Dataset Information

Chemical acetylation of ligands and two-step digestion protocol for reducing co-digestion in affinity purification-mass spectrometry

ABSTRACT: We present an effective, fast and user-friendly method to reduce co-digestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short pre-incubation of beads with Sulfo-NHS-Acetate leads to chemical acetylation of lysine residues, making ligands insusceptible to Lys-C-mediated proteolysis. In contrast to similar approaches, our procedure offers the advantage of exclusively using non-toxic chemicals and employing mild chemical reaction conditions. After binding of bait proteins to Sulfo-NHS-Acetate treated beads, we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion of the released proteins with trypsin. The implementation of this protocol results in a strong reduction of contaminating ligand peptides, which allows significantly higher amounts of sample to be subjected to LC-MS analysis, improving sensitivity and quantitative accuracy.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Wolfgang Reiter

LAB HEAD: Markus Hartl

PROVIDER: PXD043709 | Pride | 2023-10-23

REPOSITORIES: Pride

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Publications

[Evaluation of the scientific activity through bibliometric indices].

Bordons M M Zulueta M A MA

Revista espanola de cardiologia 19991001 10

The scope of bibliometric studies is the treatment and quantitative analysis of scientific publications. They belong to the so-called "social studies of science", and science policy constitutes one of its main applied fields. These studies efficiently complement the opinions and judgements of experts, thus providing objective and useful tools for evaluating the results of scientific activity. Nevertheless, given the impact that these evaluations have on the assignment of funding for research and ...[more]

PMID: 10563155

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Project description:In the past years several protocols for the proteomic profiling of plasma membrane proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach [1-3]. To pave the way to high-performance differential plasma membrane proteomics, we compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation and surface coating with silica beads to isolate plasma membrane proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of plasma membrane proteins and reproducibility parameters on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation outperformed aminooxy-biotinylation and surface coating using silica beads for most of the monitored criteria. We further simplified this procedure by introducing a competitive biotin elution strategy, for which the average plasma membrane annotated protein fraction amounted to 54 % (347 proteins). Moreover, purified non-plasma membrane annotated proteins and plasma membrane annotated proteins displayed similarly high reproducibility suggesting specific co-purification. In fact, the non-plasma membrane annotated data was extremely enriched for direct interactors of purified plasma membrane proteins. Computational analysis using additional databases and prediction tools revealed that in total over 90 % of the purified proteins were associated with the plasma membrane. The modified sulfo-NHS-SS-biotinylation protocol was validated by tracking changes in the plasma membrane proteome composition induced by genetic alteration and drug treatment. GPI-anchored proteins were depleted in plasma membrane purifications from cells deficient in the GPI transamidase component PIGS; and treatment of cells with tunicamycin significantly reduced the abundance of N-glycoproteins in surface purifications. Altogether, this study introduces an improved, filter-free sulfo-NHS-SS-biotinylation protocol and demonstrates it to be a specific, effective and reproducible method to isolate proteins associated with the plasma membrane, thus enabling future large-scale comparative cell surface mappings.

Project description:Background: The force generating mechanism of muscle is evolutionarily ancient; the fundamental structural and functional components of the sarcomere are common to motile animals throughout phylogeny. Recent evidence suggests that the transcription factors that regulate muscle development are also conserved. Thus, a comprehensive description of muscle gene expression in a simple model organism should define a basic muscle transcriptome that is also expressed in animals with more complex body plans. To this end, we have applied Micro-Array Profiling of Caenorhabditis elegans Cells (MAPCeL) to muscle cell populations extracted from developing Caenorhabditis elegans embryos. Results: Fluorescence Activated Cell Sorting (FACS) was used to isolate myo-3::GFP-positive muscle cells, and their cultured derivatives, from dissociated early Caenorhabditis elegans embryos. Microarray analysis identified 6,693 expressed genes, 1,305 of which are enriched in the myo-3::GFP positive cell population relative to the average embryonic cell. The muscle-enriched gene set was validated by comparisons to known muscle markers, independently derived expression data, and GFP reporters in transgenic strains. These results confirm the utility of MAPCeL for cell type-specific expression profiling and reveal that 60% of these transcripts have human homologs. Conclusions: This study provides a comprehensive description of gene expression in developing Caenorhabditis elegans embryonic muscle cells. The finding that over half of these muscle-enriched transcripts encode proteins with human homologs suggests that mutant analysis of these genes in Caenorhabditis elegans could reveal evolutionarily conserved models of muscle gene function with ready application to human muscle pathologies. Experiment Overall Design: Our goal is to profile gene expression in the major excitable tissues of Caenorhabditis elegans. As part of this effort, we profiled embryonic muscle cells at two timepoints: 0hr and 24hr.. To isolate transcripts from these cells we utilized the MAPCeL (Microarray Profiling Caenorhabditis elegans Cells) technique, which our lab previously developed (Fox et al 2005) in which myo-3::GFP+ cells are captured by FACS for RNA isolation. We verified these data by bioinformatic means and by in vivo validation by creating GFP reporters for a random set of genes in our enriched gene list.

Dataset Information

Chemical acetylation of ligands and two-step digestion protocol for reducing co-digestion in affinity purification-mass spectrometry

Publications

[Evaluation of the scientific activity through bibliometric indices].

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