ABSTRACT: Data files and supplementary data associated with the one-pot method. These files are the results of one-pot enrichment of both acetylated and succinylated peptides.
Project description:Protein post-translational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi-PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here we describe a streamlined, quantitative label-free proteomic workflow – “one-pot” PTM enrichment – which enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein. Coupled with a label-free, data-independent acquisition (DIA), we identified and quantified 2235 acetylated and 2173 succinylated peptides with the one-pot method and show that peak areas are highly correlated between the one-pot and traditional single-PTM enrichments. The ‘one-pot’ method makes possible detection of multiple PTMs occurring on the same peptide, and we show that it can be used to make unique biological insights into PTM crosstalk. Compared to single-PTM enrichments, the one-pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue.
Project description:We present One-Tip, a lossless proteomics methodology that seamlessly combines swift, one-pot sample preparation with narrow-window data-independent acquisition mass spectrometric analysis. With simplest sample processing, One-Tip reproducibly identifies > 9,000 proteins from ~1000 cells and ~ 6,000 proteins in a single mouse zygote with a throughput of 40 samples-per-day. This easy-to-use method expands capabilities of proteomics research by enabling greater depth, scalability and throughput covering low to high input samples.
Project description:a ‘multi-in-one’ strategy to saturate the phosphosite coverage from multiple-proteases by one-step enrichment and one-shot analysis in just over one hour, through data-independent acquisition (DIA)
Project description:Quantitative proteomic analysis of Myc-induced apoptosis in serum-deprived Rat1_Myc fibroblasts. Mitochondrial, chromatin, and soluble fractions analyzed. Original peptide data contained in Supplementary files. Keywords: proteomic, apoptosis, cell fractions
Project description:Supplementary proteome data of Vibrio alginolyticus strain: ZJ-T, the Wild-type raw files are in another project (ProteomeXchange ID: PXD035385).
Project description:Sample preparation for single-cell proteomics is generally performed in a one-pot workflow with multiple dispensing and incubation steps. These hours-long workflows can be labor intensive and lead to long sample-to-answer times. Here we report a sample processing method that achieves cell lysis, protein denaturation and digestion in one hour with a single reagent dispensing step using commercially available high-temperature-stabilized proteases. Four different one-step reagent compositions were evaluated, and the mixture providing greatest proteome coverage was compared to the previously employed multi-step workflow. The one-step preparation increases proteome coverage relative to the multi-step workflow while minimizing labor input and the possibility of human error. We also compared sample recovery between previously used microfabricated glass nanowell chips and injection-molded polypropylene substrates, and found the polypropylene provided improved proteome coverage. Combined, the one-step sample preparation and the polypropylene substrates enabled identification of an average of nearly 2,400 proteins per cell using a standard data-dependent workflow with an Orbitrap mass spectrometer. As such, these advances significantly simplify sample preparation for single-cell proteomics and broaden accessibility with no compromise in terms of proteome coverage.
Project description:Temporal analysis of Irf4 and PU.1 genome binding during B cell activation and differentiation in vitro using antigen (NP-Ficoll) CD40L and IL-2/4/5 cytokines (see Molecular Systems Biology 7:495 for details of cellular system). The results provide insight in the target genes and binding specificity of IRF4 and PU.1 during coordination of different programs of B cell differentiation. Regrettably three of the FASTQ raw sequence files in our study were corrupted during storage. FASTQ data from our experimental and control groups are available for download via GEO SRA; however, two groups are missing select raw sequence files. These include one PU.1 Day 3 group file (Sample GSM1133499) and two of four input files used to generate a concatenated “super” input file (Sample GSM1133490); the raw data provided for input consists of the two input files recovered. Importantly, FASTA sequences for both of these datasets are available as supplementary data through GEO, and we can make available upon request (rsciamma@uchicago.edu) all files in our study in the ELAND-extended alignment format. Please note that GEO no longer supports this format.
Project description:Temporal analysis of Irf4 and PU.1 genome binding during B cell activation and differentiation in vitro using antigen (NP-Ficoll) CD40L and IL-2/4/5 cytokines (see Molecular Systems Biology 7:495 for details of cellular system). The results provide insight in the target genes and binding specificity of IRF4 and PU.1 during coordination of different programs of B cell differentiation. Regrettably three of the FASTQ raw sequence files in our study were corrupted during storage. FASTQ data from our experimental and control groups are available for download via GEO SRA; however, two groups are missing select raw sequence files. These include one PU.1 Day 3 group file (Sample GSM1133499) and two of four input files used to generate a concatenated “super” input file (Sample GSM1133490); the raw data provided for input consists of the two input files recovered. Importantly, FASTA sequences for both of these datasets are available as supplementary data through GEO, and we can make available upon request (rsciamma@uchicago.edu) all files in our study in the ELAND-extended alignment format. Please note that GEO no longer supports this format.
Project description:Illumina bodyMap2 transcriptome Transcription profiling by high throughput sequencing of individual and mixture of 16 human tissues RNA. Additional supplementary files available at foot of this record. Additional information available as supplementary files at the foot of this record. ArrayExpress Release Date: 2011-03-17 Person Roles: submitter Person Last Name: Khrebtukova Person First Name: Irina Person Mid Initials: Person Email: ikhrebtukova@illumina.com Person Phone: 1-510-723-9219 Person Address: 25861 Industrial Blvd, Hayward CA 94545, USA Person Affiliation: Illumina