Project description:In this study, we developed and optimized a nanoproteomic workflow that we termed Nanogram TMT Processing in One Tube (NanoTPOT). Through the assessment of proteolytic digestion, tandem mass tag (TMT) labeling, online and offline frac-tionation strategies, our optimized workflow effectively eliminated the need for sample desalting and enabled compatible sample processing for MS analysis. We further applied the NanoTPOT workflow to examine cellular response to stress caused by dithiothreitol in Hela cells, where we identified and quantified 6935 proteins in a TMT 10-plex experiment with one microgram of starting material in each channel.
Project description:Assessment of technical error in a dual-channel, two timepoint experiment using White lab Drosophila melanogaster microarrays Keywords: repeat sample
Project description:The binding of serum immunoglobulins to proteins was compared using serum from after and before an immunotherapeutic intervention (donor lymphocyte infusion) in two patients who had relapsed chronic lymphocytic leukemia (CLL) after bone marrow transplant. One Invitrogen ProtoArray was used for each sample. Significant interactions were determined by comparing the before and after samples for each patient separately, using the Concentration-Dependent Analysis described in Marina et al., J Proteome Res, 2008. One sample from before and one sample from after immunotherapy were tested for each patient. Keywords: Immune response discovery
Project description:Despite the widespread adoption of ChIP-seq there is still no consensus on quality assessment metrics. No single published metric can reliably discriminate the success or failure of an experiment, thus hampering objectivity and reproducibility of quality control. We introduce a new framework for ChIP-seq data quality assessment that overcomes the limitation of previous solutions. Our tool called "ChIC" incorporates a novel set of quality control metrics integrated into one single score summarizing the sample quality and a reference compendium with thousands of published ChIP-seq samples, for easier evaluation of new data. This test dataset contain an example of succesfull and non-succesfull ChIP-seq sample for mouse H3K27me3.
Project description:A new workflow for metaproteomics is presented in this study and is shown to enable the analysis of a broad range of different samples in only 24 h. The standardized sample preparation includes the combined cell lysis and phenol extraction in a ball mill followed by FASP digestion. The bioinformatic workflow using the MetaProteomeAnalyzer software was expanded with new functionalities such as annotation of metaproteins via protein BLAST or an integrated compare tool. The new workflow was shown to produce at least two times the protein identifications than previous iterations. Through many individual improvements combined into a single standardized workflow, a drastic increase in the quantity and quality of generated results was achieved.
Project description:With the ever-growing need for protein-level understanding in pathological research, proteomics researchers thrive to exam-ine detailed proteome dynamics using crucial, yet often limited, primary and clinical samples. Aside from mass spectrometer instrumentation advancement, a single-tube-based high-throughput sample processing workflow is imperative to ensure sen-sitive, quantitative and reproducible protein analysis for these increasingly sophisticated studies. Leveraging the benefits of an acid-cleavable detergent, RapiGest SF Surfactant (Waters Corporation), we developed and optimized a nanoproteomic workflow that we termed Nanogram TMT Processing in One Tube (NanoTPOT). Through the assessment of proteolytic di-gestion, tandem mass tag (TMT) labeling, online and offline fractionation strategies, our optimized workflow effectively eliminated the need for sample desalting and enabled compatible sample processing for mass spectrometry analysis. We further applied the NanoTPOT workflow to examine cellular response to stress caused by dithiothreitol in HeLa cells, where we identified and quantified 6935 and 5474 proteins in TMT 10-plex experiments with one microgram of lysate protein and 2000 sorted HeLa cells (500ng lysate protein) in each channel, respectively. Our workflow has been proven to be an effec-tive alternative for current nanoproteomic sample processing to minimize sample loss in biological and clinical applications.