Mining the Secretome of C2C12 Muscle Cells: Data Dependent Experimental Approach To Analyze Protein Secretion Using Label-Free Quantification and Peptide Based Analysis

Secretome analysis faces several challenges including detection of low abundant proteins and the discrimination of bona fide secreted proteins from false-positive identifications stemming from cell leakage or serum. Here, we developed a two-step secretomics approach and applied it to the analysis of secreted proteins of C2C12 skeletal muscle cells since the skeletal muscle has been identified as an important endocrine organ secreting myokines as signaling molecules. First, we compared culture supernatants with corresponding cell lysates by mass spectrometry-based proteomics and label-free quantification. We identified 672 protein groups as candidate secreted proteins due to their higher abundance in the secretome. On the basis of Brefeldin A mediated blocking of classical secretory processes, we estimated a sensitivity of >80% for the detection of classical secreted proteins for our experimental approach. In the second step, the peptide level information was integrated with UniProt based protein information employing the newly developed bioinformatics tool “Lysate and Secretome Peptide Feature Plotter” (LSPFP) to detect proteolytic protein processing events that might occur during secretion. Concerning the proof of concept, we identified truncations of the cytoplasmic part of the protein Plexin-B2. Our workflow provides an efficient combination of experimental workflow and data analysis to identify putative secreted and proteolytic processed proteins

High-Fat Diet Induced Isoform Changes of the Parkinson’s Disease Protein DJ‑1

Genetic and environmental factors mediate via different physiological and molecular processes a shifted energy balance leading to overweight and obesity. To get insights into the underlying processes involved in energy intake and weight gain, we compared hypothalamic tissue of mice kept on a high-fat or control diet for 10 days by a proteomic approach. Using two-dimensional difference gel electrophoresis in combination with LC–MS/MS, we observed significant abundance changes in 15 protein spots. One isoform of the protein DJ-1 was elevated in the high-fat diet group in three different mouse strains SWR/J, C57BL/6N, and AKR/J analyzed. Large-scale validation of DJ-1 isoforms in individual samples and tissues confirmed a shift in the pattern of DJ-1 isoforms toward more acidic isoforms in several brain and peripheral tissues after feeding a high-fat diet for 10 days. The identification of oxidation of cysteine 106 as well as 2-succinyl modification of the same residue by mass spectrometry not only explains the isoelectric shift of DJ-1 but also links our results to similar shifts of DJ-1 observed in neurodegenerative disease states under oxidative stress. We hypothesize that DJ-1 is a common physiological sensor involved in both nutrition-induced effects and neurodegenerative disease states

Minichromosome Maintenance Complex Is a Critical Node in the miR-183 Signaling Network of <i>MYCN</i>-Amplified Neuroblastoma Cells

MYCN and HDAC2 jointly repress the transcription of tumor suppressive miR-183 in neuroblastoma. Enforced miR-183 expression induces neuroblastoma cell death and inhibits xenograft growth in mice. Here we aimed to focus more closely on the miR-183 signaling network using a label-free mass spectrometric approach. Analysis of neuroblastoma cells transfected with either control or miR-183 expression vectors identified 85 differentially expressed proteins. All six members of the minichromosome maintenance (MCM) complex, which is indispensable for initiation and elongation during DNA replication and transcriptionally activated by MYCN in neuroblastoma, emerged to be down-regulated by miR-183. Subsequent annotation category enrichment analysis revealed a ∼14-fold enrichment in the “MCM” protein module category, which highlighted this complex as a critical node in the miR-183 signaling network. Down-regulation was confirmed by Western blotting. MCMs 2–5 were predicted by in silico methods as direct miR-183 targets. Dual-luciferase reporter gene assays with 3′-UTR constructs of the randomly selected MCMs 3 and 5 experimentally confirmed them as direct targets of miR-183. Our results reveal the MCM complex to be a critical and directly regulated node within the miR-183 signaling network in <i>MYCN</i>-amplified neuroblastoma cells

Additional file 1 of The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin

Additional file 1: Figure S1. Schematic representation of the thermal proteome profiling temperature range (TPP-TR) workflow. HeLa wt cells were treated with 100 nM prodigiosin or DMSO for 6 h. After the incubation, cells were harvested, washed and aliquots of the cell suspensions were exposed to short (3 min) treatments at different temperatures in the range between 36.5 °C and 67 °C. Cells were lysed and the non-denatured protein fraction was recovered after centrifugation. Quantitative protein analysis was performed by immunoblotting (CETSA) or MS (TPP). For MS, proteins underwent tryptic digest and the resulting peptides were labeled using TMT 10plex. The samples were combined such that prodigiosin treated and corresponding control samples belonging to the same temperature were analyzed within the same TMT set (similarly as described before for RTSA), allowing for studying not only thermal stability but also abundance effects upon prodigiosin treatment