategies to discover less abundant proteins present in the sample, are also briefly discussed along with label-free and label-incorporated methods for protein quantification.

Epithelial-mesenchymal transition-associated secretory phenotype predicts survival in lung cancer patients.

In cancer cells, the process of epithelial-mesenchymal transition (EMT) confers migratory and invasive capacity, resistance to apoptosis, drug resistance, evasion of host immune surveillance and tumor stem cell traits. Cells undergoing EMT may represent tumor cells with metastatic potential. Characterizing the EMT secretome may identify biomarkers to monitor EMT in tumor progression and provide a prognostic signature to predict patient survival.

Preimplantation factor (PIF*) reverses neuroinflammation while promoting neural repair in EAE model.

Introduction: Embryo-derived PIF modulates systemic maternal immunity without suppression. Synthetic analog (sPIF) prevents juvenile diabetes, preserves islet function, reducing oxidative stress/protein misfolding. We investigate sPIF effectiveness in controlling neuroinflammation/MS.

Preimplantation factor (PIF) analog prevents type I diabetes mellitus (TIDM) development by preserving pancreatic function in NOD mice.

Preimplantation factor (PIF) is a novel embryo-secreted immunomodulatory peptide. Its synthetic analog (sPIF) modulates maternal immunity without suppression. There is an urgent need to develop agents that could prevent the development of type 1 diabetes mellitus (TIDM).

Ion trap collision-induced dissociation of human hemoglobin alpha-chain cations.

Multiply protonated human hemoglobin alpha-chain species, ranging from [M + 4H]4+ to [M + 20H]20+, have been subjected to ion trap collisional activation. Cleavages at 88 of the 140 peptide bonds were indicated, summed over all charge states, although most product ion signals were concentrated in a significantly smaller number of channels. Consistent with previous whole protein ion dissociation studies conducted under similar conditions, the structural information inherent to a given precursor ion was highly sensitive to charge state.

Gas-phase ion/ion reactions of multiply protonated polypeptides with metal containing anions.

Gas-phase reactions of multiply protonated polypeptides and metal containing anions represent a new methodology for manipulating the cationizing agent composition of polypeptides. This approach affords greater flexibility in forming metal containing ions than commonly used methods, such as electrospray ionization of a metal salt/peptide mixture and matrix-assisted laser desorption. Here, the effects of properties of the polypeptide and anionic reactant on the nature of the reaction products are investigated.

A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system.

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass.

Whole protein dissociation in a quadrupole ion trap: identification of an a priori unknown modified protein.

A protein mixture derived from a whole cell lysate fraction of Saccharomyces cerevisiae, which contains roughly 19 proteins, has been analyzed to identify an a priori unknown modified protein using a quadrupole ion trap tandem mass spectrometer. Collection of the experimental data was facilitated by collision-induced dissociation and ion/ion proton-transfer reactions in multistage mass spectrometry procedures. Ion/ion reactions were used to manipulate charge states of both parent ions and product ions for the purpose of concentrating charge into the parent ion of interest and to reduce the product ion charge states for determination of product ion mass and abundance.